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10477754 | A Matchmaker rat brain cDNA library in pGAD10 was purchased from CLONTECH Laboratories and screened according to the manufacturer's instructions. The construct used as bait was full-length γ-adaptin in the vector pGBT9 . The colonies were allowed to grow for 8 d on plates lacking tryptophan, leucine, and histidine, and then replica plated and assayed for β-galactosidase activity. Positive colonies were grown in medium lacking tryptophan but containing leucine and histidine and tested for loss of the pGBT9-γ-adaptin plasmid, and then reassayed for β-galactosidase activity. Only those colonies that tested negative were analyzed further. Plasmid DNA was isolated from eight representative colonies, transformed into Escherichia coli TG2 cells, and analyzed by sequencing and Southern blotting . To find out whether the remaining yeast colonies contained the same plasmids as the test colonies, colony blots were prepared using Hybond-N+ nylon membranes. The membranes were placed on a plate of colonies, transferred (colony side up) onto filter paper soaked in 1 M sorbitol, 0.1 M sodium citrate, pH 7.0, 50 mM EDTA, and 15 mM DTT containing 2 mg/ml yeast lytic enzyme (ICN Biochemicals), and incubated overnight at 308C. Lysis of the cells was completed by incubating the membranes on filter paper soaked with 10% SDS for 5 min. The membranes were treated with NaOH to denature the DNA, neutralized, and alkali fixed . Prehybridization, hybridization, and washing were carried out as previously described . The constructs obtained from the library screen were all tested to determine whether they interacted with any of the other adaptor subunits , and the p34 clone was found to interact with α-adaptin as well as γ-adaptin. To determine whether the binding sites for p34 on the two adaptins mapped to their NH 2 -terminal domains, both constructs were digested with ApaI, which cuts just upstream from the hinge, end repaired, and digested with EcoRI, which cuts at the 5′ end, and ligated into pGBT9 digested with EcoRI and SmaI. The resulting constructs were cotransformed with the p34 clone (in pGAD424) into host cells, and the colonies were assayed for β-galactosidase activity. To obtain the complete coding sequence of γ-synergin, a probe was prepared from the clone obtained in the two-hybrid library screen by random priming and was used to screen a rat brain cDNA library in λgt10 (CLONTECH Laboratories). Library screening, subcloning, and characterization of the clones were all carried out as previously described . The expressed sequence tag (EST) database was also searched and three human cDNAs were identified . These three clones were obtained from the IMAGE Consortium and sequenced in their entirety in both directions, using oligonucleotide primers. In addition, a search of the nonredundant database revealed a partial human genomic sequence for γ-synergin , which enabled us to confirm the EST sequences and to identify the exon-intron boundaries, including the alternative splice sites. A rat multiple tissue Northern blot was purchased from CLONTECH Laboratories and probed according to the manufacturer's instructions. The probe for p34 was the insert from one of the clones from the two-hybrid screen, containing the full coding sequence, whereas the probe for γ-synergin was the insert from the single clone identified in the two-hybrid screen, which encodes the NH 2 -terminal portion of the protein (see below). Both inserts were labeled with [ 32 P]dCTP by random priming . Antibodies were raised against a GST-γ-synergin fusion protein, using the expression vector pGEX3X (Pharmacia). PCR was used to introduce SmaI and EcoRI sites into the insert from the original rat γ-synergin clone (corresponding to amino acids 168–786 of the full-length protein, but missing amino acids 197–274, presumably because the protein is alternatively spliced) so that it could be expressed in the appropriate reading frame. The construct was soluble and was purified on GSH-Sepharose (Pharmacia) according to the manufacturer's instructions. Immunization and affinity purification of the resulting antisera were carried out as previously described . In brief, two rabbits were injected with 0.5 mg of fusion protein in complete Freund's adjuvant, followed by boosts of 0.5 mg fusion protein in incomplete Freund's adjuvant at 2 and 8 wk after the primary injection. 10 d after the final injection, the rabbits were bled out and preimmune and immune sera were tested on blots of the fusion protein. Both immune sera were found to give a strong signal, whereas no signal was obtained with the preimmune sera. The antisera were absorbed with GST and affinity-purified with GST-fusion protein, and then the affinity-purified antisera were absorbed again with GST . Immunoprecipitation of coat proteins from rat liver cytosol was carried out under nondenaturing conditions as previously described . The antibodies included affinity-purified anticlathrin heavy chain, anti–γ-adaptin, and anti–α-adaptin , and the affinity-purified antibody against γ-synergin described above. Western blotting was carried out as previously described . The affinity-purified anti–γ-synergin was used at 1:500. Madin Darby bovine kidney cells were grown on multiwell test slides, fixed with methanol and acetone, and prepared for immunofluorescence as previously described . For some experiments, before fixation the cells were treated with 5 μg/ml brefeldin A (Sigma Chemical Co.), and then either fixed immediately or washed in fresh medium and allowed to recover. To examine the distribution of γ-synergin in cells expressing the chimeric adaptin γγα, stably transfected Rat1 cells were fixed as above. The fixed cells were incubated with mouse anti–γ-adaptin mAb100/3 (Sigma Chemical Co.) , which interacts both with the endogenous (nonrodent) γ-adaptin in the MDBK cells and with the chimeric construct in the transfected Rat1 cells, together with affinity-purified rabbit anti–γ-synergin, followed by fluorescein-labeled donkey anti–rabbit IgG and Texas red-labeled sheep anti–mouse IgG, both obtained from Amersham. The slides were examined in a Zeiss Axioplan fluorescence microscope. The binding sites on γ-adaptin for γ-synergin and on γ-synergin for γ-adaptin were identified using both the yeast two-hybrid system (described above) and GST pulldown experiments. The γ-adaptin ear GST fusion protein has already been described and the a-adaptin ear GST fusion protein , used as a control, was a gift from David Owen (MRC Laboratory of Molecular Biology, Cambridge, UK). The γ-synergin GST fusion proteins were constructed from the clone isolated in the original two-hybrid library screen. The construct GST-γs1 contains the rat sequence corresponding to amino acids 168–517 of the human sequence (but missing amino acids 197–274, presumably because of alternative splicing), the construct GST-γs2 contains the rat sequence corresponding to amino acids 385–661 of the human sequence, and the construct GST-γs3 contains the rat sequence corresponding to amino acids 518–786 of the human sequence. The construct GST-EH contains the EH domain and several amino acids on either side of it, corresponding to amino acids 188–390 of the human sequence (but missing amino acids 197–274). All of the constructs were soluble and were prepared as previously described . For the GST pulldown experiments, 1 ml of rat liver cytosol prepared in PBS containing 0.1% NP-40 and a protease inhibitor cocktail (Complete Mini; Boehringer Mannheim) at a protein concentration of 1.5 mg/ml was first precleared by incubating for 8 h at 4°C on a rotating wheel with 10 μg GST and 60 μl of a 50% suspension of glutathione-Sepharose 4B (Pharmacia). The samples were incubated with 10 μg of the appropriate fusion protein and 60 μl of the 50% suspension of glutathione-Sepharose overnight at 4°C. The beads were pelleted, washed five times in PBS containing 0.1% NP-40, eluted with SDS-PAGE sample buffer, and subjected to SDS-PAGE and Western blotting. A blot overlay assay was used to demonstrate that g-adaptin and γ-synergin bind directly to each other. First, the insert from the GST-γs3 construct was ligated in-frame into the vector pQE30 to introduce a histidine tag at the NH 2 terminus (Qiagen). Expression and purification of the resulting His-γs3 construct were carried out as instructed by the manufacturer. A control construct, His-DHFR (supplied with the kit), was also expressed and purified. Equivalent amounts of the two His-tagged fusion proteins were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane, and the blot was blocked with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 0.5% BSA, 3 μM reduced glutathione for 30 min. This buffer was used in all the following steps. The blot was incubated with 10 nM GST or γ-ear-GST for 45 min, washed for 30 min, and then labeled as described above using anti-GST followed by 125 I–protein A. Full-length mouse γ-adaptin cDNA was used as bait to screen a yeast two-hybrid library containing inserts derived from rat brain cDNA. Out of ∼6 × 10 6 transformants, 62 clones were isolated that exhibited a strong and specific interaction between the g-adaptin construct and the library construct. To determine the identity of each of the 62 clones, both colony blotting and sequencing were carried out. The results are shown in Table . Six of the clones were found to contain plasmids encoding σ1 (σ1A), the small chain subunit of the AP-1 adaptor complex. We have previously shown that σ1 and γ-adaptin interact strongly in the yeast two-hybrid system, so the isolation of a σ1-encoding plasmid confirmed that the library screen had worked . Another six plasmids were found to encode a protein closely related to s1, with 87% amino acid identity. This protein has been independently identified by Takatsu et al. 1998 and named σ1B. Eleven plasmids were found to encode β-spectrin. The significance of this interaction is at present unclear (see Discussion). The other 39 plasmids were found to encode two unknown proteins, and these cDNAs were subjected to further analysis. The first of the two unknown cDNAs was isolated with very high frequency in the two-hybrid screen, accounting for more than half of all the clones. A representative clone with an insert of ∼2.5 kb was sequenced and was found to encode a protein of 315 amino acids with a deduced size of ∼34 kD (p34) . There are several mammalian ESTs in the database encoding p34, but no homologues were found that might help to establish the protein's function. Northern blotting demonstrated that p34 is expressed ubiquitously and that the mRNA has a size of ∼2.75 kb . Unlike most of the other proteins identified in the screen, p34 was found to interact not only with γ-adaptin but also with α-adaptin in the two-hybrid system, and this interaction was mapped to the NH 2 -terminal domains of the two adaptins . Attempts were made to raise antisera against p34, but unfortunately the protein proved to be a very poor antigen. Thus, although two different domains were expressed as fusion proteins for antibody production, and although the resulting antisera were affinity-purified, all of the antisera labeled multiple bands on Western blots. However, one of the antisera labeled a band of around the expected size (∼37 kD), and this protein could be immunoprecipitated in substoichiometric amounts with cytosolic AP-1 and AP-2, suggesting that the interactions detected in the two-hybrid system are physiologically relevant (data not shown). But because we were looking for proteins that interact specifically with the AP-1 complex, p34 was not characterized further. The other unknown cDNA came up with the least frequency in the library screen, accounting for only one of the clones. Northern blotting showed that the transcript is expressed ubiquitously and has a size of ∼4.4–5.6 kb, with bands of different mobilities labeled in different tissues . The original clone contained an insert of only ∼1.6 kb and the sequence appeared to be all open reading frame, so this clone was used as a probe to screen a rat brain λgt10 cDNA library to try to obtain a full-length sequence. Three additional clones were isolated and sequenced . A comparison of the four sequences revealed that the mRNA is alternatively spliced, consistent with the heterogeneity seen on the Northern blot. A putative start was identified in one of the clones, but none of the clones had an in-frame stop codon at the 3′ end. However, when the EST database was searched with the rat sequences, three human sequences were found, and from the corresponding cDNAs it was possible to assemble a contiguous human open reading frame. The nonredundant database was also searched with both the rat and the human sequences, and the human genomic sequence encoding the 3′ end of the mRNA was found. Fig. 2 c shows the genomic structure, including the alternative splice sites. An analysis of the open reading frame revealed that the protein contains an EH domain at amino acids 295–377 . EH domains, which bind to proteins containing the sequence NPF, have now been found in a large number of proteins, including the mammalian proteins Eps15, Ese1, and Ese2, and the yeast proteins End3p and Pan1p, all of which are involved in endocytosis . γ-Adaptin contains no NPF sequences, so it is likely that the novel protein serves as a linker between γ-adaptin and some other unidentified protein. We propose that this protein be called γ-synergin (from the Greek, synergos, meaning partner or workmate). A schematic diagram of γ-synergin is shown in Fig. 3 a, indicating the positions of the EH domain, some of the alternative splice sites, and the γ-adaptin–binding domain (see below). Fig. 3 b shows an alignment of the EH domains from γ-synergin, Eps15, Ese1, End3p, and Pan1p. The EH domain of γ-synergin can be seen to contain all of the highly conserved amino acids found in other, well characterized EH domains. However, apart from the EH domain, γ-synergin shows no significant sequence homology to any other proteins in the database, and it does not share any of the other features found in the α-adaptin binding partner Eps15 such as a coiled-coil domain or a proline-rich region. To learn more about the function of γ-synergin, the original clone identified in the two-hybrid library screen was expressed as a fusion protein for antibody production. Fig. 4 shows a Western blot of equal protein loadings of homogenate from both brain and liver as well as various subcellular fractions from liver probed with the affinity-purified antibody. Two bands are labeled in the brain of ∼110 and ∼150 kD, whereas in the liver, a single band is labeled of ∼190 kD. This is consistent with the Northern blot in which a single band was labeled in liver, whereas two bands were labeled in brain, indicating that the different protein species might represent different spliced variants, although we cannot rule out the possibility that the differences might also be due to proteolysis. γ-Synergin is found in both a high speed supernatant and membrane-containing pellet, indicating that it is peripherally associated with membranes. It is somewhat enriched in a TGN-enriched fraction from liver and it is strongly enriched in liver clathrin-coated vesicles. The association between γ-synergin and γ-adaptin was confirmed by immunofluorescence microscopy. Double labeling of MDBK cells with anti–γ-synergin and anti–γ-adaptin revealed a striking degree of colocalization of the two proteins . Next, we investigated whether the membrane association of g-synergin is affected by the drug brefeldin A (BFA). This drug causes ARF to dissociate rapidly from membranes , leading to the dissociation of other peripheral membrane proteins whose membrane association is ARF-dependent, such as the AP-1 adaptor complex . In Fig. 5c and Fig. d , MDBK cells were treated with BFA for 2 min, and then double labeled for γ-synergin (c) and γ-adaptin (d). Both proteins can be seen to have redistributed to the cytoplasm. To examine the behavior of the two proteins upon BFA washout, we treated cells with the drug for 30 min, and then allowed them to recover for 2 min . Both proteins can be seen to have reassociated with the membrane, which now has a more tubular appearance as a result of the BFA treatment. Thus, γ-synergin, like the AP-1 adaptor complex, appears to associate with the TGN membrane in an ARF-dependent manner. The immunofluorescence data demonstrate that γ-synergin is associated with AP-1 on TGN membranes. To find out whether the two proteins are also associated in the cytosol, immunoprecipitations were carried out under nondenaturing conditions . Rat liver cytosol was immunoprecipitated with anti–γ-adaptin followed by protein A–Sepharose, and Western blots were probed with anti–γ-synergin or anti–γ-adaptin. As a control, cytosol was also immunoprecipitated with anti–a-adaptin. Cytosolic γ-synergin was found to coprecipitate with γ-adaptin but not with α-adaptin. Thus, γ-synergin is associated with AP-1 in the cytosol as well as on membranes. To identify the domain on γ-adaptin that binds to γ-synergin and the domain on γ-synergin that binds to γ-adaptin, two approaches were used: yeast two-hybrid analysis and GST pulldown experiments. The NH 2 -terminal domain construct of γ-adaptin, which was found to interact with p34 in the two-hybrid system , did not interact with γ-synergin nor did a γ-adaptin construct with the a-adaptin ear, indicating that g-synergin binds to the ear domain of γ-adaptin (data not shown). This was confirmed using GST fusion proteins to isolate binding partners in rat liver cytosol, followed by Western blotting and probing with anti–γ-synergin. Fig. 7 a shows that GST fused to the γ-adaptin ear binds γ-synergin, whereas GST alone or GST fused to the α-adaptin ear do not. The same general strategy was used to identify the domain of γ-synergin that binds to γ-adaptin. Four sets of constructs were made from the original γ-synergin clone isolated in the two-hybrid library screen: one containing just the EH domain (GST-EH); one containing the NH 2 -terminal half, including the EH domain (GSTγs-1, corresponding to amino acids 168–517 of the human sequence but missing amino acids 197–274, presumably because of alternative splicing); one containing the middle portion of the protein (GSTγs-2, amino acids 385–661); and one containing the COOH-terminal half (GSTγs-3, amino acids 518–786). Only the construct containing the more COOH-terminal portion of g-synergin (GSTγs-3) bound γ-adaptin in the GST pulldown experiments , and this interaction was confirmed using the two-hybrid system (data not shown). The GST pulldown experiments were carried out using whole cytosol, and, thus, they cannot distinguish between a direct interaction between γ-synergin and γ-adaptin and an indirect one, mediated by another protein or proteins. The ability of the two proteins to interact in the yeast two-hybrid system strongly suggests that the interaction is direct, but to prove this formally, we also carried out Western blot overlay experiments, a technique that has been used to demonstrate that the α-adaptin ear domain binds directly to proteins such as amphiphysin and epsin . For these experiments, the portion of γ-synergin that contains the γ-adaptin–binding domain, amino acids 518–786, was expressed as a histidine-tagged construct (His-γs3) and purified on a nickel affinity column. A control histidine-tagged construct was also expressed and purified (His-control). The two constructs were subjected to SDS-PAGE, blotted, and probed either with GST alone followed by anti-GST, with GST-γ ear followed by anti-GST, or with anti–γ-synergin. Fig. 7 c shows that the GST-γ ear construct, but not GST alone, binds to the His-γs3 band on the Western blot. Thus, the interaction between γ-adaptin and γ-synergin must be a direct one. The binding site on γ-synergin for γ-adaptin is indicated in Fig. 3 a. We have previously shown that the COOH-terminal ear domains of γ- and α-adaptin contain weak targeting signals for recruitment onto the TGN and plasma membranes, respectively . Thus, a construct containing mostly a-adaptin, but with the γ-adaptin COOH-terminal domain, coassembles with the three subunits normally found in the AP-2 complex and is mainly associated with the plasma membrane, although a small fraction is seen on the TGN. Similarly, a construct containing mostly γ-adaptin, but with the α-adaptin COOH-terminal domain, coassembles with the subunits normally found in the AP-1 complex and is mainly associated with the TGN, although a small fraction is seen on the plasma membrane. We and others have long been interested in identifying the membrane docking sites for coat proteins, and although it is clear that γ-synergin cannot be the only docking site for AP-1, since the a-adaptin chimera with the γ-adaptin ear goes mainly to the plasma membrane, it is possible that it might participate in AP-1 recruitment. Alternatively, γ-synergin may be localized to the TGN because of its association with AP-1 rather than vice versa. To distinguish between these two possibilities, i.e., to determine whether γ-synergin leads AP-1 onto the TGN or follows it there, we examined the distribution of γ-synergin in cells expressing a chimera consisting of the α-adaptin NH 2 -terminal domain with the γ-adaptin hinge and ear (agg). If γ-synergin helps to recruit AP-1, we would expect its distribution to be unchanged in such cells, However, if AP-1 recruits g-synergin, we would expect some of the γ-synergin to be rerouted to the plasma membrane. Fig. 8 clearly shows that the latter is the case. In the transfected cell expressing the chimeric adaptin, much of the γ-synergin labeling shows the characteristic punctate plasma membrane pattern (b), colocalizing with the chimera (a). Thus, γ-synergin follows AP-1 onto the appropriate membrane rather than leading it there. The yeast two-hybrid system has proved to be a powerful way of investigating protein–protein interactions that may be difficult to study by more conventional biochemical methods. Among the advantages of the two-hybrid system are that it can detect interactions that may occur only transiently in the cell, and that it can be used not only to identify but also to clone the binding partners of a protein of interest. Its disadvantages are that it sometimes fails to pick up protein–protein interactions that normally occur in the cell, while at the same time revealing interactions that may occur in the two-hybrid system, but not under more physiological conditions. In the present study, we have used this approach to search for γ-adaptin binding partners and have cloned cDNAs encoding five different proteins: σ1A, σ1B, β-spectrin, p34, and γ-synergin. The cloning of s1A acts as a positive control, since we previously showed that it interacts strongly with γ-adaptin in the two-hybrid system . The finding that σ1B also binds to γ-adaptin is consistent with the findings of Takatsu et al. 1998 , who also found that both isoforms of s1 can interact with the same isoform of γ. Northern blotting reveals that both isoforms of s1 are expressed ubiquitously . Although we attempted to raise monospecific antibodies that recognize σ1B but not σ1A by immunizing and cross-absorbing with different fusion proteins, so far we have not succeeded, presumably because of the high degree of homology between the two proteins. Thus, at present we do not know whether there are any functional differences between the two σ1 isoforms. In our previous study in which we investigated interactions between neighboring adaptor subunits using the two-hybrid system, we found that γ-adaptin binds not only to σ1, but also to β1 and (to a lesser extent) to β2 . However, we did not pick up either of the two β-adaptins in the library screen. This appears to be because under the more stringent conditions used to screen a two-hybrid library, the interaction between γ-adaptin and β1 or β2 is not strong enough to produce a signal (data not shown). Another potential γ-adaptin binding partner is p75, which can be cross-linked to membrane-associated γ-adaptin . However, none of the proteins that we identified in the screen has a molecular mass of ∼75 kD. It is not clear why we failed to clone this protein, but one possibility is that the interaction between p75 and γ-adaptin does not occur when the two are expressed as fusion proteins in yeast (e.g., if p75 only interacts with γ-adaptin when it is incorporated into the AP-1 complex). Alternatively, if p75 is an integral membrane protein, the presence of a transmembrane domain may prevent it from entering the nucleus, which is where it must be to be detected by the two-hybrid system. The cloning of β-spectrin was unexpected and it is not yet clear whether its interaction with γ-adaptin is physiologically relevant. Although spectrin was initially assumed to be associated only with the plasma membrane, a number of immunofluorescence studies using certain antibodies against erythrocyte β-spectrin have indicated that an isoform of the protein is associated with the Golgi apparatus, and recently a novel member of the β-spectrin family, bIII spectrin, was cloned and localized to the Golgi region of the cell . However, sequencing indicates that the β-spectrin isoform that we cloned as a γ-adaptin binding partner is not βIII spectrin but βII spectrin, which has been localized to the plasma membrane. Future studies should show whether γ-adaptin can bind to βIII spectrin as well as to βII spectrin, and whether the two proteins can associate with each other in the cell. The protein that came up most frequently in the screen, p34, is unusual in that it interacts with both γ-adaptin and α-adaptin. This interaction was mapped to the NH 2 -terminal domains of the two adaptins, which is where γ and α show the most homology, although even here they are only 32% identical. Another clue as to the function of p34 comes from the observation that it can be coimmunoprecipitated with soluble adaptor complexes, both AP-1 and AP-2, although it is not enriched in purified clathrin-coated vesicles. This suggests that p34 may play some sort of chaperone role. For instance, it could help to prevent the soluble adaptors from coassembling with soluble clathrin, or it could participate in uncoating by helping to remove the adaptors from the coated vesicle. Another possibility is that p34 may aid in the recruitment of soluble adaptors onto the membrane. However, it is clear that it cannot be involved in the specificity of adaptor recruitment, since it appears to interact equally well with both adaptor complexes. Potentially the most interesting of the proteins that we isolated is γ-synergin. This protein colocalizes with AP-1 by immunofluorescence, and it can be coimmunoprecipitated with cytosolic AP-1. It binds specifically to the COOH-terminal ear domain of γ-adaptin, the same domain that, on a-adaptin, binds to at least three different partners. The COOH-terminal ear domains of both γ and α have also been implicated in the recruitment of the AP-1 and AP-2 complexes onto their respective membranes, although the major targeting information appears to reside in the adaptor heads. However, γ-synergin is a peripheral membrane protein, not an integral membrane protein; its sensitivity to BFA indicates that it associates with the TGN in an ARF-dependent manner, and we have previously shown that the only soluble proteins required for AP-1 recruitment are the AP-1 itself and ARF-1 . In addition, in cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, a substantial amount of the γ-synergin also goes to the plasma membrane. These observations indicate that γ-synergin is recruited onto the membrane through its interaction with AP-1 rather than vice versa, and, thus, that it does not play any part in targeting the AP-1 complex to the appropriate membrane. What, then, is the function of γ-synergin? The presence of an EH domain indicates that, like Eps15 (the first EH domain-containing protein to be characterized) it is an adaptor for an adaptor. Eps15 interacts with the ear domain of the α-adaptin subunit of the AP-2 complex although, like γ-synergin, its adaptin binding site is distinct from its EH domain. The α-adaptin binding site on Eps15 is quite large, comprising over 100 amino acids and including multiple repeats of the tripeptide DPF . The γ-adaptin binding site on g-synergin shows no homology to the α-adaptin binding site on Eps15, and no DPF sequences are present in γ-synergin. However, it may be relevant that there are five repeats of the sequence DDFXD/EF, three of which (at positions 668–673, 689–694, and 774–779) are within amino acids 518–786, which we mapped as the γ-adaptin binding site . The other two copies of this sequence are outside of this region ; however, preliminary evidence suggests that the true γ-adaptin binding site may encompass more than amino acids 518–786. Although only the construct containing this sequence interacted with the γ-adaptin ear domain in GST pulldown experiments, when interactions between γ-adaptin and γ-synergin were assayed using the two-hybrid system, clones containing amino acids 168–517 and 385–661 as well as 518–786, but not the clone containing the EH domain alone, produced positive results (data not shown). We now intend to investigate whether the DDFXD/EF sequence is required for g-adaptin binding. How much of the γ-adaptin and γ-synergin in the cell are associated with each other? Western blots of AP-1 immunoprecipitated from cytosol under nondenaturing conditions show that γ-synergin coprecipitates ; however, under conditions where AP-1 subunits can be seen by Coomassie blue staining, no γ-synergin band can be seen, indicating that the interaction is substoichiometric (Sowerby, P.J., and M.S. Robinson, unpublished observation). When we immunoprecipitate γ-synergin under nondenaturing conditions, we are unable to detect any γ-adaptin by Western blotting (Sowerby, P.J., and M.S. Robinson, unpublished observations). This is presumably because the anti–γ-synergin antibody, which was raised against the portion of the protein that we isolated in the two-hybrid screen, binds to the same site on γ-synergin as γ-adaptin. Thus, it is clear that although some of the AP-1 and γ-synergin are associated with each other in the cytosol, there are also unoccupied pools of both proteins. At present, we do not know whether the γ-synergin and AP-1 that are associated with each other are stably bound, or whether the interaction is more dynamic. In addition to its association with γ-adaptin, we would predict that γ-synergin has at least one additional binding partner, which would interact with its EH domain. So far, attempts to screen a yeast two-hybrid library with the EH domain of γ-synergin have been unsuccessful, nor have we identified any candidates by GST pulldown or blot overlay experiments using the γ-synergin EH domain. However, when we have carried out GST pulldown experiments with the γ-adaptin ear construct, we find that other proteins come down in addition to γ-synergin (Liu, W.W., P.J. Sowerby, and M.S. Robinson, unpublished observations), which could interact either directly with γ-adaptin or indirectly, via γ-synergin or another γ ear binding partner. We now intend to identify and characterize these proteins, to see whether they are related to any of the AP-2 interacting proteins, or whether they contain NPF sequences. One potential (indirect) γ-adaptin binding partner might be an isoform or homologue of dynamin, such as dynamin 2, which has been implicated in trafficking from the TGN . However, the lack of sequence homology between the γ ear and the α ear suggests that there may also be binding partners that are entirely specific for one or the other of the two AP complexes. Some of the proteins associated either directly or indirectly with AP-2, including Eps15 , amphiphysin , dynamin , and epsin , have been used to create dominant negative mutants by overexpressing truncated or mutated forms of the protein. Thus, for instance, a construct consisting of just the α-adaptin binding portion of Eps15 is a potent inhibitor of clathrin-mediated endocytosis . It should be possible to use the same strategy with γ-synergin. Preliminary experiments in which we transiently transfected cells with a truncated form of γ-synergin, consisting of amino acids 168–786 (the original two-hybrid clone), indicate that this construct is toxic to cells, so we are now developing inducible systems. These studies should help to define both the role of g-synergin and the role of the AP-1 pathway in general. Although there is abundant evidence that AP-1 is involved in the trafficking of newly synthesized lysosomal proteins from the TGN to an endosomal or prelysosomal compartment, it may have other functions as well. AP-1 has been localized not only to the TGN, but also to early/recycling endosomes and (in cells with a regulated secretory pathway) to immature secretory granules , and it has been proposed that it may participate not only in trafficking to prelysosomes, but also in transcytosis , the recycling of proteins from the endosome back to the plasma membrane , transport from the early endosome to the TGN , and the removal of nongranule proteins from the immature secretory granule . Another possibility is that the AP-1 pathway may be used to transport not only lysosomally directed proteins, but also some of the proteins destined for the plasma membrane, from the TGN to an endosomal compartment. It has also been proposed that AP-1 may play a specialized role in polarized epithelial cells, and interestingly there is an isoform of the m1 subunit, m1B, which is expressed exclusively in epithelial cells and tissues . So far, most of our knowledge of the AP-1 pathway has come from immunolocalization and in vitro binding experiments. If we can inactivate the AP-1 pathway experimentally, it should be possible to carry out the same types of functional studies, making use of living cells, that have been so informative in the case of AP-2 and AP-3. | Study | biomedical | en | 0.999998 |
10477755 | All stocks were grown at 258C in cornmeal media. Standard techniques of fly manipulation were used. w 1118 or Oregon R was used as wild-type. Details of balancer chromosomes and mutations are described in Lindsley and Zimm 1992 . A reversion test of msps P was carried out as described in Ohkura et al. 1997 . yw;msps P /TM6C females were crossed with l(3)/TM3 , P (Δ2-3) males to obtain jumpstarter males, yw;msps P /TM3 , Sb P (Δ2-3). Individual males were crossed back to yw; msps P /TM6C females. From 21 crosses, 18 gave viable revertants without balancer chromosomes marked with Sb . Out of 26 viable revertants examined from 17 independent crosses, 22 have lost the w + marker derived from the P-lacW at 89B in msps P , indicating the P-lacW is responsible for the mutation. To isolate new alleles of the msps mutant, remobilization of the P-lacW was performed as above. Each independent w − revertant chromosome was tested over the msps P mutation. Squashed preparations of central nervous systems from third instar larvae were examined by orcein staining as previously described . Whole mount preparations of larval central nervous systems were prepared for immunostaining according to Gonzalez and Glover 1993 with the following modifications. Central nervous systems were dissected in 0.7% NaCl with 5 mM EGTA (pH 8) and fixed in 10% formaldehyde in 0.7% NaCl followed by two washes in 0.7% NaCl. This method gave an identical spindle structure in wild-type or the msps P mutant to that obtained by other methods, such as by incubation in taxol followed by 3.7% formaldehyde fixation, or by dissection and fixing in 10% formaldehyde. Immunostaining of embryos was carried out using methanol or 37% formaldehyde as a fixative according to Gonzalez and Glover 1993 with omission of taxol. Although both fixation methods gave identical staining, methanol fixation consistently gave stronger and clearer Msps staining and was routinely used. TAT1 , YL1/2 or YOL1/34 were used for α-tubulin staining. Rb188 or Bx63 were used for CP190 staining. Monoclonal and polyclonal antibodies were used at 1/10–1/100 and 1/500–1/1,000, respectively. FITC-, Cy3-, Cy5- (Jackson Lab), or Alexa488-conjugated (Molecular Probes) secondary antibody was used at 1/500–1/2,000. Absence of cross-species reaction was confirmed by ourselves. DNA was counterstained with 0.2 μg/μl of DAPI or 1 μg/μl of propidium iodide with 100 μg/μl of RNase. Microtubules were artificially induced in embryos by incubation with PBS containing 10 μM of paclitaxel (ICN) and heptane for 5 min before fixation. To depolymerize microtubules, embryos were incubated with 30 μg/ml of colchicine (Sigma Chemical Co.) for 10 min after permeabilization by octane according to Gonzalez and Glover 1993 . Weak tubulin staining remained around centrosomes even after longer incubation, treatment with nocodazole, 1 mM calcium, or cold temperature, alone or in combination. Samples were examined and images were collected using an Optiphot (Nikon) microscope with a confocal scan head , or an Axioskop or Axioplan2 (Zeiss) attached with CCD camera (Princeton or Hamamatsu). Figures were prepared using Photoshop (Adobe). Standard DNA manipulation techniques were followed. A genomic fragment flanking the P-lacW was isolated by plasmid rescue from msps P /TM6C adult flies after digestion of the genomic DNA with EcoRI. This was then used to isolate two overlapping cosmids (104B04, 129C05) from a wild-type library . In situ hybridization to wild-type polytene chromosomes was carried out according to Saunders et al. 1989 to confirm that the plasmid-rescued fragment and the cosmids were derived from chromosomal region 89B. The 12-kb SpeI fragment and the 4-kb EcoRI fragment around the P-lacW insertion site was subcloned and sequenced. One of four transcription units in the region was shown to correspond to the msps gene through rescue of msps P mutation by the transgenic wild-type genomic fragment. The 5′ end of the transcript was determined using 5'/3'RACE kit (Boehringer). EST cDNA clones made by the Berkeley Drosophila Genome Project were obtained through Genome Systems or Research Genetics. DNA sequences were determined using the ABI dye termination kit and automatic sequencer. Database searches were carried out using BLAST and prediction of coiled-coil structure was carried out using the method by Lupas et al. 1991 . The SpeI–NotI 12-kb fragment from cosmid 104B04 was subcloned into pW8 . The resulting plasmid (pHN267) was used for germline transformation of w 1118 flies by coinjection with Δ2-3 plasmid (pπ25.1) into embryos. Two independent insertions on the 2nd chromosome, were used to test for rescue of the msps P mutation. P [ w + , HN267]/+; msps P /TM6B males (or msps P /TM6B sibling males as a control) were crossed with msps P /TM6C females. Both rescue mitotic defects in central nervous systems and fully restored growth of imaginal discs in homozygous third instar larvae of msps P , although one of them supported development only until the pharate stage, suggesting the insertion site may affect expression of the gene. A control plasmid (pHN276 containing a SpeI-EcoRI 3.3-kb fragment) that lacks most of the msps gene was tested for rescue of the msps P mutation by the same procedure. Two independent insertions tested failed to restore viability, disc growth or normal mitosis. Presence of the transgenic construct in each larvae examined for mitotic defects was positively identified by PCR. Standard protein and immunological techniques were followed. For immunoblotting, peroxidase-conjugated secondary antibodies (Jackson Lab) were used and detected using the ECL kit (Amersham). Total protein samples from Drosophila tissues were prepared by homogenization in SDS sample buffer. Preheating the samples at 1008C helps to prevent protein degradation. Embryos were dechorionated by bleach before preparation of samples. pHN264 containing the BamHI-XhoI 1.5-kb fragment of cDNA in pET-23a (Pharmacia) was used to express amino acids 1,349–1,784 of Msps protein in E. coli . The polypeptide was initially purified in inclusion bodies then further purified by elution from an SDS gel. A rabbit was immunized with the antigen every 4 wk and antisera were taken a week after immunization by Scottish Antibody Production Unit. The antisera collected on 24.9.98 was used after affinity purification throughout the experiments described in this paper. The antibody was affinity-purified from antisera by incubation with the antigen on nitrocellulose strips followed by low pH elution according to Smith and Fisher 1984 . Crude antiserum and affinity-purified antibody both gave similar staining predominantly on mitotic spindles and centrosomal regions in embryos. Affinity purification of the antibodies using Msps protein resulted in reduced staining of cytoplasm without affecting the intensity or pattern of staining of the mitotic spindles or the centrosomal region. Preimmune serum taken from the same rabbit did not stain mitotic spindles or the centrosomal region even at a high concentration of the serum. Exclusion of the primary antibody against Msps eliminates the staining even when it was costained with other primary and secondary antibodies, indicating that the secondary antibody alone, cross reaction of secondary antibodies or leaking through between the channels is not responsible for the staining. Microtubule preparation was carried out according to Barton et al. 1995 and Saunders et al. 1997 with some modification. After dechorionation with bleach, 0–6-h-old embryos were homogenized in PEM buffer containing a cocktail of protease inhibitors and 1 mM dithiothreitol. The homogenate was incubated at 4°C for 30 min and spun at 140,000 g at 4°C for 30 min. A final concentration of 20 μM of taxol (paclitaxel; ICN) and 1 mM GTP was added before incubation at room temperature to polymerize microtubules. The microtubules and associated proteins were pelleted by spinning at 80,000 g for 30 min through a 30% sucrose cushion in the buffer. The supernatant and pellet fractions were analyzed by SDS–polyacrylamide gel electrophoresis. The mini spindles ( msps ) mutant was identified through a cytological screen for mutants with mitotic defects in the larval central nervous systems from ∼1,000 lethal or semi-lethal lines in a collection of third chromosome P-insertion mutants . As this mutant is induced by a P-lacW insertion, we call this allele msps P . msps P homozygotes die around the larval/pupal transition. Mutant third instar larvae are superficially normal in size and behavior (they were capable of feeding and crawling) although they grow more slowly than wild-type. They die before or after pupation and no development beyond early pupal stage is observed. Dissection of late third instar larvae from msps P homozygotes revealed that imaginal discs are missing or very small and the size of the central nervous systems is also reduced. Polytenised tissues, such as the salivary glands and fat bodies, do not appear to be affected. These observations suggest that msps gene activity is essential for mitotic cycles but is required at significantly lower levels or not at all for endoreduplication cycles. To examine mitosis in the msps P mutant, we dissected central nervous systems from late third instar larvae and stained chromosomes of squashed preparations with aceto-orcein. In most mitotic cells, the degree of chromosome condensation is considerably higher than that seen in wild-type , and sister chromatids are attached together at heterochromatic regions. A high level of chromosome condensation is typical when the cells are blocked in mitosis as chromosome condensation continues during the arrest. Consistent with this, higher frequencies of mitotic cells are detected in central nervous systems from the msps P mutant , where an average of the mitotic index is roughly twice that of wild-type. The most striking feature observed in the msps P mutant is the very low frequency of anaphases . The frequency of anaphases among mitotic cells is only 3% in comparison to the 23.5% seen in wild-type. In addition, polyploid cells were found but the frequency is low (2%) in msps P mutants. The high degree of chromosome condensation, high mitotic index, and low frequencies of anaphases indicate that cells are blocked in mitosis before sister chromatid separation. Among the rare anaphases we observed V-shaped configurations in which chromosomes appeared to be distributed to three poles in a significant proportion (25%) of cells . These anaphase cells appear to have a diploid complement of chromosomes that have undergone separation of sister chromatids. Although one set of sister chromatids moves to one pole, the other set of sister chromatids appear to be divided in their movement to two distinct poles. All chromatids appear to move synchronously and none of the chromosomes are left behind. To examine mitotic spindle structure, we fixed whole central nervous systems dissected from late third instar larvae for immunostaining to visualize microtubules, chromosomes, and the centrosomal antigen, CP190 . We found that only 28% of mitotic cells formed an apparently normal bipolar spindle while the rest showed abnormal structures . The most common abnormalities (36% of total mitotic cells) are cells containing more than one bipolar spindle forms . As shown in Fig. 2 b, most chromosomes are aligned at the metaphase plate and associated with a bipolar spindle, but a few of them have become separated from other chromosomes and associate with an additional smaller bipolar spindle. The two bipolar spindles typically share one of the poles. The major bipolar spindle usually has CP190 staining at both poles, whereas the unshared pole of the other spindle typically has no CP190 staining. The poles of both bipolar spindles are focused. In some cases, chromosomes appeared still to be aligned on a common metaphase plate even though the spindle had bifurcated and the metaphase plate appears to be kinked . In other cases, a large bipolar spindle is no longer observed, but instead there are multiple small bipolar spindles associated with individual chromosomes . Multipolar spindles are often observed in polyploid cells which can be caused by either failure of mitosis or cytokinesis. However, the multiple bipolar spindle phenotype seen in the msps P mutant is unlikely to be a consequence of the cell becoming polyploidy because orcein staining of squashed samples from the same strain indicates that polyploid cells are rare (2%). 12% of mitotic cells have one bipolar spindle and one monopolar spindle , but a monopolar spindle is rare (3%). A significant proportion (16%) of mitotic cells have mitotic spindles that have disintegrated so that their exact structure cannot be determined. They contain many short but thick microtubule bundles associated with the chromosome mass . Often no discrete CP190 staining was observed. These could represent different classes of structural abnormality, or alternatively such cells may belong to the class with small bipolar spindles associated with the mass of chromosomes. To gain insight of the molecular nature of the msps mutation, we intended to clone the wild-type msps gene. First we mapped the position of the P-lacW to 89B by in situ hybridization to polytene chromosomes. To confirm that the P-lacW insertion at 89B is responsible for the mutation we remobilized the P-lacW in msps P and obtained viable revertants at a high frequency (see Methods and Materials). Reversion of the lethality correlates with loss of the w + marker derived from the P-lacW insertion at 89B. The revertants have no mitotic defects or disc abnormalities. These results indicate that the P-lacW at 89B is responsible for the lethality and the mitotic defects seen in msps P mutants. A fragment of genomic DNA flanking the P-lacW was isolated by plasmid rescue and used as a probe to isolate overlapping cosmid clones from a genomic library . Two cosmid clones covering over 30 kb around the P-lacW insertion were identified. We have sequenced a total of 14 kb around the P-lacW insertion site and identified four transcription units in the region . To determine which transcription unit is responsible for the msps phenotype, we undertook rescue of the msps phenotype by wild-type genomic fragments. A 12-kb wild-type genomic fragment containing two of the transcription units was introduced into the germline by P element–mediated transformation. This construct HN267 was able to restore the viability and fertility of the msps P mutant. In contrast HN276, in which one of the transcription units is truncated, was not capable of rescuing the viability and fertility of the msps P mutant. We then examined whether HN267 can rescue the mitotic defects of msps P and found the frequency of anaphases to be restored from 4 to 24% comparable to wild-type levels and all other mitotic defects rescued. In contrast, the control, HN276, failed to rescue the mitotic defects of msps P . These results indicate that one of the transcription units corresponds to the msps gene. RACE (rapid amplification of cDNA end) analysis indicates that msps gene has a 0.9-kb intron in the 5′ nontranslated region and that the P-lacW in msps P is inserted in the intron. It is likely that the P-lacW insertion interferes with the transcription or splicing in the msps P mutant, and therefore disrupts the production of msps protein (see below). The sequence of the msps gene predicts it to encode a protein of 2,050 amino acids, with an estimated molecular mass of 227 kD and an isoelectric point of 8.4. Database searches revealed that the entire region of the Msps protein has striking similarity to the human TOG protein . Further analysis identified a family of proteins which share small regions of similarity. This family, which here we call the dis1-TOG family, consists of proteins encoded by Schizosaccharomyces pombe dis1 , Saccharomyces cerevisiae STU2 , Caenorhabditis elegans zyg-9 , Xenopus laevis XMAP215 , and human ch-TOG, all of which have been reported to have microtubule binding activity. The common signature among all members of the family is limited to four separate motifs within a repeated sequence unit . The repeats are each ∼200 amino acids long and tandemly arranged in the amino terminus with ∼100 residue spacers. Interestingly the two yeast proteins and the C. elegans ZYG-9 have only two repeats, while those from Drosophila and vertebrates have four. The similarity between human TOGp and Drosophila Msps extends along nearly the entire COOH-terminal half, while there is no similarity beyond the repeats between the yeast proteins and the higher eukaryotic proteins . The C. elegans ZYG-9 protein shares similarity with only half of the COOH-terminal region of the higher eukaryotic proteins. Although there are no sequence similarities in the COOH-terminal half at the primary sequence level between Dis1 and STU2, the entire region is predicted to have extensive coiled-coil structure. In contrast the Msps and TOG proteins do not have an extensive predicted coiled-coil structure. These sequence characteristics suggest that Drosophila Msps and vertebrate proteins form a distinct higher eukaryotic subfamily of the dis1-TOG family. To characterize the msps gene product, we raised a polyclonal antibody against Msps protein (Materials and Methods). The affinity-purified antibody recognizes mainly one polypeptide of ∼220-kD in immnoblots. To confirm that the antibody specifically recognizes Msps protein, protein samples were prepared from wild-type and msps P homozygous late third instar larvae . The 220-kD band and other minor bands are greatly reduced in the msps P homozygote. It can be argued, however, that as the sizes of imaginal discs are reduced in the mutant, any proteins highly expressed in discs can be reduced. To eliminate this possibility we used a semi-lethal allele, msps MJ15 which we created by P-element remobilization. Adult males from this allele are morphologically normal except for weak rough eyes and a few missing bristles, and have normal testis with no spermatogenesis defects. The quantity of 220-kD protein and smaller proteins recognized by the antibody is significantly reduced . These minor smaller proteins sometimes recognized by this antibody are likely to be degradation products as their quantity is greatly reduced in msps mutants and is variable in one preparation to another. These results indicate that the affinity-purified antibody specifically recognizes Msps protein. To examine the expression of Msps protein during wild-type development, protein samples were prepared from various stages of Drosophila development, and comparable amount of total protein was loaded for immunoblotting . As we expected, a large amount of Msps protein is found in embryos that undertake rapid cycles of nuclear and then cell divisions. However, a significant amount of Msps protein was also detected throughout larval, pupal, and adult stages, when cell division is limited to certain tissues. Next, protein samples were prepared separately from head, thorax, and abdomen of adult females for immunoblotting . A high level of Msps protein was detected in the abdomen, probably reflecting high expression in gonads. It is surprising to see that a significant level of Msps protein is detected in both the thorax and heads, especially considering that the amount of protein from the head is relatively small and that there are supposed to be no cell divisions in the central nervous system at this stage. These results raise the possibility that Msps protein may have some function in non dividing cells. Further analysis will be required to test this possibility. As the predicted sequence of the Msps protein showed high similarity to microtubule-associated proteins from other organisms, we wished to determine whether it was capable of binding to microtubules. We therefore prepared a soluble protein extract from embryos under conditions that depolymerize microtubules. Taxol (paclitaxel) and GTP were then added to repolymerize microtubules, and after 30 min incubation at room temperature, the microtubule fraction (P2) and the soluble fraction (S2) were separated by centrifugation. These protein fractions were run on an SDS–polyacrylamide gel and visualized by Coomassie blue staining. The protein profiles of soluble fractions before and after taxol addition are almost identical, indicating the specific effect of taxol. The microtubule fraction consisted predominantly of proteins of ∼55 kD corresponding to tubulins, together with a number of minor proteins that can be seen by silver staining (data not shown). Immunoblotting indicates that both α-tubulin and Msps are found exclusively in the microtubule fraction (P2), confirming that Msps has microtubule binding activity. To examine whether Msps protein is a constituent of the mitotic spindle, we used the antibody against Msps to localize Msps protein with respect to microtubules in wild-type embryos. We first examined mitotic cycles 11–13 which take place at the cortex of syncytial embryos . In prophase, when chromosomes start condensing, we observed microtubules radiating from two discrete regions around the nucleus. That these discrete regions correspond to centrosomes was confirmed using an antibody against a centrosomal antigen, CP190 (data not shown). Affinity-purified antibody against Msps protein revealed that Msps protein localizes predominantly around the centrosomes at this stage. In metaphase, mitotic chromosomes are fully condensed and aligned on the metaphase plate, and the bipolar spindle has formed between the centrosomes. Msps protein appears to spread over the mitotic spindles although it is still concentrated in the polar regions or in the vicinity of the centrosomes. This concentration at the centrosomal regions is more evident in merged images, where the polar region appears orange rather than yellowish green seen along the spindles . During anaphase when sister chromatids are separated and move to each pole, Msps staining is still observed on the mitotic spindles and the centrosomal region. In telophase the chromosomes become decondensed, the nuclear membrane reformed, and the mitotic spindle disassembles except for the midbody at the equator. In syncytial cycles, the centrosomes duplicate and start separating at telophase. Msps protein still appears to be tightly localized in the vicinity of the centrosomes, while a lower level of staining is observed at the midbody. During interphase, chromatin is decondensed, except for the heterochromatin which lies in the apical region of the nuclei. Thin microtubules emanate from two centrosomes that have already separated at this time. Intense staining of Msps protein is observed in the vicinity of these centrosomes. Interphase embryos without discrete Msps staining at the centrosomal region were not observed, suggesting Msps protein localizes to the centrosomal region throughout interphase in these cycles. In syncytial embryos, there is often a gradient of mitotic progression as mitotic waves start from the two ends of the embryos. This allowed us to follow the detailed changes in Msps distribution between the different mitotic stages in neighboring nuclei . From prophase to metaphase, Msps staining is strong at the centrosomal regions throughout, gradually spreading out along the mitotic spindles. There is little change in Msps staining at the metaphase/anaphase transition. In particular, the midzone between separating chromatids is stained throughout. This suggests that Msps protein is predominantly localized on pole to pole microtubules. At late anaphase to telophase, Msps staining at the mitotic spindles gets weaker but staining at the centrosomal region stays strong and becomes discrete. The cell cycles in syncytial embryos are unique in several respects. The cell cycle is the shortest among any known eukaryotic cells, there are no gap phases in interphase, no DNA replication checkpoints, and cytokinesis does not take place after nuclear division. To test whether the observed localization of Msps is unique to the syncytial cycles, we also examined its localization in cellularized embryos. Cellularization takes place after completion of the 13th mitosis, after which the length of interphase dramatically increases from 10 min to more than an hour. In cellularized embryos, mitotic activity is seen in domains of incompletely synchronized cell divisions. During mitosis, the localization of Msps in cellularized embryos is basically the same as that observed in syncytial embryos . In prophase (p) the anti-Msps antibody strongly stains the centrosomal region. This persists through metaphase (m) and anaphase (a) when Msps spreads along the mitotic spindles. At telophase (t), it is accumulated on centrosomal regions and weakly on the midbody. The significant difference between syncytial and cellularized embryos is observed in interphase (I). No accumulation of Msps staining at the centrosomal regions is observed in cellularized embryos, contrasting with syncytial embryos in which Msps protein is associated with centrosomal regions throughout interphase. This may simply be a reflection of short interphase in syncytial embryos. To determine whether the localization of Msps protein is dependent on microtubules, we looked at the effects of microtubule inhibitors. When we treated syncytial embryos with colchicine, microtubules appeared depolymerized but weak tubulin staining remained around the centrosomes . This is the case even when microtubules were depolymerized by other methods (Materials and Methods). In all cases a significant amount of Msps protein was detected around the centrosomes. This result indicates that long microtubules are not essential for the centrosomal localization of at least some of Msps protein. It is possible, however, that short microtubules or tubulin may be required for Msps localization on centrosomes. The dynamic changes of Msps protein localization in the embryonic cell cycles may reflect cyclical regulation of this process. Alternatively, it may simply be a reflection of changes in concentration, distribution, and dynamics of microtubules. In an attempt to address this question, we artificially induced the formation of microtubules in both mitotic and interphase syncytial embryos using the microtubule stabilizing drug, taxol (paclitaxel). In interphase embryos, taxol treatment induces extra microtubules from one of two discrete regions around the nuclei, which are likely to be centrosomes . Msps protein remained tightly associated with centrosomal regions in interphase, and there was little staining along the microtubules. In mitotic embryos, extra microtubules appear to be polymerized around the centrosomal regions . In contrast to interphase embryos, Msps protein follows the distribution of microtubules faithfully, as the tubulin and Msps staining overlaps. Thus Msps protein shows a higher affinity for taxol-induced microtubules during mitosis, while during interphase it shows a higher affinity for centrosomes. We then followed the distribution of CP190 in relation to Msps protein. In untreated embryos, CP190 localizes on centrosomes during mitosis (not shown). CP190 also has microtubule binding activity, so in addition to a strong accumulation on centrosomes a small proportion of the protein localizes on the mitotic spindle during metaphase . After incubation with taxol, CP190 was still tightly associated with centrosomes during mitosis , confirming that centrosomes are not disrupted by taxol treatment. In contrast only weak Msps staining was observed in the vicinity of centrosomes. Although both Msps protein and CP190 are capable of binding to microtubules and localize on or around centrosomes, both proteins behave in different ways when microtubules are induced by taxol during mitosis. Msps protein appears to have a higher affinity to taxol induced microtubules than centrosomes while CP190 has a higher affinity for centrosomes. We have identified a new Drosophila gene, msps , which is essential for spindle formation and function. It encodes a protein belonging to a family of MAPs that can bind to microtubules and associates with the mitotic spindle and centrosomal regions in a cell cycle–dependent manner. Molecular cloning revealed that the msps gene encodes a protein which belongs to the dis1-TOG family. This family of proteins is very divergent at the primary sequence level. Conserved residues are limited to four motifs that form part of repeating regions in the NH 2 -terminal portion of the molecule. These repeats with their limited conservation could be a structural or functional module in the sense of the tetratrico peptide repeats (TPR), which are found in proteins with various functions . In support of this idea, it was shown that the repeats are dispensable for in vivo function of S. pombe Dis1 . This raises the question as to whether these repeats do indeed have a common function and whether the dis1-TOG family shares a conserved function in vivo. The vertebrate and fly proteins share features that are distinct from lower eukaryotic proteins. They have four repeats in the NH2-terminal region, while the yeast and C. elegans have only two repeats. The Msps protein is highly homologous to human TOGp along its entire length. Their COOH-terminal regions are conserved and share some homology with the C. elegans protein. In contrast, the COOH-terminal regions of the yeast protein has a coiled-coil structure bearing no sequence homology. Drosophila genetics should provide a unique opportunity to study in vivo function of higher eukaryotic members of this MAP family. Considering the divergence of protein sequence of the family members and structure of the mitotic apparatus among eukaryotes, localization of the dis1-TOG proteins is surprisingly similar among dis1-TOG family from yeasts to human . They concentrate in the vicinity of the centrosome or SPB during the early stages of mitosis, transiently spread along the whole length of the mitotic spindles during mid-mitosis, before localizing back to the vicinity of the centrosome/SPB region in late mitosis. The cell cycle–dependent interaction between Msps and microtubules or centrosomes could occur as a result of posttranslational modification of Msps protein. Although we have no evidence for such modification of Msps, cell cycle–dependent phosphorylation of the XMAP215 and Dis1 proteins has been observed . In the case of Dis1, cdc2 kinase appears responsible for this phosphorylation. However, the effect of phosphorylation on the interaction of these proteins with microtubules and in vivo function remains to be determined. As we expected, Msps protein is abundant in tissues that contain many dividing cells. However, we also found a significant amount of Msps protein in nonproliferating tissues, such as the adult head. This is also seen in vertebrates. Both human ch-TOG and Xenopus XMAP215 are highly expressed in adult brains . As these proteins can regulate microtubule dynamics, it is an attractive possibility that these proteins may also function in post mitotic cells, a question that could in future be addressed in vivo in Drosophila . The msps mutation affects only a limited aspect of spindle formation. It does not appear to have a strong impact on microtubule nucleation, bipolarity of the spindle, focusing of the poles, or chromosome alignment. Rather the mutant appears defective in holding the mitotic spindle together. As the Msps protein is localized to centrosomal regions, it is possible that it is involved in the nucleation of microtubules around centrosomes. If centrosomal microtubule nucleation were defective, the effects of chromosomes on stabilizing microtubules would become dominant, resulting in the mini spindles phenotype. Such chromosome driven bipolar spindle formation has been demonstrated in centrosome-free systems. Beads coated with DNA are capable of organizing a bipolar spindle in Xenopus egg extracts and single meiotic chromosomes expelled from the spindle in various mutants can organize a bipolar mini spindle during Drosophila female meiosis . Moreover, mini spindle formation is triggered when chromosomes are detached by micromanipulation from the Drosophila male meiotic spindle which contains centrosomes . However, this model is not consistent with the phenotypes seen in either Drosophila γ-tubulin mutants or asp mutants. The γ-tubulin complex and the Asp protein appear essential for the integrity of microtubule nucleation activity of centrosomes . The Drosophila γ-tubulin mutant shows a variety of defects but no mini spindles phenotype has been reported . Similarly the poles of asp mutant spindles are highly disorganized but the spindles are largely intact and bipolar. Alternatively, it may be possible that Msps protein is required for anchoring spindle microtubules to centrosomes. It is known that many microtubules are not directly attached to centrosomes , and partial loss of Msps protein may cause a set of spindle microtubules to detach from centrosomes. However, neither this nor the previous model explain the conserved localization of Msps protein along the spindle microtubules during mid-mitosis. We have also found that Msps protein localizes on the female meiotic spindle at metaphase I (Cullen, C.F., and H. Ohkura, unpublished data). As the female meiotic spindle at metaphase I lacks centrosomes and its formation is driven by chromosomes , this observation supports the possibility that Msps protein has functions that are independent of centrosomes. The msps phenotype may be best explained by a failure in the microtubule bundling that holds the mitotic spindle together. Microtubule bundling combined with minus end motors has been proposed as a mechanism to focus the spindle at the polar region. The dynein–dynactin complex in association with NuMA , Ncd , and CTK2 are proposed to fulfil such roles. The msps phenotype may suggest that focusing of the polar region requires two steps. In one step, the microtubules that emanate from each chromosome are bundled together, whereas in the second these microtubule bundles are held together. Msps protein may be required mainly for the second step. Although human TOGp and Xenopus XMAP215 have high sequence similarity to Msps protein, no such in vitro activity has been reported for either of the purified proteins. However, it is possible that interaction with other proteins is required for this activity. The most simple model is that Msps protein is required for formation of long microtubules during mitosis. When cells fail to make long microtubules, the mitotic spindle cannot hold all of its chromosomes and it collapses to form small spindles. This model is supported by the in vitro activity of purified XMAP215 and human TOGp. Purified XMAP215 dramatically increases elongation and shortening velocity and decreases the frequency of the rescue at the plus ends of microtubules while effects on the minus end are much less dramatic. In total, it promotes plus end assembly and turnover, resulting in a population of extremely long but highly dynamic microtubules . In contrast, it was reported that TOGp increases the elongation rate of both ends equally and appears to inhibit catastrophes. As a result, TOGp promotes microtubule assembly . Although it is not clear whether these differences reflect experimental approaches, it is evident that both proteins can promote microtubule assembly. Apart from a limited sequence similarity, dis1-TOG family members in eukaryotes share an in vitro microtubule binding activity, localization to the spindle and SPB or centrosomes, and function in mitosis. Do lower eukaryotic proteins have the same function in mitosis as Msps protein, and do the studies on lower eukaryotic proteins give clues to the function of Msps? C. elegans ZYG-9 shows intermediate organization between the higher eukaryotic and yeast proteins at the primary sequence level. Crucially, C. elegans has similar mitotic apparatus to the higher eukaryotes. zyg-9 mutant embryos exhibit disorganized spindles and numerous cytoplasmic clusters of short microtubules during meiosis. Subsequently, pronuclear migration and the migration and rotation of the centrosome-nuclear complex fails . Zyg-9 gene activity is less important for the second or subsequent mitosis and dispensable after gastrulation . Based on these observations, it was proposed that zyg-9 is required for the formation of long microtubules during the first division. This is supported by the observation that nocodazole, which destabilizes microtubules, mimics the zyg-9 phenotype . In contrast to the zyg-9 gene, msps gene activity appears to be universally required in mitotic cells throughout the development. We observed mitotic defects in the central nervous system of third instar larvae in the msps mutant and severe growth defects of imaginal discs. In addition, mutants with semi-lethal alleles of msps show defects indicative of the failure of cell division of sensory mother cells or histoblast cells. Moreover, female homozygous for those alleles laid eggs that showed mitotic defects during the embryonic divisions (Cullen, C.F., and H. Ohkura, unpublished data). Although the developmental requirement for msps and Zyg-9 differs, an attractive model that reflects the cellular phenotypes of both mutants is that both gene products promote the formation of long microtubules during mitosis. S. cerevisiae STU2 was originally identified as a dominant chromosomal suppressor of tub2-423 , a cold sensitive allele of the β-tubulin gene . Although these dominant mutations on their own do not affect growth, the disruption of the gene is lethal. The cytological phenotype of this disruptant has not been studied, so the in vivo function of STU2 has not been established. As STU2p was shown to bind microtubules laterally and localizes on the SPB in a microtubule-independent manner, it was proposed that STU2p tethers the microtubules to the SPB while allowing exchange of tubulin subunits at their minus ends. In contrast to the msps mutant, S. pombe dis1 mutation does not affect the integrity of the mitotic spindle, at least at the light microscope level , but sister chromatids fail to separate. Real time analysis showed it to be defective in the oscillation of centromeres during metaphase and in restraining spindle elongation . It was proposed that Dis1 may be required for interactions of microtubules with the kinetochore and SPB, consistent with a microtubule anchoring model. This model is also consistent with observation that NH 2 -terminally truncated Dis1 protein localizes only on SPB but not on the mitotic spindle and can complement the cold sensitivity of dis1 deletion mutants . It will be interesting to see whether this is also true for the higher eukaryotic proteins. Studies of the relationship between domain structure, localization, and in vivo function in higher eukaryotic protein is an exciting future possibility in Drosophila . It should be noted that the dis1 + gene is not an essential gene. The dis1 deletion mutant is viable but shows cold sensitive lethality . This may suggest a role in promoting the assembly of microtubules that are unstable at low temperatures. Studies of the dis1-TOG family members indicate that they share many common features as well as some apparent differences. At this moment, it is not clear whether they execute exactly the same function. Further studies are needed to understand their exact molecular function and the regulation of localization with respect to protein structure. The availability of Drosophila mutants and the feasibility of genetic manipulation will be invaluable for studying the higher eukaryotic forms in vivo. | Study | biomedical | en | 0.999996 |
10477756 | The yeast pheromone α-factor peptide was synthesized by Research Genetics and stored as a 2 mg/ml stock solution in H 2 O at −20°C. Hydroxyurea (Sigma Chemical Co.) was added directly to yeast media from solid. Nocodazole (Sigma Chemical Co.) was stored as a 15 mg/ml stock solution in DMSO at −20°C. Rhodamine-conjugated phalloidin (Molecular Probes) was stored as a 200 U/ml stock solution in methanol at −20°C. 4′,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) was stored as a 1 mg/ml stock solution in H 2 O at −20°C. Latrunculin-A (Lat-A; Molecular Probes) was stored as a 20 mM stock solution in DMSO at −20°C. Monoclonal rat anti–yeast tubulin, YOL1/34 (Accurate Chemical and Scientific Corp.), was used at 1:100 dilution, and goat anti–rat Cy2 secondary antibody (Jackson ImmunoResearch Laboratories) was used at 1:25 dilution. The yeast strains used in this study are listed in Table . Strains DLY1, DLY5, and SBY153 (a gift from Steven B. Haase, The Scripps Research Institute, La Jolla, CA) are in the BF264-15DU background , and JMY6-10 (John N. McMillan, Duke University Medical Center, Durham, NC) contains the cdc31-1 allele backcrossed six times into the same strain background. Strains 5050 and 5105 were gifts from D. Koshland (Carnegie Institute of Washington, Baltimore, MD) and are described in Palmer et al. 1992 . Strain KBY62 was made by transforming the dyn1::LEU2 plasmid described in Li et al. 1993 into W303a. Strains KBY1010 and KBY1011 were made by disrupting KAR9 and BNI1 , respectively, in the YEF473 background. The kar9::LEU2 plasmid described by Miller and Rose 1998 was used to disrupt KAR9 , and the entire open reading frame of BNI1 was deleted using the PCR strategy of Baudin et al. 1993 with pRS305 ( LEU2 ) as template and the primers 5′-gatgtttgttttggtattactgttgtcataattttttggtttaatattttatttgaaacttagcctgttacct-gtgctatgcggtatttcacaccg-3′ and 5′-gccatttgtatct-atcttctgtattgaggagaaacattttaactcaagcctag-ttaaattctaaatacacgccgattgtactgagagtgcacc-3′. Strains ABY971 and ABY973 were gifts from D. Pruyne and A. Bretscher (Cornell University, Ithaca, NY) and are described in Pruyne et al. 1998 . Cells were grown in YEPD (1% yeast extract, 2% bacto-peptone, 2% dextrose, 0.01% adenine), YEPS (containing 2% sucrose instead of 2% dextrose), or YEPG (containing 2% galactose instead of 2% dextrose) medium at 30°C, except for the experiments using temperature-sensitive mutants, in which cells were grown at the permissive temperature (23–24°C) and shifted to the indicated restrictive temperatures. Synchronized cell cultures were obtained in a variety of ways. For hydroxyurea-induced synchrony, cell were grown in YEPD to 5 × 10 6 cells/ml, harvested, and resuspended at 2 × 10 7 cells/ml in YEPD containing 100–200 mM hydroxyurea, and the cells were incubated for 3 h at 23–30°C, as indicated. For α-factor–induced synchrony, α-factor was added to cell cultures in YEPD at 5 × 10 6 cells/ml to a final concentration of 25 ng/ml, and the cells were incubated for 3 h at 30°C. Centrifugal elutriation of cells grown in YEPS or YEPG was performed as described , except for the temperature in the cdc31-1 experiments, in which the cell cultures and collection flasks were kept in a 37°C water bath, the rotor was prewarmed by spinning the rotor until the chamber warmed up to 37°C, and then using the cooling feature of the centrifuge to keep the temperature constant. Nocodazole was added to a final concentration of 15 μg/ml. For the cdc31-1 cells, nocodazole was added directly to the collection flasks from the elutriation, whereas in the clb1 Δ -6 Δ experiment, the cells were first harvested by centrifugation and resuspended in fresh YEPD with nocodazole. Lat-A was added to the indicated concentration from DMSO stock solution, and an equal amount of DMSO was added to the control samples in all experiments (final DMSO concentration did not exceed 1%). Cells were fixed in 4.5% formaldehyde for 2 h at 23°C. Immunofluorescence procedures for visualizing tubulin distribution with the YOL1/34 antibody were performed according to Pringle et al. 1991 . DNA was visualized by staining with DAPI. F-actin was visualized by staining with 10 U/ml rhodamine-phalloidin added to the secondary antibody incubation for the immunofluorescence procedure. Cells were washed, mounted, and examined using a Zeiss Axioskop microscope equipped with epifluorescence and differential interference contrast (DIC) optics. Images were captured using a Pentamax cooled CCD camera (Princeton Instruments), interfaced with MetaMorph software (Universal Imaging Corp.). Spindle orientation was scored as described by Palmer et al. 1992 : a spindle was considered properly oriented if an imaginary line drawn through the long axis of the spindle traversed the mother-bud neck. This scoring criterion contains information on both the position and orientation of the preanaphase spindle. Given that the z-axis was sometimes hard to assess in this scoring, it is possible that some spindles were misscored (in either direction), but this should not affect our conclusions because the misscoring should not be affected by Lat-A treatment. SPB position was inferred from the tubulin staining on the assumption that the SPB is at the focus of the cytoplasmic microtubule asters: only cells where this focus was obvious were scored. SPB position was classified into one of three categories: neck, indicating a distance of <0.5 μm from the mother-bud neck; mother, or bud, indicating a distance of >0.5 μm from the mother-bud neck on either side. In the experiment of Fig. 5 , the bud category is expanded to include all cells with the SPB on the bud side of the neck. Mothers were distinguished from buds by morphological criteria: in Fig. 5 , mothers were larger and had a distinct vacuole; in Fig. 6 , mothers were shmoo-shaped; and in Fig. 9 , buds were elongated. The reliability of these criteria in identifying mother and bud was tested by comparison to the actin-staining pattern in the same cells (actin patches are predominantly in the bud). In all cases, there was excellent agreement between morphological and actin-based criteria. The statistical significance of the differences we observed in the proportion of cells that had correctly oriented spindles or that had undergone SPB migration in the presence or absence of Lat-A was calculated with standard sampling theory for proportions . In brief, the standard error (s) of our estimates was calculated based on the sample size (N) and observed proportion (p), according to the binomial distribution formula: s = √p(1−p)/N. Differences between samples were tested against the null hypothesis that the samples were derived from the same population, using a two-tailed test. In all cases mentioned in the text, the null hypothesis (i.e., no effect of Lat-A) was rejected at a <0.01 significance level. In the course of studies using the actin-depolymerizing drug Lat-A , we were surprised to find that Lat-A did not perturb spindle orientation in cells released from hydroxyurea arrest. An example is shown in Fig. 1 , where spindle orientation was examined in formaldehyde-fixed cells by indirect immunofluorescence using the monoclonal antitubulin antibody YOL1/34. Following Palmer et al. 1992 , we score a spindle as being properly oriented if an imaginary line drawn through the long axis of the spindle traverses the mother-bud neck. This scoring criterion contains information on both the position and orientation of the preanaphase spindle. Wild-type cells were arrested by a three hour incubation in medium containing hydroxyurea, which inhibits DNA replication, but allows bud formation and proper orientation of a preanaphase spindle. After release from the arrest into medium containing Lat-A, the cells did not display significant defects in spindle orientation either before or during anaphase, even though F-actin was completely depolymerized, as judged by rhodamine-phalloidin staining . In contrast to the prevailing view, we conclude that F-actin is not required for maintenance of spindle orientation. Possible reasons for the discrepancy between our results and previous studies include strain differences, different effects of the agents employed to perturb actin (Lat-A versus act1-4 ), and differences in synchronization or temperature regimes. We have repeated the experiment above in strain 5050 , with identical results, indicating that this result is not strain-specific. To address whether Lat-A versus temperature-shift of act1-4 strains produced differing results, we performed an experiment to compare their effects using a single protocol. For consistency with the previous study, we followed the same synchrony and temperature shift protocol reported by Palmer et al. 1992 . Compared with the untreated wild-type controls, both Lat-A and temperature-shift of act1-4 caused only mild spindle misorientation in large-budded cells . We conclude that both actin perturbing agents have similar effects on spindle orientation. One difference in the protocol of Fig. 2 , compared with that of Fig. 1 , lies in the degree of synchrony attained by the hydroxyurea treatment. In Fig. 1 , the cells were treated with 200 mM hydroxyurea at 30°C, whereas in Fig. 2 , they were treated with 100 mM hydroxyurea at 23°C. This led to significantly better arrest in the experiment of Fig. 1 (89% compared with 67% large-budded cells for the wild-type cultures). In addition, the arrest of the act1-4 mutants was significantly less homogeneous (51% large-budded). In the data reported above, we attempted to compensate for this synchrony difference by scoring only large-budded cells. In fact, examination of the sizable population of smaller budded cells in the act1-4 population revealed a much greater degree (64% compared with 20%) of spindle misorientation . Thus, the apparent discrepancy between our results and those of Palmer et al. 1992 can be fully accounted for on the assumption that the act1-4 cells scored in that study included a substantial proportion of smaller budded cells. In sum, these data suggest that whether or not perturbation of actin results in spindle misorientation may depend on when the perturbation occurs during the cell cycle. In the well-arrested cells examined in Fig. 1 , no errors occurred upon actin perturbation. In the less well-arrested wild-type cells (or large-budded act1-4 cells) examined in Fig. 2 , a moderate degree of spindle misorientation was induced (20%), whereas in the poorly arrested act1-4 population, frequent errors were induced (64%). Thus, cells that are truly arrested by hydroxyurea maintain correct spindle orientation, despite the absence of F-actin, whereas less well-arrested cells apparently do not. To examine whether cells traversing the cell cycle require actin to establish and/or maintain spindle orientation, a proliferating asynchronous population of wild-type cells was treated for 30 min with Lat-A. This induced a dramatic spindle misorientation in 53% of the cells . Misaligned preanaphase spindles were observed in small- and medium-budded cells, but at lower frequency in large-budded cells . In addition, postanaphase spindles in medium-budded cells were often misaligned, probably resulting from elongation of short spindles that had become misoriented soon after Lat-A addition . Thus, spindle orientation is actin-dependent in some fraction of a proliferating cell population, but not in hydroxyurea-arrested cells. Intriguingly, a similarly dramatic spindle misorientation was induced even by treatment with a low dose of Lat-A, a point we return to below. In the experiments above , the conclusion that maintenance of correct spindle orientation was actin-independent relied on perturbation of the cell cycle: hydroxyurea triggers arrest through the DNA replication checkpoint . Therefore, it was conceivable that actin independence was somehow induced in response to the checkpoint arrest, rather than constituting a normal part of the unperturbed cell cycle. To address this issue, we monitored the effect of Lat-A on spindle orientation in wild-type cells progressing through a synchronous cell cycle. Cells were synchronized in G1 with α-factor and then harvested and resuspended in fresh medium. Lat-A (or DMSO for controls) was then added to separate aliquots of cells at 15 min intervals, and the cells were fixed 75 min after release from arrest to examine spindle orientation. Cell cycle synchrony in this experiment was good, with most cells budding between 30 and 45 min after release from pheromone arrest . When Lat-A was added to small-budded cells early in the cell cycle (30 min sample), preanaphase spindles became misoriented in 35% of the cells . A similar degree of spindle misorientation was observed after only 15 min in Lat-A (data not shown). In addition, subsequent spindle elongation occurred entirely within the mother in 33% of the cells . This confirms the requirement for F-actin in spindle orientation early in the cell cycle. In contrast, when Lat-A was added to large-budded cells late in the cell cycle (60 min sample), preanaphase spindles did not become significantly misoriented . Furthermore, anaphase spindle elongation was completely unaffected . This implies that after a certain point (at least 15 min before anaphase) in the normal cell cycle, maintenance of spindle orientation becomes actin-independent. When Lat-A was added to medium-budded cells at an intermediate time (45 min sample), preanaphase spindles became misoriented in many cells , but anaphase spindle elongation was not significantly perturbed . We interpret this to mean that at this intermediate time the population was mixed, with some cells still early enough in the cell cycle to require actin (hence misorienting their spindles in response to Lat-A), and others, later in the cycle, being insensitive to actin perturbation (hence executing a correctly oriented anaphase). The proportion of cells displaying Lat-A induced errors in either preanaphase or postanaphase spindle orientation at different times is summarized in Fig. 4 D. In aggregate, these data strongly support the conclusion that spindle orientation becomes insensitive to actin perturbation at some time during G2/M phase of the cell cycle. A simple explanation for the difference in the sensitivity of spindle orientation to actin perturbation at early versus late times in the cell cycle might be that actin is required for the initial establishment, but not for the subsequent maintenance, of spindle orientation. The earliest step in spindle orientation is thought to be the orientation of the SPB (or duplicated side by side SPBs) towards the bud site before spindle assembly . Some studies have suggested that the SPB also undergoes a concerted migration to the bud site or bud neck , although this remains controversial . Addressing whether these early steps in spindle orientation are actin-dependent is complicated by the fact that actin is also required to construct the bud towards which the SPBs must align. To circumvent this difficulty, we devised a protocol to delay SPB orientation until after a bud had been formed. We used the antimicrotubule drug nocodazole to prevent polymerization of microtubules, and hence SPB orientation or migration, until a bud had been formed. In addition, we used cdc31-1 mutant cells to prevent SPB duplication, so that spindle formation would be blocked and we could examine SPB position directly. cdc31-1 is a temperature-sensitive mutation that prevents SPB duplication at the restrictive temperature . cdc31-1 cells were grown at 23°C and shifted to 37°C for 1 h to inactivate Cdc31p. G1 daughter cells were then isolated by centrifugal elutriation and incubated in the presence of 15 μg/ml nocodazole to prevent SPB orientation during bud formation. After a majority of cells had formed small- or medium-sized buds, cells were harvested by centrifugation and resuspended in medium lacking nocodazole (all steps including the elutriation were carried out at 37°C; see Materials and Methods). At this point, the nuclei were distributed randomly within the mother portion of the cell . Within 30 min of nocodazole wash-out, the SPB had moved to the vicinity of the mother-bud neck in 76% of the cells . This indicates that the cytoplasmic microtubules emanating from the unduplicated SPB were able to recognize asymmetric cues and promote appropriate SPB migration. To address whether this SPB migration was actin-dependent, cells were synchronized as above, and Lat-A was added to the culture immediately after washing out the nocodazole. Staining with rhodamine-phalloidin confirmed that the Lat-A caused complete depolymerization of F-actin (data not shown). As shown in Fig. 5 A, this led to a partial disruption of SPB migration to the neck (56% compared with 76% SPB migration in controls). The fact that many SPBs did migrate to the neck suggests that the migration was not actin-dependent in all cells (see also below), though the effect of Lat-A was statistically significant (see Materials and Methods for evaluation of statistical significance). We conclude that SPB migration, an early step in spindle orientation, requires F-actin in at least a fraction of the cells. One model compatible with the data presented thus far would be that spindle orientation involves a single two-step process in which the first step requires F-actin, and the second step does not. Alternatively, the data might reflect the acquisition of a novel actin-independent mechanism for spindle orientation late in the cell cycle. In this latter model, all steps in spindle orientation could be accomplished in an actin-independent manner late in the cell cycle. To distinguish between these models, we examined whether the SPB migration step was differentially sensitive to perturbation of actin at early versus late times in the cell cycle . We isolated cdc31-1 cells synchronized in G1, as before, and incubated them in the presence of nocodazole for different times before wash-out to allow them to reach different stages of the cell cycle. To distinguish the buds from the mother cells at later stages, we marked the mother cells by inducing them to form shmoo projections during a brief exposure to α-factor in G1. Curiously, this pretreatment slightly altered the behavior of the SPB after nocodazole wash-out, so that in most cells, the SPB migrated all the way into the bud, rather than stopping near the neck. The basis for this effect of α-factor is unclear. When nocodazole was washed out 60 min after α-factor treatment , the SPB migrated to the neck or into the bud. Addition of Lat-A partially disrupted this migration (62% SPB migration in Lat-A treated cells versus 82% in controls). In contrast, Lat-A had little effect when nocodazole was washed out at 120 min after α-factor treatment : the SPB migrated to the neck or into the bud in all cases (93% SPB migration in Lat-A treated cells versus 98% SPB migration in controls). Staining with rhodamine-phalloidin confirmed that the Lat-A caused complete depolymerization of F-actin in all cases (data not shown). Thus, even this early SPB migration step becomes actin-independent late in the cell cycle. At earlier times, Lat-A had statistically significant effects on SPB migration and spindle orientation, but the effect of Lat-A was not complete . This may indicate that, even at early times, some cells can orient their spindles in an actin-independent manner. However, it is probable that the imperfect synchrony of the cell populations assayed led to the inclusion of some G2 cells in our early samples, reducing the apparent effect of actin perturbation. Similarly, random microtubule-powered movements might lead to a seemingly correct orientation in some cells, further reducing the apparent effect of actin perturbation. For these reasons, we do not know whether the requirement for actin at early times is absolute. Regardless, these experiments demonstrate that both SPB migration and maintenance of spindle orientation can occur in an actin-independent manner late in the cell cycle. Previous studies have implicated a number of proteins, including the putative microtubule capturing protein Kar9p , the cortical protein Bni1p , and the microtubule-based motor protein dynein in the process of spindle orientation. To address whether these proteins were important for spindle orientation late in the cell cycle, we arrested kar9 Δ, bni1 Δ, or dyn1 Δ mutant strains with hydroxyurea. As shown in Fig. 7 , none of these mutants differed from isogenic wild-type strains in terms of their ability to orient spindles. The wild-type W303 cells were not as proficient in this regard, as the wild-type YEF473 cells, presumably reflecting strain background effects. The fact that spindle orientation did (eventually) occur in these strains suggests that, given sufficient time, both the establishment and maintenance of spindle orientation can occur in the absence of these proteins. To ask whether these proteins might play a redundant role with F-actin–dependent processes to orient the spindle late in the cell cycle, we treated the hydroxyurea-arrested mutant cells with Lat-A. However, no errors were induced by Lat-A in any of the mutants . In the SPB migration experiments, the movement of the SPB to the mother-bud neck (or bud) in a majority of the cells implied that cytoplasmic microtubules successfully interacted with asymmetric determinants to orient and move the SPB. Indeed, in the experiment of Fig. 5 , 81% of the cells contained cytoplasmic microtubules extending from the SPB into the bud . Surprisingly, however, in almost half of these cells the SPBs were also associated with microtubules reaching back from the neck into the mother . Thus, SPB migration was associated with orientation of some, but not necessarily all, microtubules towards the bud. Treatment of cells with Lat-A caused a significant reduction (47% compared with 81%) in the proportion of cells displaying cytoplasmic microtubules extending into the bud . This result suggests a role for F-actin in either guiding cytoplasmic microtubules into the bud or in keeping them there. In proliferating cells, Lat-A induced very rapid spindle misorientation, with a maximal effect by 5 min of treatment . In addition, even low doses of Lat-A were capable of inducing maximal levels of spindle misorientation . Unlike cells treated with 100 μM Lat-A, which lacked detectable F-actin as judged by rhodamine-phalloidin staining, cells treated with 6.25 μM Lat-A lacked detectable actin cables, but retained many cortical actin patches, which were sometimes polarized to the bud tip in small-budded cells . However, these cells displayed similar degrees of spindle misorientation . This suggests that actin cables, rather than actin patches, may be important for spindle orientation. To examine this issue further, we took advantage of the tpm1-2 tpm2 Δ strain described recently by Pruyne et al. 1998 . Shift of this strain to the restrictive temperature of 34.5°C results in the complete disappearance of actin cables within 1 min, while cortical actin patches remain polarized for over 15 min . We compared the effects of a 5 min Lat-A treatment (eliminating all F-actin) to a 5 min tpm1-2 tpm2 Δ temperature shift (eliminating only actin cables) with regard to spindle orientation. As shown in Fig. 8 , both treatments produced comparable degrees of spindle misorientation . This suggests that actin cables, rather than cortical actin patches, are important for spindle orientation. Many cell cycle events are thought to be triggered by a master cell cycle clock centered around a set of cyclins and cyclin-dependent kinases . In S . cerevisiae , events in the post-G1 cell cycle are governed by the B-type cyclins Clb1p-6p, together with the cyclin-dependent kinase Cdc28p . Thus, a plausible hypothesis would be that Clb1p-6p/Cdc28p complexes are responsible for triggering the observed switch between actin-dependent and actin-independent behavior in terms of spindle orientation. To test this hypothesis we used a strain in which CLB1-6 had been deleted, containing CLB1 driven from the regulatable GAL1 promoter. On galactose medium, Clb1p is overexpressed and the strain is viable. Upon addition of dextrose, the GAL1 promoter is repressed and the cells arrest lacking Clb1p-6p. As previously described , the arrested cells initiate bud formation and duplicate the SPB, but cannot replicate DNA or separate the SPBs to form a spindle. In addition, they cannot perform the apical-to-isotropic switch in bud growth: actin remains highly polarized to the bud tip and the buds become elongated . To address whether Clb1p-6p function was required to trigger the switch from actin-dependent to actin-independent spindle orientation, we employed a protocol similar to that described above for cdc31-1 mutants. Dextrose was added to a proliferating culture of clb1 Δ -6 Δ GAL1:CLB1 cells to shut off Clb1p synthesis, and 1 h later newborn G1 cells were isolated by centrifugal elutriation. Since Clb1p is degraded as cells exit mitosis , these G1 cells should uniformly lack Clb1p-6p, as indeed was apparent from the ensuing elongated-bud arrest morphology . After elutriation, nocodazole was added to prevent SPB migration during bud formation. At different times, the nocodazole was washed out and cells were resuspended in media containing Lat-A (or DMSO for controls). 30 min later, the cells were fixed and scored for SPB migration (without Clb1p-6p function, the duplicated SPBs remain side by side and move as a unit). As shown in Fig. 9 , a majority of the SPBs migrated either to the vicinity of the neck or all the way into the bud, and most cells displayed cytoplasmic microtubules extending from the duplicated SPBs into the bud. Note that this result implies that Clb1p-6p function is not required for SPB migration. About half of the cells contained multiple microtubules pointing in different directions, extending both into the bud and back towards the mother. Thus, as was also evident in the cdc31-1 experiments, SPB migration was associated with orientation of some, but not necessarily all, microtubules towards the bud. When nocodazole was washed out early in the cell cycle (predominantly small-budded cells), the SPBs migrated to the neck or into the bud. As expected, addition of Lat-A significantly perturbed this migration . In contrast, Lat-A had little effect when nocodazole was washed out later (predominantly large-budded cells): the SPBs migrated to the neck or into the bud in all cases . Thus, a switch to actin-independent SPB migration was observed even in cells lacking Clb1p-6p. We conclude that this switch is not triggered by the cyclin/Cdc28p clock and occurs regardless of DNA replication or other Clb1p-6p–dependent events. The behavior of the SPB was very similar in the cells lacking Clb1p-6p compared with cdc31-1 mutants. However, these cells differed in the overall lengths of the cytoplasmic microtubules: in particular, only 55% of cdc31-1 cells contained microtubules extending all the way to the cortex, compared with 83% for the clb1 Δ -6 Δ cells. A subset (18%) of the clb1 Δ -6 Δ cells also displayed extra-long microtubules that reached the cortex and were bent around the surface of the cell . We report that, contrary to the prevailing view, maintenance of spindle orientation does not require F-actin in budding yeast. The apparent discrepancy between our results and those of Palmer et al. 1992 can be fully explained by differences in the synchrony of the hydroxyurea-treated cultures analyzed. In principle, actin-independent maintenance of spindle orientation could be due to a spindle docking process, whereby spindles that have already been correctly positioned and aligned through an actin-dependent process are immobilized at the mother-bud neck. However, even events that normally occur early in spindle orientation, such as SPB orientation and migration to the neck, occurred in an actin-independent manner if they were delayed until later in the cell cycle. This argues against the docking model and supports the hypothesis that, late in the cell cycle, all steps in spindle orientation can be carried out in the absence of F-actin. A further surprise was that deletion of KAR9 , BNI1 , or DYN1 , which are important for spindle orientation at other times in the cell cycle, did not prevent spindle orientation in hydroxyurea-arrested cells. Furthermore, simultaneous loss of F-actin (upon Lat-A treatment) and any one of Kar9p, Bni1p, or Dyn1p still did not perturb spindle orientation. In a previous study , inactivation of a temperature-sensitive kip3 allele was similarly ineffective in misorienting the spindle in hydroxyurea-arrested cells, in contrast to its effect earlier in the cell cycle . These findings suggest that the mechanism responsible for orienting the spindle late in the cell cycle is quite distinct from that occurring earlier in the cell cycle. Intriguingly, actin-independent spindle orientation was not observed until G2/M phase of the cell cycle. Thus, there was effectively a switch between actin-dependent and actin-independent behavior on the part of the spindle. This switch did not require spindle formation, because we observed a similar switch to actin-independent behavior on the part of the unduplicated SPB in cdc31-1 mutants. Furthermore, the switch did not require continued cell cycle progression, since cells lacking Clb1p-6p still displayed a switch to actin-independent SPB behavior. Thus, actin independence seems to develop with time, rather than being switched on by a cell cycle cue. This conclusion stems from experiments in which actin was perturbed using Lat-A after a large bud had been formed. In experiments where Lat-A was added to small-budded cells early in the cell cycle, spindles in many cells failed to orient along the mother-bud axis. If an actin-independent orientation capability were to develop with time under these conditions, we would expect that the initial spindle misorientation would be corrected after a suitable time interval. However, we found that spindle misorientation persisted, and that eventual anaphase frequently occurred entirely within the mother cell under these circumstances. Thus, actin independence did not develop in cells that had been treated with Lat-A early on. This suggests that F-actin is itself required, early in the cell cycle, for the development of a spindle orientation mechanism that is then insensitive to perturbation of F-actin. It is unclear whether this requirement is related to the F-actin–dependent spindle orientation process observed early in the cell cycle or involves a distinct role for F-actin. Indeed, since these experiments required >30 min treatments with Lat-A, it remains possible that the failure to develop actin independence was a secondary consequence of prolonged loss of F-actin. Nevertheless, one simple explanation for these observations would be that, during early stages of bud formation, actin is required for the delivery of cortical determinants into the bud: these determinants could later interact with astral microtubules in an actin-independent fashion to promote spindle orientation. Perturbing F-actin early in the cell cycle significantly decreased the proportion of cells displaying astral microtubules extending into the bud, and similarly decreased the proportion of cells displaying SPB migration to the mother-bud neck. This suggests that F-actin is important for guiding astral microtubules into the bud, or perhaps for anchoring of astral microtubules within the bud. F-actin displays a polarized distribution that makes it well suited to these tasks: the mother cell contains a cortical basket of actin cables converging on the bud neck, whereas cortical actin-rich patches are largely restricted to the bud . Selective disruption of actin cables in tpm1-2 tpm2 Δ mutants led to spindle misorientation without affecting the polarized distribution of cortical actin patches. This suggests that it is the actin cables, rather than the actin patches, that are important for spindle orientation (although it remains possible that actin patches were affected in some manner in the tpm1-2 tpm2 Δ mutants). One hypothesis consistent with these findings would be that astral microtubules interact via cross-linking proteins with actin cables in the mother cell to be guided into the bud. An attractive candidate for such a cross-linking protein might be the recently described coronin Crn1p, which binds to microtubules and actin filaments . However, Crn1p was predominantly localized in cortical actin patches rather than cables, and mutants lacking Crn1p did not display major spindle misorientation, indicating that other proteins must be capable of orienting astral microtubules in its absence. Perhaps the most attractive candidate for such a protein currently known is Kar9p , although it is not known whether Kar9p interacts with microtubules or actin filaments directly. Astral microtubules in kar9 Δ cells frequently fail to extend into the bud , a phenotype similar to that displayed upon actin perturbation of cells early in the cell cycle . Thus, one role for Kar9p might be to promote interactions between astral microtubules and actin cables, leading to frequent penetration of the microtubules into the bud. Kar9p is predominantly localized to a single spot on the bud cortex, where it has been proposed to anchor cytoplasmic microtubules . The formin Bni1p is localized at the cell cortex, is required for localization of Kar9p in a cortical dot, and is important for proper spindle orientation . However, Kar9p retains a reduced ability to promote correct spindle orientation in bni1 Δ cells, suggesting that Kar9p localization may not be essential for its function . Conceivably Kar9p functions (independent of Bni1p?) to guide astral microtubules along actin cables into the bud, and also (together with Bni1p?) to anchor them within the bud. In summary, spindle orientation is very sensitive to perturbation of actin cables early in the cell cycle, and at this stage the actin cables play a role in either guiding astral microtubules into the bud, anchoring them within the bud, or both. In addition, F-actin is required early in the cell cycle for the development of actin independence later in the cell cycle. Although SPB migration occurred even in cells lacking Clb1p-Clb6p, the astral microtubules were longer in these cells, suggesting that astral microtubule behavior may be regulated by B-type cyclins. Indeed, a recent study proposed a role for Clb5p in regulating astral microtubule behavior , based on the observation that cells with a partial loss of Clb/Cdc28p activity exhibited spindle migration all the way into the bud, rather than stopping at the neck. An unexpected finding from our SPB migration assays was that in ∼50% of the cells, SPBs were associated with astral microtubules extending back into the mother cell, as well as into the bud. This suggests that the net movement of SPBs to the mother-bud neck or into the bud was not simply due to asymmetric orientation of the attached microtubules. Rather, there may be some asymmetry in force production between bud-directed and mother-directed microtubules. Several microtubule motor proteins have been shown to affect astral microtubule behavior and spindle orientation in yeast . In particular, dynein (and dynactin) mutants frequently undergo anaphase with the spindle entirely within the mother portion of the cell , even after release from hydroxyurea arrest , suggesting that dynein is important for spindle orientation late in the cell cycle. However, we found that dynein was not required for spindle orientation in hydroxyurea-arrested cells. One hypothesis that reconciles these observations is that dynein is not required for spindle orientation per se, but is important for pulling one pole of the spindle through the neck during anaphase. This would be consistent with the observation that initial spindle elongation during anaphase generally occurs along the mother-bud axis in dynein mutants, albeit within the mother cell . We have found that the mitotic spindle is able to orient along the mother-bud axis in an F-actin–independent manner late in the cell cycle of budding yeast. We suggest that actin plays a role in two aspects of spindle orientation early in the cell cycle. First, actin cables are required to guide astral microtubules into the bud or possibly to anchor them within the bud, allowing SPB positioning near the mother/bud neck. Second, F-actin is required for the development of a cortical asymmetry that subsequently maintains correct spindle orientation in an actin-independent manner late in the cell cycle. | Study | biomedical | en | 0.999998 |
10477757 | Goldfish fin fibroblasts (line CAR, no. CCL71; American Type Culture Collection) were maintained in basal Eagle medium with Hanks' BSS and nonessential amino acids and with 15% fetal calf serum at 25°C. For experiments, cells were plated onto coverslips coated with human serum fibronectin (Boehringer Mannheim GmbH) for at least 48 h. Fibronectin was coated onto polylysine-treated coverslips by incubation on a drop of 50 μg/ml fibronectin in PBS at 4°C overnight; after rinsing in PBS these coverslips were used without drying. Fibronectin was stored as a stock solution in 2 M urea at 4°C. For polylysine coating, coverslips were incubated on a drop of aqueous 100 μg/ml polylysine for 30 min at room temperature (RT) 1 , rinsed with water, dried, and UV sterilized. Injections were performed with sterile Femtotips (Eppendorf) held in a Leitz Micromanipulator with a pressure supply from an Eppendorf Microinjector 5242. Cells were injected with a continuous outflow mode from the needle under a constant pressure of between 20 and 40 hPa. For local application of drugs, performed with the same system, a constant pressure of 50–100 hPa was used. Tetramethyl rhodamine (5-TAMRA; Molecular Probes) conjugated vinculin from turkey gizzard was kindly provided by Mr. K. Rottner and Dr. M. Gimona (Institute of Molecular Biology, Salzburg, Austria). Small aliquots in 2 mg sucrose/mg protein were stored at −70°C. Before use, the fluorescent vinculin was dialyzed against 2 mM Tris-Acetate, pH 7.0, 50 mM KCl, 0.1 mM DTE, and used at a concentration of ∼1 mg/ml. Cy3-conjugated tubulin was kindly provided by J. Peloquin and Dr. G. Borisy (University of Wisconsin, Madison, WI). It was stored at a concentration of 10 mg/ml in aliquots at −70°C. Rhodamine-conjugated rat tubulin was kindly provided by Drs. R. Tournebize and T. Hyman (EMBL, Heidelberg) and stored at −70°C in 5-μl aliquots (∼20 mg/ml) in BRB80 buffer (80 mM Pipes, pH 6.8, 1 mM MgCl 2 , and 1 mM EGTA). For microinjection, rhodamine tubulin aliquots were diluted 1:3 with Tris-acetate injection buffer (2 mM Tris-acetate, pH 7.0, 50 mM KCl, and 0.1 mM DTE) and used on the same day. For coinjections, tubulin and vinculin were mixed after separate centrifugation for 10 or more min at 18,000 g in a proportion of 1:4 and used immediately. Recombinant L61Rac was kindly provided by K. Rottner (using a construct originally provided by Professor A. Hall), dialyzed into 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl 2 , and 1 mM DTE for microinjection , and injected at a concentration of 2 mg/ml. For local application through a microneedle, drugs were dissolved in microinjection buffer (2 mM Tris-Acetate, pH 7.0, 50 mM KCl, and rhodamine dextran as a marker): the inhibitor of myosin light chain kinase, ML-7 (Alexis Corporation) was used at a concentration of 2 mM; the actomyosin inhibitor 2,3-butanedione 2-monoxime (BDM) was used as a saturated solution (∼500 mM); and nocodazole (Sigma Chemical Co.) was used at a concentration of 160 μM. Complete depolymerization of microtubules for spreading experiments was achieved using a concentration of 2.5 μg/ml. Cells were preincubated with nocodazole for 1–3 h and replated in the presence of the drug. Nocodazole was stored as a 16-mM stock solution in DMSO. A low concentration (20 nM) of taxol (paclitaxel; Sigma Chemical Co.) was used for suppression of microtubule dynamics. Taxol was stored as an 10 mM stock solution in DMSO. The inhibitor of p160ROCK, Y27632 , was added to culture medium at a concentration of 100 μM, obtained by dilution from a 10-mM stock solution in DMSO. For coexpression of GFP-fused proteins, mouse β 3 tubulin in a pEGFP-C2 vector and human zyxin in a pEGFP-N1 vector were used. Both probes were kindly provided by Professor J. Wehland and coworkers (BGF, Braunschweig, Germany). Subconfluent monolayer cultures on 30-mm petri dishes were used for transfection. For each dish, the transfection mixture was prepared as follows: 1 μg of EGFP-zyxin DNA and 2 μg of EGFP-β-tubulin DNA and 14 μl of Superfect lipofection agent (Qiagen) were mixed in 400 μl of serum-free medium. After 30 min incubation at RT a further 1.2 ml of medium containing 5% serum was added. Cells were incubated in this mixture for 4 h at 25°C and the medium then replaced by normal medium containing 15% serum. After 24 h, cells were replated at a dilution of 1:15 onto coverslips for microscopy (see Cells). The EGFP-zyxin expressing stable cell line was produced by transfection as above using 3 μg EGFP-zyxin DNA, followed by selection in 1 mg/ml G418 (GIBCO)-containing medium. Positive clones were identified in the fluorescence microscope and maintained in 0.4 mg/ml G418-containing medium. Cells were injected and observed in an open chamber at RT on an inverted microscope (Axiovert 135TV; Zeiss) equipped for epifluorescence and phase contrast microscopy. Injections were performed at an objective magnification of 40× (NA 1.3 Plan Neofluar) and video microscopy with a 100×/NA 1.4 Plan-Apochromat with or without 1.6 optovar intermediate magnification. Filters blocking wavelengths below 590 nm were used for phase contrast illumination in order to avoid excitation of the fluorescent probe. Tungsten lamps (100 W) were used for both transmitted and epi-illumination. Data were acquired with a back-illuminated, cooled CCD camera from Princeton Research Instruments driven by IPLabs software (both from Visitron Systems) and stored as 16-bit digital images. The microscope was additionally equipped with shutters (Optilas GmbH) driven through a homemade interface to allow separate recordings of video sequences in phase contrast and fluorescence channels. Times between frames were 27 or 37 s. These time intervals are longer than used in the previous study , but were chosen so as to allow the recording of video sequences (50–100 frames) long enough to observe substrate contact turnover. Under these compromise conditions, not all targeting events would have been recorded. The video sequences were analyzed and processed on a Macintosh Power PC 7100/80 using IPLabs (Visitron Systems) and Adobe Photoshop 2.5.1 (Adobe Systems, Inc.) software. For phalloidin stainings, cells were fixed for 10 min in 3% paraformaldehyde in cytoskeleton buffer (CB: 10 mM MES, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, and 5 mM MgCl 2 , pH 6.1) and extracted for 1 min in 0.25% Triton X-100 in the same buffer. Cy3-Phalloidin was a kind gift of Professor H. Faulstich (Max-Plank Institute, Heidelberg, Germany) and was used at a concentration of 0.2 μg/ml for 30 min at RT. Pictures of cells that were fixed and stained on the microscope were taken in 100 mM DTE in CB to avoid photobleaching. For calculation of targeting frequency, 20-μm-wide regions of retracting and protruding edges of the same cells were compared. Contacts within 5 μm of the cell edge were analyzed, and the number of targeting events for each contact in a time period of 15 min recorded. For comparison of the size of focal adhesions contact length was measured using the IPLabs Measure Length tool. Focal contact turnover in spreading cells was determined by following the fate of contacts newly formed at 15 min after a further 15-min period. All calculations and statistics were performed using KaleidaGraf version 2.3.1. (Synergy Software, Inc.). The online version of this article includes videos that accompany several of the figures presented here. The video number, related figure, and URL are listed below. Video 1: Fig. 1 . http://www.jcb.org/cgi/content/full/146/5/1033/F1/DC1 Video 2: Fig. 3A–C ; Video 3: Fig. 3D–F . http://www.jcb.org/cgi/content/full/146/5/1033/F3/DC1 Videos 4 and 5: Fig. 4A–C , http://www.jcb.org/cgi/content/full/146/5/1033/F4/DC1 Video 6: Fig. 5 and Fig. 7 A; Video 7: Fig. 5 C. http://www.jcb.org/cgi/content/full/146/5/1033/F5/DC1 Video 8: Fig. 6 . http://www.jcb.org/cgi/content/full/146/5/1033/F6/DC1 Video 9: Fig. 7 A. http://www.jcb.org/cgi/content/full/146/5/1033/F7/DC1 Video 10: Fig. 8 . http://www.jcb.org/cgi/content/full/146/5/1033/F8/DC1 The spreading of a cell, when freshly plated onto a substrate may be likened to a state of unpolarized locomotion, in that protrusive activity occurs in all radial directions. Since freshly plated cells are more or less impossible to inject with fluorescent probes, we used goldfish fibroblasts (CAR cells) that had been stably transfected with EGFP-zyxin to follow contact dynamics during the spreading process. Fig. 1 shows typical examples of the contact dynamics of spreading cells in which the microtubule system was either intact , or disassembled by treatment before and during plating with nocodazole . For comparison, video frames are shown for the same times after plating. The first, evident difference is the greater rate of spreading in the presence of an intact microtubule cytoskeleton; after 30 min, the total spread area of the cell in A was almost twice that achieved without microtubules (B). A second, notable difference was seen in the rate of turnover of the formed contact sites. This is best illustrated by referring to the contact sites marked with arrowheads for each of the series shown in Fig. 1 . In A, the two marked contacts formed at the cell periphery at time 10′00″ after plating were no longer existent at 30′48″, having been superceded by new contacts formed under the advancing cell front. The same turnover of contacts was not seen in the presence of nocodazole. Instead, contacts formed at an early stage of spreading remained unchanged or elongated outwards . Analysis of contact fate was made in 9 cells by counting the number of contacts that disappeared between 15 and 30 min of spreading. In control cells, 75% of a total of 91 contacts (4 cells) disappeared during this period, whereas only 22% of a total of 99 contacts (5 cells) disappeared in cells spreading in nocodazole. An influence of microtubules on substrate contact turnover could also be observed during the reassembly of microtubules in cells that had first been allowed to spread to their maximum extent in the presence of nocodazole. In this case cells were used that had been transiently transfected with both EGFP-zyxin and EGFP-tubulin. As seen in Fig. 2 , the recovery of the microtubule network was associated with the disassembly of a large proportion of contacts in the perinuclear region of the cell. Out of 61 nonperipheral contacts, 29 disappeared during microtubule recovery and 32 were retained, but even these became smaller or less intense. Video sequences from a further 3 cells showed similar results. Since the active growth of microtubules was essential for targeting to occur, we modified microtubule growth kinetics to establish how this affected contact dynamics. Microtubules were first stabilized by brief treatment with taxol (20 μM, 30 min) and then, still in the presence of taxol, selected cells were injected with constitutively active L61Rac to induce the protrusion of lamellipodia and membrane ruffling. Microtubules and contacts were labeled in this case by the preinjection of cells (before taxol) with rhodamine-conjugated tubulin and vinculin. An example of such an experiment is shown in Fig. 3 . At the time of Rac injection , microtubules extended to the cell periphery but were no longer dynamic, according to the video analysis. Rac injection induced an advance of the cell edge as well as active rearward cytoplasmic flow and ruffling activity. The combination of the block in microtubule polymerization and the centripetal flow, sweeping microtubules rearwards , created a microtubule-free border behind the lamellipodium. In this region, contact sites that were only just visible at time 0′00″ (white arrowheads in A) had become enlarged and elongated at the end of the sequence . Notably, one peripheral contact (indicated by hollow arrow) that had remained associated with the end of a microtubule throughout did not increase in size. Measurements of 10 cells made 1 h after injection with L61Rac, 5 in the absence and 5 in the presence of taxol, revealed an average length of peripheral contacts, formed after Rac injection, of 0.67 ± 0.07 μm for the control set (total 472 contacts) and 1.29 ± 0.12 μm for the taxol-treated cells (total 344 contacts). An example of a control cell injected with L61Rac is shown in Fig. 3D–F , with a comparison in the inset (in F) of the peripheral contacts in C (taxol) at the same magnification. In a parallel study on Swiss 3T3 cells , it has been shown that the Rho kinase inhibitor, Y27632 , causes the dissolution of Rho-dependent focal adhesions, but not of Rac-dependent peripheral “focal complexes” that are associated with lamellipodium protrusion and membrane ruffling. As in Swiss 3T3 cells and HeLa cells , similar Rho-kinase independent focal complexes were found in CAR cells. Since many of these were elongated and associated with filopodia it is likely that their formation is not only dependent on Rac but also on Cdc42. Analysis of a total of 14 cells treated with the Rho-kinase inhibitor showed that focal complexes were also targeted by microtubules. For these experiments, cells were doubly transfected with EGFP-tubulin and EGFP-zyxin. In the example shown in Fig. 4 , the cell was fixed after the video sequence and stained with phalloidin to confirm that the concentration of Y27632 used (100 μM) had caused the disassembly of stress fiber bundles. As shown in the video sequence , one contact (arrowhead) that first appeared at 17′24″ was targeted between 45′09″ and 45′46″ and had then dissociated 10 min later (55′38″). Additional, new contacts were formed as the cell edge protruded beyond this contact site and these were also targeted by microtubules (arrows at 64′53″). During cell motility, the trailing parts of a cell develop strong contacts with the substrate and must be retracted to support the locomotory efforts of the cell front. It was therefore pertinent to analyze targeting activities in retracting regions, carried out in this case with fibroblasts coinjected with fluorescent tubulin and vinculin. An example of a moving cell is shown in Fig. 5 in which a lateral flank was retracted, in concert with the protrusion of the anterior lamellipodium. The video sequences revealed that the contact targeting frequency in the trailing flank was several fold higher than in the protruding front. 10 peripheral contacts between the two asterisks shown in A were all multiply targeted by microtubules. The number of targeting events in total were 57, amounting to an average of 5.7 targeting interactions for each contact. The sequence of events for one of the contacts is shown in Fig. 5 C. This contact, indicated at time 0′0″ with an open arrow, was targeted a total of 6 times, shown in the following 6 frames, and was retracted inwards after time 7′42″, along with the rest of the cell edge. The frequency of contact targeting in the protruding cell front during the same sequence was 0.75, that is eightfold less than at the retracting flank. For two other cells, corresponding targeting frequencies at retracting, versus protruding edges were 4.1 versus 0.28 (cell 2) and 4.3 versus 0.81 (cell 3). To further assess the consequences of contact targeting, we examined the effect of short term recovery of microtubules from depolymerization by nocodazole. This was most conveniently achieved through the temporal application of nocodazole (50 μg/ml) through a micro-pipette to one side of a cell. When locally applied for 15–20 min, nocodazole caused the depolymerization of peripheral microtubules in approximately one cell quadrant, adjacent to the needle tip (not shown). The phase of recovery was associated with either the retraction or protrusion of the cell edge and in both cases was accompanied by the dissolution of peripheral contact sites. A typical example of retraction (total of 5 cells examined: total of 80 contacts) is shown in Fig. 6 . In this example, 8 peripheral contacts were targeted by individual microtubules that grew out to the cell periphery. Each targeting event was followed by a decrease in vinculin label in the contact, culminating with contact release and retraction of the cell edge. Two further aspects of contact targeting are shown in Fig. 7 . In Fig. 7 A, the fate is shown of the medially situated contact outlined by a circle in Fig. 5 . This contact, originally oriented in the direction of cell protrusion , was subsequently divided into two contacts oriented along the axis of the retracting cell edge. Division of the contact was associated with microtubule targeting from opposite directions . 11 similar remodeling events were observed in 7 other cells. The spatially selective effect of targeting is likewise illustrated in the example shown in Fig. 7 B. Here, the fate of a closely packed group of contact sites is shown during microtubule recovery in a cell that had initially spread in the presence of nocodazole . The cell was doubly transfected with EGFP-tagged tubulin and zyxin. During recovery, one of the contacts (position marked by arrowhead) disappeared from the group and this contact alone was targeted by microtubules, at times 3′42″ and 21′35″. The selective dissolution of contact sites after microtubule targeting during recovery from nocodazole was observed for 25 contacts in 11 cells. Since a link between microtubules and the tension developed by stress fiber bundles has already been suggested (see introduction), we considered that strain-dependent signals may play a role in the targeting process. If so, it was to be expected that the relaxation of tension by external means would affect microtubule-contact interactions. To test this presumption, relaxants of actomyosin contractility were applied locally to cells that had been preinjected with rhodamine-tagged tubulin and vinculin. As antagonists, we used the myosin light chain kinase inhibitor, ML-7, and the myosin antagonist, BDM. Concentrations in the needle for local application were 2 mM for ML-7 and ∼0.5 M (saturated), for BDM. Since each inhibitor gave essentially the same result, only one example, with ML-7, is shown . In this figure the center of the ellipse corresponded to the point of application of the drug, with the needle directing the flow of inhibitor away from the cell edge. Two effects were observed. First, microtubules depolymerized from affected cell edge and this was followed by the release of contacts and their inward retraction. Outside the region of ML-7 influence , contact sites and microtubules were unaffected. The same result was obtained with a total of 5 cells treated with BDM (total of 157 contacts analyzed) and for 3 cells treated with ML-7 (total of 81 contacts analyzed). In parallel experiments, we treated CAR cells on coverslips with 50 mM BDM, fixed them after different times, and performed immunolabeling for tubulin and vinculin. Under these conditions, microtubules shrank towards the cell center so that after 30 min only a remnant of the microtubule cytoskeleton remained around the centrosome. Based on the observation that microtubule ends invaded vinculin positive contact sites in fibroblast lamellipodia, we earlier speculated that microtubules act to stabilize or potentiate the growth of substrate adhesions. Our present findings indicate that quite the opposite is the case, namely that microtubules exert a negative influence on contact development. As we have recently demonstrated , the interaction of microtubules with contact sites is by no means fortuitous, but entails a direct engagement that can involve a modulation in microtubule dynamics. The present results show that microtubules, in a reciprocal manner, influence the development and turnover of substrate contact sites. We propose that by this route microtubules exert the influence on the actin cytoskeleton which underlies their control of cell polarization. By virtue of their fibrillar nature and dynamic growth characteristics, microtubules are ideally suited for the point delivery of molecular complexes. Since contact formation is under the control of the small Rho-GTPases it is most likely that microtubules intervene in one or more of their signaling pathways. Indeed, in vitro experiments have already revealed the association with microtubules of a number of potential candidates that could be involved, including Rac , the Rac/Rho GEFs, H1 , and Vav , as well as the oncoprotein Lfc . Since only the immediate proximity of microtubules affects the development of contact sites, we presume that the effectors responsible for modulation are specifically concentrated at microtubule ends. In yeast, the accumulation or acquisition of molecular assemblies at the ends of microtubules is evident during mitosis and the concentration of tea1 at the ends of interphase microtubules appears to play a role in the determination of cell polarity . Further, in vertebrate fibroblasts, CLIP-170, whose Drosophila homologue binds myosin VI , accumulates at the growing, but not at the shrinking ends of microtubules . Regulatory factors could be delivered to the end of microtubules by molecular motors, one possible candidate being the Rac and Cdc42 effector MLK2, which associates with KIF3 kinesin . A primary conclusion from this study is that factors concentrated at the ends of microtubules are brought by microtubule growth into the proximity of substrate contacts to influence their development, by modulating acto-myosin contractility. Acto-myosin–based contractility, stimulated via phosphorylation of the myosin II regulatory light chain, has been shown necessary for the formation of stress fiber bundles and focal adhesions . Accordingly, inhibitors of contractility, such as BDM, or of myosin light chain kinase, cause the dissociation of focal adhesions, or block their formation in response to the upregulation of Rho . Other studies have shown that the Rho-associated kinase, p160ROCK or Rho-kinase, acts downstream of Rho and stimulates myosin phosphorylation by inhibiting the myosin light chain phosphatase. At the same time, it has become apparent that not all substrate contact sites depend on Rho-kinase for their formation. In particular, peripheral contact assemblies similar to focal complexes induced by Rac are formed in cells in which Rho-kinase is specifically inhibited . Despite their independence of Rho-kinase, these contact sites are likewise dissociated by both BDM and ML-7 and therefore also depend on myosin II–based contractility for their maintenance. Thus, the development of Rho-kinase–independent contacts, which serve to support the protrusive activity of the cell front may also be regulated via modulations in contractility. Since targeting events correlated with the diminution, retraction, or dissociation of contact sites, it is evident that microtubule targeting serves to antagonize contact development. This was supported by our finding that a block in targeting activity caused the uncontrolled growth of peripheral contacts. Former studies have linked microtubule disassembly with the enhancement of contractility. We propose that a converse mechanism exists whereby the targeted growth of microtubules into contact sites mediates a highly localized relaxation and that this is the mechanism by which contact growth is reversed or retarded. Consistent with this conclusion was the finding that intensive targeting of contacts at cell edges and the application of myosin inhibitors produced similar effects. As we have seen, the retraction of an adhesion site at a trailing cell edge may be preceded by multiple targeting events. These findings are generally supported by the statistical characterization of regional microtubule dynamics by Wadsworth 1999 ; her studies showed that microtubule ends close to nonmotile edges exhibited shorter excursions and frequent catastrophes, whereas longer excursions and fewer catastrophes occurred in protruding regions. The shorter and more frequent excursions would correspond to what we observe at cell edges destined for retraction. We conclude that a single targeting event serves to provide a quantum relaxation dose and that further doses, as required, must be supplied by freshly charged microtubule ends in subsequent targeting forays. By modulating the number of targeting events, differential relaxation effects may then be exerted on contact assemblies in different regions of a cell. In this way, microtubule signaling at contact sites could modulate the substrate contact pattern of a cell and thereby determine its polarity . In a recent study, Pelham and Wang 1999 provided the most detailed tension patterns yet of a locomoting fibroblast. Their results show a high, but fluctuating level of tension at the cell front and a lower but constant tension in the trailing cell rear. It is further proposed that mechanical forces play an important role in signal transduction. In this context, we suggest that microtubules are important players in these signal transduction events. In response to tension-sensing cues propagated through the actin cytoskeleton, microtubules could be guided to contact sites to control tension according to the requirements for protrusion or retraction. Indeed, the rapid depolymerization of microtubules in response to the treatment of cells with contractility inhibitors may reflect a general dependence of microtubule stability on a stressed actin cytoskeleton. It is noteworthy that the microtubule destabilizing effect we observed with myosin inhibitors was not detected in previous studies . This can be explained for BDM by the use here of both higher concentrations and longer times; a higher sensitivity of CAR cells to BDM can also not be excluded. That the effect was not simply an artefact induced by high concentrations of BDM is suggested by the observation that the myosin light chain kinase inhibitor, ML-7, produced the same result. Chrzanowska and Burridge showed that the alternative inhibitor, H-7, applied at a concentration of 300 μM for 30 min dissociates focal adhesions in REF-52 fibroblasts; under these same conditions, we also observed microtubule destabilization, culminating in the shrinkage of the microtubule cytoskeleton to the perinuclear area (not shown). In line with our own studies, Cook et al. 1998 found that elevated levels of Rho, which increases contractility, lead to a stabilization of microtubules. An alternative view of the way microtubules may determine cell polarity has been put forward by Waterman-Storer et al. 1999 , who showed that the polymerization of microtubules during the recovery of Swiss 3T3 cells from nocodazole was associated with the upregulation of Rac and the corresponding induction of membrane ruffling. They suggested that this effect may be explained by the generation of Rac-GTP at microtubule ends. Such a mechanism was, however, difficult to reconcile with the continued elevation of Rac-GTP long after microtubule polymerization was complete . As in earlier studies , these authors noted a penetration of microtubules into regions of active protrusion and further noted a correlation between protrusive activity and dynamic microtubule growth, rather than with polymer mass. However, protruding cell edges in rapidly motile cells can have very few associated microtubules , making it unlikely that active Rac is generated from microtubule ends at the cell front. An alternative explanation of their result is provided by the finding that Rac and Rho antagonize each others activities . As has previously been demonstrated, microtubule depolymerization leads to the upregulation of Rho and microtubule polymerization to its downregulation, reflected here in the dissolution of focal adhesions during recovery from nocodazole. We surmise that the upregulation of Rac observed during microtubule polymerization is an indirect result of the downregulation of Rho and not to the microtubule-dependent generation of Rac at the cell front. Further work will be required to demonstrate the means of guidance of microtubules to contact sites and the nature of the relaxing signal they convey to regulate contact dynamics. As regards the signal, since both Rho-kinase–dependent focal adhesions and Rho-kinase–independent focal complexes are modulated by microtubule targeting, we conclude that the point of intervention of microtubule-bound modulators in the pathway leading to contractility is downstream of Rho-kinase. | Study | biomedical | en | 0.999997 |
10477758 | Explant and dissociated cultures of superior cervical ganglion (SCG) 1 neurons were prepared from embryonic rats (E19) or postnatal mice (P0) as described previously . For microinjection and gene gun experiments, cells were plated on substrates coated with 0.1–0.2 mg/ml polyornithine followed by laminin (16 μg/ml). Microinjection was performed 1–3 d after plating. For cultures grown in nocodazole or latrunculin, the drug was added from stock solutions prepared in DMSO directly to the plating medium (unless indicated), and then exchanged for fresh medium + drug every 48 h. The final DMSO concentration did not exceed 0.01%. Latrunculin A was used at a concentration of 0.1 μg/ml . Cytochalasin E was used at a concentration of 20 μg/ml . Nocodazole was used at a concentration of 3.3 μg/ml unless indicated. SCG neurons from dilute-lethal mice were more sensitive to nocodazole than cells from rats or heterozygous mice. Less than 10% of the cells plated directly in medium containing nocodazole-developed processes after 3 d in culture (compared with ∼70% in controls). Therefore, a modified approach was used. SCG neurons from dilute-lethal and heterozygous littermates were plated in normal medium and grown for either 4 or 16 h. The cultures were then switched to medium containing nocodazole. Two different concentrations of nocodazole were used to depolymerize microtubules from neurites. Cultures grown for 4 h received 0.1 μg/ml, while cultures grown for 16 h received 0.15 μg/ml nocodazole. An affinity-purified antiserum to rat myosin Va was used on immunoblots of homogenized whole brain to identify dilute-lethal (d l-20J /d l-20J ) P0 mouse pups. The methods were the same as previously described . The antiserum used for microinjection was made against a maltose binding protein—rat myosin Va tail fusion protein. The myosin Va peptide corresponded to amino acids 1005–1830 of the mouse myosin Va sequence, which includes part of the rod and the entire globular tail . The affinity purification and specificity of the antiserum has been described previously . An IgG fraction of the affinity-purified antiserum was used for conjugation with indocarbocyanine (Cy3). The conjugation was carried out according to instructions provided by the manufacturer of the dye (BDS). The conjugate was separated from free dye on a G25 column and eluted in injection buffer (100 mM KCL, 10 mM Na 2 HPO 4 , pH 7.2). The concentration of dye compared with protein was estimated from absorbance at 556 and 280 nm; the ratio was 5:1. The resulting protein solution was concentrated threefold using a Centriprep-30 concentrator. The activity was tested by immunofluorescence labeling of cultured SCG neurons. The distribution of label was identical to that obtained with the nonconjugated purified antiserum. Before micropipette filling, the solution was centrifuged for 15 min at maximum speed in a microcentrifuge. An Eppendorf microinjection system equipped with Femtotips was used for pressure microinjection. Considering the appearance in phase contrast and ability to retain dye, ∼60–80% of the rat cells survived the injection processes. Mouse neurons were more difficult to inject and so most microinjection experiments used rat neurons. Healthy cells were imaged 15 min to 5 h after microinjection unless otherwise indicated. A rat myosin Va cDNA clone containing the coil–coil and globular tail, but lacking the IQ repeats and motor domain was inserted into a green fluorescent protein (GFP) vector (Clontech) and amplified in bacteria. Purified plasmid DNA was used for transfection. 1- or 1.6-μm gold particles were coated with 2 μg DNA per mg gold according to the manufacturer's instructions. SCG cultures were transfected with a hand-held Helios gene gun (Bio-Rad Laboratories) using a pressure of 70–80 psi 1–3 d after plating. Neurons microinjected with myosin Va-Cy3 antibodies, or expressing the GFP-myosin Va tail construct, were imaged at 37°C on an inverted microscope (Olympus Corp.) with a 100× 1.4 NA lens (Nikon Inc.), unless otherwise indicated. Before imaging, the cultures were switched to a Hepes-based culture medium (+ or − the appropriate drug). A 100-W mercury arc lamp was used for illumination. The light was attenuated with a neutral density filter (25% transmittance) and shuttered between exposures. Images were collected using a cooled, slow scan CCD (C250; Photometrics) containing a 512 × 512 pixel chip (thinned, back illuminated; Tektronix Inc.). The images were binned (2 × 2). Exposures were 0.25 or 0.5 s at 6- or 5-s intervals. Cultures were fixed for 20–30 min with a warm (37°C) solution of 4% paraformaldehyde (EM grade; EM Sciences) in 0.1 M cacodylate buffer, pH 7.4, containing 10 mM CaCl 2 and 10 mM MgCl 2 . Cells were permeabilized with 0.1% saponin or 0.1% Triton in the same buffer for 30 min. They were incubated with blocking solution (8 mg/ml BSA, 5% goat serum, 0.5% fish gelatin) for 20–30 min, and then labeled with primary antibodies diluted in a 1/5 dilution of the blocking solution for 1 h. The anti–rat myosin Va affinity purified antiserum was used at a concentration of 0.5 μg/ml. The SV2 mAb (gift of Dr. K. Buckley) was used at a 1/200 dilution. The anti–synaptophysin mAb (Sigma Chemical Co.) was used at a 1/1,000 dilution. Secondary antibody (minimal species cross-reacting antibodies; Jackson Immunochemicals) incubations were for 45–60 min; dilutions ranged from 1/200 (FITC) to 1/1,000 (Cy3). An antifade agent (Vectashield) was used to minimize photobleaching during imaging. To determine the degree of colocalization of fluorescent spots, the 16-bit images were converted to 8 bits and superimposed in two different colors. They were scored as “colocalized” if their fluorescent area overlapped and the peak brightness of the SV2 or synaptophysin staining was at least 20% of the brightness of the myosin Va staining. Although this may underestimate the degree of association between the two labels, it prevents the possibility of scoring spots for colocalization that show apparent association due to fluorescence filter bleed-through. For quantitation of intensity, values were taken directly from flat field–corrected 16-bit images and were expressed in relative intensity units (iu) with a maximum possible value of 65,000. For each comparison, the same exposure time and excitation light intensity were used. Absolute values of intensity units obtained in different comparisons depended on the intensity of the excitation light and exposure time, which was varied between experiments. The movement of either Cy3-labeled antibody or GFP-myosin Va (tail) fluorescent spots was analyzed with the following method. Time-lapse images were adjusted for optimal contrast, and then played in series using the movie tool available in either Isee (Inovision Corp.) or Iplab Spectrum (Scanalytics). Sequences from thin neurites with relatively few clearly visible spots were selected for analysis. This allowed us to follow the same spot between time points because most spots moved in predictable trajectories within a single focal plane. The sequences were zoomed (2×) and played forward, and then in reverse so that moving spots could be selected and followed over multiple frames. For each frame, a mark was placed over the center of an individual spot on a clear acetate overlay on the computer monitor screen. Spots that could not be followed for at least four frames (elapsed time of 15 or 18 s) were not used in the final analysis. This limited the analysis to ∼30% of the spots that appeared to move in a sequence. The distance between marks was measured in millimeters, and then converted to micrometers using a calibration slide (Leica Inc.) imaged with the same optics and CCD detector, and then displayed on the monitor. The number of time points analyzed for individual spots was highly variable (4–18). For comparison purposes, the average rate of displacement was defined as the displacement that occurred between the first and fifth time point divided by the total elapsed time (20 or 24 s). The maximum rate was defined as the largest displacement between two time points divided by the time interval. Methods were modified from published methods . Cultures grown on aclar coverslips containing neurons that had been microinjected with the anti–myosin Va-Cy3–conjugated antibody were returned to the incubator and allowed to grow for 30–90 min. Cells were fixed with a warm mixture of 0.25% glutaraldehyde and 0.32% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH = 7.4, containing 10 mM CaCL 2 and MgCL 2 . The cells were reidentified by fluorescence and phase microscopy and the images recorded. The cultures were preincubated with cold 1.4 mg/ml DAB (Sigma Fast DAB; Sigma Chemical Co.) in phosphate buffer for 15 min. After exchange with fresh cold solution, the microinjected cells were irradiated using a 100-W mercury arc lamp on an inverted microscope (Olympus Corp.). A 25×, 0.75 NA oil immersion lens with high transmittance (Leica Inc.) was used for the irradiation. Irradiation times were varied from 15 to 35 min. Fluorescence and bright field observations were periodically made to check for photobleaching and appearance of a dark brown reaction product. Darkening was detected by 15 min, but photobleaching was not complete until ∼35 min. For controls, cells that were not microinjected, but on the same coverslip, were irradiated for the same times in fresh DAB solution. Cells were further fixed with 2.5% glutaraldehyde for 30 min. They were post-fixed with 3% osmium tetroxide for 1 h. Before dehydration and embedding, the cells were stained with 1% uranyl acetate dissolved in 1% sodium acetate for 1 h. The areas containing the photoconverted cells were cut from the larger aclar coverslip to allow identification before thin sectioning. The coverslip piece was flat embedded. Once the coverslip was removed from the polymerized resin, the cells could be identified by phase contrast microscopy using the recorded images as guides, and then further trimmed for sectioning. After microinjection and recovery in the incubator, cells were fixed and images were recorded as described above. Cells were treated with 1 mg/ml sodium borohydride in PBS for 20 min. The cells were permeabilized with 0.05% filipin in PBS for 30 min. They were incubated with the antibody blocking solution for 30 min, followed by incubation with 6 nm colloidal gold conjugated to an anti–rabbit antibody overnight at 4°C. The procedure was modified from that described by Mrini et al. 1995 . Mice were perfused with 0.25% glutaraldehyde + 4% paraformaldehyde in cacodylate buffer, pH 7.4 (+ 2 mM CaCl 2 ). The brain was removed and immersed in the same fixative for at least 4 h. The cerebellum was dissected free and embedded in agar for vibratome sectioning. 50-μm sections were cut. The sections were treated with freshly made 1 mg/ml sodium borohydride in PBS for 20 min. They were equilibrated with 35% sucrose + 14% glycerol in PBS. The sections were frozen for 15 s in isopentane cooled with liquid nitrogen and thawed in PBS. This was repeated once. The sections were incubated for 2 h with PBS containing 0.1% saponin. They were incubated for 2 h in block solution containing (freshly made) 0.1% saponin. The incubation was changed to block solution without saponin and continued for another 30 min. They were incubated overnight at room temperature with the primary antibodies (1/2,000 antimyosin Va; 1/200 anti–SV2 mAb) in a 1/5 dilution of the block. The incubation was continued at 4°C for another 48 h. After washing with PBS on rocker for 1 h, the sections were incubated overnight on a rocker with gold-conjugated antibodies diluted 1/50 in a 1/5 dilution of the block. The incubation was continued at 4°C overnight. The sections were washed on a rocker for 1 h in PBS. They were fixed with 2.5% glutaraldehyde in PBS for 1 h. They were postfixed with osmium-potassium ferricyanide and stained en block with 1% uranyl acetate for 1 h. They were dehydrated and embedded using standard procedures. EM images were digitized using a high-resolution scanner (Agfa Duoscan). The areas of presynaptic terminals were determined by tracing the perimeter of terminals in Iplab Spectrum. The area in pixels was converted to square micrometers using the calibration bars printed on the EM negatives. Because the number of mitochondria in terminals were highly variable and could influence the calculation of synaptic vesicle density, their area was also determined and subtracted from the measured terminal area for the calculation of synaptic vesicle density. The numbers of synaptic vesicle profiles were counted and the density calculated from the corrected terminal area. Initially, a series of antibody concentrations and injection pressures were tested in microinjection experiments. It was determined that at a minimal injection pressure (<4 psi) and low antibody concentrations (<2 mg/ml) small bright myosin Va-Cy3 spots appeared in superior cervical ganglion neurons. The size and distribution of these spots were consistent with previous results using myosin Va antibody staining on fixed SCG neurons . The bright spots were not apparent immediately after injection, but appeared at the earliest time point (15 min) for time-lapse observations after injection and persisted at least 16 h (longest time after injection of observation). Higher concentrations of antibody (4 mg/ml) or injection pressures produced much larger bright spots . The small bright spots were highly dynamic, while the large bright spots never made rapid movements [but in neurites did make slow (0.28 ± 0.08 μm/min, n = 15) retrograde movements]. The absence of larger spots from the dimmer microinjected cells combined with the differences in dynamics suggests that the large spots represented aggregates of the smaller spots formed by antibody cross-linking. SCG neurons cultured from dilute-lethal mice grow and extend neurites normally in culture . Neurons injected ( n = 8) with the different concentrations of the antibody showed only a diffuse distribution of fluorescence in cell bodies and neurites . The lack of bright spots in dilute-lethal neurons indicates that the spots are likely to result from specific interactions with myosin Va–containing structures rather than nonspecific antibody aggregation or sequestration. The rapid movements of individual small bright spots observed in neurites of rat (21 cells) or heterozygous (d v /d l ) mouse (nine cells) SCG neurons injected with low concentrations of antibody at reduced pressures were measured and analyzed in several different ways. Most frequently, during a 20-s time period, ∼10% of the spots moved and the remaining were stationary. Movement of spots was bi-directional; spots sometimes paused or transiently reversed direction, but usually made progress either towards or away from the cell body . For analysis, movements away from the cell body are designated with positive values, while movements toward the cell body are indicated by negative values. Among the 40 spots selected for analysis, there was an apparent overall bias toward anterograde movement (69%). To determine whether our selection process influenced this result, the direction was scored in three 6-min time-lapse sequences from different cells for every moving spot ( n = 53). An anterograde bias (62%) was still detected. An anterograde bias is consistent with previous reports on axonally transported vesicles in cultured neurons . However, chi-square analysis using the assumption that transport was equal in both directions (null hypothesis) indicated that the apparent bias was not statistically significant ( P > 0.2). The maximum rates of movement achieved by small spots were widely distributed . Because we could not make recordings of spot movement with delays between exposures <5 s, it is not possible to be certain of the reason for the variation in maximum rate. Either the spots achieve highly variable maximum rates or movements between time-lapse intervals are sometimes interrupted by short pauses at irregular intervals. The time-lapse data is clearly influenced by pauses and transient direction reversals, since the average rate of particle movement was considerably less (0.18 ± 0.09 μm/s, n = 24) than the mean maximum rate (0.46 ± 0.28 μm/s, n = 40). To determine whether the movements of the microinjected myosin Va-Cy3–labeled antibodies were characteristic of a set of structures that are uniquely associated with myosin Va, some rat SCG neurons were microinjected with an affinity purified myosin 1α-Cy3 antiserum . The pattern of fluorescent spots that formed was identical to that observed in fixed cells stained with the myosin 1α antiserum. Fluorescent spots were both stationary and moving. The movement was bi-directional. The mean maximum rate of particle movements from myosin 1α labeled structures was significantly slower (0.09 ± 0.08 μm/s, n = 12; t test, P < 0.001) than those labeled with antibodies to myosin Va. The slower rate of movement of the myosin 1α-Cy3 fluorescent spots suggest that the two different antibodies are reacting specifically with different populations of transported structures. To determine the identity of the myosin Va-Cy3–labeled spots in microinjected cells, some SCG cultures (six) were fixed, permeabilized, and labeled with mAb antibodies to the synaptic vesicle proteins, SV2 and synaptophysin . SV2 label showed partial colocalization (49%) with the small spots of myosin Va-Cy3 antibody ( n = 129) in neurites . This partial colocalization was apparent in cell bodies, varicosities, and enlarged neurite terminations (possible presynaptic terminals), but was difficult to quantitate because of the increased thickness. The partial colocalization may result from the limited amount of myosin Va-Cy3 antibody microinjected. Consistent with this possibility, neurons injected with higher concentrations of antibody that showed large bright spots of antimyosin Va-Cy3 antibody also showed large bright spots of SV2 label . Superimposition of images showed that all of the large bright spots in neurites colocalized and smaller SV2 spots were no longer detectable. This suggests that the higher concentration of the myosin Va antibody coaggregated most, if not all, of the available SV2. SV2 and synaptophysin have been reported to be axonally transported in different organelle compartments . To determine whether both of these organelle compartments are transported in association with myosin Va, a mAb to synaptophysin was also used for labeling. In contrast to the results with the SV2 labeling, immunofluorescence labeling of injected neurons with a monoclonal antibody to synaptophysin produced only very limited colocalization between the two labels. In neurites, the colocalization was minimal (≤4%). Neurons with large bright spots of myosin Va-Cy3 label occasionally showed corresponding bright spots of synaptophysin label. However, these spots were usually smaller than the myosin Va-Cy3 spots. In addition, many small spots of synaptophysin label still filled the cell body, indicating that most of the synaptophysin was not coaggregated by the microinjected antibody. In cultures that were 3 d or older, enlarged terminations formed in close opposition with other neurons (possible synaptic contacts). In neurons injected with low concentrations of antibody, bright staining with both synaptophysin and myosin Va-Cy3 labels was observed within these structures, but it was difficult to determine the degree of overlap because of their thickness. The colocalization of the microinjected myosin Va-Cy3– and SV2-labeled antibodies provides support for the vesicle association of myosin Va. To test this assumption, two types of ultrastructural analysis were used. First, the microinjected myosin Va-Cy3–labeled antiserum was photoconverted to an electron-dense reaction product, visualized by thin section EM. This method avoids the permeabilization of the injected cells. Second, a gold-conjugated secondary antibody was applied to microinjected neurons that had been permeabilized with saponin. In photoconversion experiments, neurons microinjected with low concentrations of myosin Va-Cy3 antibody that were clearly intact after fixation (noninjured or degenerating as indicated by phase contrast and fluorescence microscopy) showed dark reaction product surrounding individual vesicle profiles in neurites . Neurons injected with higher concentrations of the myosin Va-Cy3 antiserum that showed the large bright spots by fluorescence microscopy also contained larger discrete areas of dark granular reaction product in cell bodies and proximal neurites . These areas were always associated with multiple organelle profiles, suggesting that the antibody aggregated vesicles through its cross-linking ability. Controls (noninjected cells subjected to irradiation) showed no reaction product. In one experiment, a microinjected cell showing only the small spots of myosin Va-Cy3 fluorescence was fixed, permeabilized, and then incubated with a secondary antibody conjugated to 6 nm colloidal gold. Thin sections from this cell showed discrete labeling of individual structures that resembled organelle profiles . These results indicate that the rapidly moving spots in neurites of cells microinjected with the myosin Va-Cy3 antiserum are likely to be individual vesicles. A different method of marking myosin Va–associated organelles was necessary to: (a) control for the possible effects of surface antibody binding on myosin Va function, and (b) allow labeling of myosin Va–associated structures in dilute-lethal neurons. There is evidence that the globular tail portion of the myosin Va protein is responsible for its putative interaction with organelle surfaces . Expression of a GFP–myosin Va construct containing this domain would be expected to produce a fusion protein capable of binding to organelles. In melanocytes, cultured cells and yeast expression of similar constructs have been shown to target the tail truncate to the full-length myosin V's normal location . At appropriate expression levels, this would provide a marker for individual organelles that normally associate with native myosin Va. To determine if a rat GFP–myosin Va tail construct could act as an appropriate marker, we expressed it (designated as GFP–myosin Va-t) in cultured SCG neurons. We then used immunofluorescence with a myosin Va antibody (Dil-2, gift of John Hammer) that was treated so that it lacked reaction with the expressed fusion protein to determine if it was correctly targeted in mouse neurons. Most heterozygous cell bodies appeared bright and fluorescence could be detected at low magnification in all neurites emerging from the cell body. At higher magnification, the fluorescence consisted mainly of bright spots of varying size and intensity. In cell bodies, bright irregular shaped spots were frequently seen in the perinuclear region and near the plasma membrane. Large bright spots were also seen in proximal portions of some neurites, but most neurites contained only small spots. Terminal varicosities in older cultures (>2 d) contained many small dim spots and an occasional bright spot of fluorescence. Nearly complete overlap in the distribution of GFP–myosin Va-t and myosin Va immunofluorescence label was observed in thin neurites . Similar overlap was seen in cell bodies, growth cones, or terminal, but the increased thickness made it more difficult to determine the precision of the colocalization. The exception was very bright large GFP spots (possibly vacuoles) located in a few cell bodies that increased in size and number with time in culture. Similar large very bright spots were seen in most nonneuronal cells. The large very bright spots showed weak or no colocalization with myosin Va antibody staining (not shown). We assume that the very bright spots in these cell bodies (and nonneuronal cells) represent sequestered and/or degraded GFP–myosin Va-t fusion protein. Cells showing such large bright spots were not used for analysis of spot movement or cell body brightness. Dilute-lethal neurons showed a similar distribution of the GFP–myosin Va-t. However, no specific DIL-2 antibody staining was seen in the cells expressing GFP–myosin Va-t, indicating that the antibody did not recognize the expressed myosin Va tail fusion protein . Colocalization of the GFP-myosin Va-t small spots of fluorescence with SV2 immunofluorescence were also observed in cells fixed and then stained with a monoclonal antibody to SV2 . Although we did not quantitate the degree of colocalization, it resembled that seen with the microinjected myosin Va-Cy3 antibody. To compare the dynamics of myosin Va–associated organelles in normal and dilute-lethal neurons, we used time-lapse recording to obtain quantitative data on their movements in neurons transfected with myosin Va-t. Unexpectedly, expression of the GFP-myosin Va-t construct in neurons cultured from dilute-lethal mice resulted in bright spots with similar characteristics to those observed in neurons from rats and heterozygous mice . Small bright fluorescent spots were distributed along the entire length of both dilute-lethal and control neurites. No detectable accumulation of bright spots was observed in cell bodies of dilute-lethal neurons. Consistent with this observation, the peak brightness of neuronal cell bodies from heterozygous or dilute-lethal mice showed no significant difference ( P > 0.2; heterozygous = 9,982 ± 3,220 iu, n = 4, dilute-lethal = 7,765 ± 2,424 iu, n = 5). Surprisingly, ∼10% of the spots in any one sequence recorded from neurites of dilute-lethal neurons were undergoing rapid movements. Since myosin Va cannot be the motor in dilute-lethal neurons, other motor proteins must be responsible for the movement. Similarly, recordings from transfected SCG neurons from rat embryos or heterozygous mice revealed that ∼10% of the fluorescent spots in neurites were undergoing movement . To determine whether small spots that remained stationary in neurites during an entire sequence of recording might represent a different population than moving spots, we compared their peak brightness. Consistent with this idea, the stationary spots gave a brighter mean value (2,865 ± 1,089 iu, n = 21) than moving spots (1,065 ± 426 iu, n = 14), and the difference was significant ( t test, P < 0.001). Similar to the microinjected myosin Va-Cy3 antiserum, sometimes spots that were stationary at the start of recording began to move and motile spots stopped moving (and were therefore categorized as moving spots). Moving spots also showed short pauses and direction reversals characteristic of saltatory movement . Movement was bi-directional and was biased toward anterograde movement. In dilute-lethal neurons, 88% of the spots analyzed for speed (21/24) moved in the anterograde direction. We also scored the direction of movement of all spots in eight sequences. Again, the movement was predominantly in the anterograde direction (26/30 or 87%). In contrast, in the rat or heterozygous neurons, only 64% percent of the spots selected for quantitative analysis made anterograde movements. In five 6-min time-lapse sequences, the directions of all moving particles were scored. 68% (23/34) made anterograde movements. While chi-square analysis indicated that the apparent anterograde bias observed in neurites from rat or heterozygous mice was not statistically significant, the anterograde bias observed in dilute-lethal neurites was significant ( P < 0.01). There was no detectable difference in maximum or average rates observed in SCG neurons from rats compared with heterozygous mice and so the results have been pooled . The maximum rate was significantly greater ( t test, P < 0.02) than that obtained with the microinjected myosin Va-Cy3 antibody. However, the average transport rates were the same (myosin Va-Cy3 antibody = 0.18 ± 0.09, n = 40 vs. GFP-myosin Va-t = 0.15 ± 0.07 μm/s, n = 23). Comparison of the maximum rates of particle movements in cells derived from rats and heterozygous mice to those in cells from dilute-lethal mice indicated that the latter were significantly faster . Thus, myosin Va activity may dampen the speed of organelle transport similar to that observed in melanocytes . However, the average rates were not significantly different (0.15 ± 0.07 μm/s, n = 23 vs. 0.22 ± 0.21 μm/s, n = 21, P > 0.1), because the pauses and direction reversal have more impact on the average rate than the small difference in maximum rate. The above results indicate that motors other than myosin Va must contribute to the movement of myosin Va–associated organelles. The high rates of movement suggest that microtubule motors might be involved. To determine whether microtubules were necessary for the rapid movements of myosin Va–associated organelles, chronic application of nocodazole was used to depolymerize microtubules. Cells were plated and then grown in the continuous presence of the nocodazole (3.3 μg/ml) for 3–6 d . Microtubules were completely eliminated from the broad lamellae and short processes produced by the treated neurons under these conditions . Despite the difference in morphology, nocodazole-treated cells microinjected with myosin Va-Cy3 antibody showed a similar distribution of fluorescent spots to untreated cells . Fluorescent spot movements were still apparent in time-lapse recordings, but moved at reduced rates . Fluorescent spots (particles) paused and reversed direction more frequently than in untreated cells . A sizable and significant decrease ( t test, P < 0.001) occurred in maximum rate (0.1 ± 0.05 μm/s, n = 12) in the absence of microtubules . Similarly, SCG neurons from embryonic rats or P0 heterozygous mouse pups cultured for 3 d in medium containing nocodazole and then transfected with GFP-myosin Va-t showed fluorescent spots distributed throughout the entire length of their short processes. Time-lapse recordings revealed that small spots in the short neurite-like processes moved at significantly reduced average and maximum rates ( t test, P < 0.001) compared with untreated cells . After recordings, cultures were fixed and immunofluorescently stained for actin and microtubules. Identical to cells used for antibody microinjection, short microtubules segments were sometimes observed in the perinuclear region, but most cells lacked microtubules in the broad lamellae and short neurite-like processes used for time-lapse recordings. In cells from dilute-lethal mice that were grown under a modified nocodazole treatment protocol, GFP-myosin Va-t fluorescent spots were absent from the distal portions of processes, but were present at the base and in more proximal portions. Time-lapse recordings indicated that the small fluorescent spots within the processes appeared to make only short back and forth movements. Quantitative analysis indicated that the net displacement of spots during the recordings were extremely small . The calculated rates of movement (maximum = 0.04 ± 0.01 μm/s, average = 0.005 ± 0.004 μm/s, n = 19) were significantly less ( t test, P < 0.001) than the maximum and average rates of movements seen in heterozygous cells treated with nocodazole. This suggests that the small back and forth movements that were observed were not directed, but are likely to be Brownian-like. The very small rate of displacement was the same as the slow movement of large bright spots observed in cells microinjected with high concentrations of myosin Va-Cy3 antibody. This movement may result from other non–myosin Va-dependent mechanisms. Similar slow movements have been reported in dilute-lethal melanocytes . Dilute-lethal cultures used for recordings were fixed and labeled with rhodamine phalloidin and a monoclonal antibody to tubulin to reveal the distribution of F-actin and microtubules. Similar to cells plated directly in nocodazole, microtubules were sometimes present in the perinuclear region, but were absent from the lamellae and most short processes that extended from the cell body. Unlike cells plated directly in nocodazole, very small, discontinuous microtubule segments were occasionally observed in the shortened processes, suggesting that some microtubule segments formed in processes before nocodazole exposure were stabilized. However, in the processes of nocodazole-treated cells from dilute-lethal mice, we never observed movements of GFP-myosin Va-t that were clearly directed. To determine whether actin-based motors might contribute to the rapid movements of myosin Va–associated organelles, SCG neurons from heterozygous mice were grown for 3 d in medium containing latrunculin A. Neurons had altered morphology , and lacked detectable actin filaments when stained with rhodamine phalloidin (not shown). Neurons expressing GFP-myosin Va-t showed fluorescent spots distributed along the entire length of their processes. Time-lapse recordings revealed the rapid movement of small GFP-myosin Va-t spots in neurites . The maximum rates of movement were significantly greater ( t test, P < 0.001) than those observed in nocodazole-treated cells . Although the maximum rate of spot movement was slightly less than the maximum rate observed in untreated cells, the difference was not significant ( t test, P = 0.1). This indicates that actin-based motors are not necessary for rapid movements in axons If the capture model for melanosome accumulation in peripheral dendrites of melanocytes holds for myosin Va–associated organelle distribution in neurons from dilute-lethal mice, then one would predict: (a) a concentration of myosin Va–associated organelles within cell bodies of neurons, and (b) a depletion of the same organelles from their peripheral target. We had not observed accumulation of GFP-myosin Va-t label in cell bodies of dilute-lethal neurons. However, because the observations were done at relatively short times (<20 h) after the beginning of the GFP-myosin Va-t expression, it is possible that accumulation would only be detected with increased time. To address this possibility, the distributions of anti–SV2 label in 3-d-old dissociated cultures of SCG neurons from heterozygous and dilute-lethal mice were compared. Because SV2 shows a high degree of colocalization with bright spots of myosin Va, it should provide a good independent marker of myosin Va–associated organelles. The peak brightness of SV2 label in cell bodies of dilute-lethal neurons was slightly greater than those from heterozygous controls ( dilute-lethal = 778 ± 45 iu, n = 4 vs. heterozygous = 639 ± 48 iu, n = 8), but the difference was not significant ( P > 0.1). This indicates that SV2 does not accumulate in cell bodies of dilute-lethal neurons, consistent with the observation that transport of myosin Va–associated organelles is biased towards anterograde movement. The brightness of SV2 label was also compared in regions that showed the brightest SV2 label in neurites: varicosities, branch points, and axon terminations. The comparison was done under two different culture conditions: in 3-d-old low-density dissociated cell culture (cells were not contacting each other), and in explants grown for 5 d. In the low-density dissociated cultures, varicosities, branch points, and terminations of dilute-lethal neurons showed a significant increase ( P < 0.001) in peak brightness ( dilute-lethal = 1,068 ± 220 iu, n = 11 vs. heterozygous = 678 ± 89 iu, n = 14), indicating that SV2 does accumulate peripherally within these regions. The regions showing accumulation of SV2 label also colabeled with a monoclonal antibody to tyrosinated tubulin . Since tyrosinated tubulin is associated with dynamic plus ends of microtubules, it suggests that these regions contain dynamic microtubule ends. The degree of colocalization was 100%. In other words, the presence of bright SV2 label could predict the occurrence of bright tyrosinated tubulin label and vice versa. To insure that the low density of cells were not affecting the accumulation of SV2, the peak brightness of SV2 label was also compared in neurites of 5-d-old explant cultures. Under these conditions, neurites grow out radially from explants and do not form synapses (outside the body of the explant). Dilute-lethal neurons showed significant increases ( P < 0.001) in the peak brightness of staining in varicosities, ( dilute-lethal = 6,378 ± 1,289 iu, n = 14 vs. heterozygous = 4,155 ± 400 iu, n = 12). Second, the distribution of anti–SV2 antibody label was compared on cryostat sections taken from the cerebellum of either heterozygous or dilute-lethal mice. We compared the peak intensity of label in bright puncta in three regions of individual sections: the granule cell layer, the region surrounding Purkinje cell bodies, and the outer portion of the molecular layer. The granule cell layer of the heterozygous sample had a significantly ( P = 0.05) brighter staining than the dilute-lethal sample (heterozygous = 2,905 ± 423, n = 13 vs. dilute-lethal = 2,538 ± 367, n = 10). In contrast, the portion of the molecular layer immediately adjacent to Purkinje cells had significantly ( P < 0.001) brighter staining in the dilute-lethal sample ( dilute-lethal = 3,342 ± 197, n = 10 vs. heterozygous = 2,171 ± 367, n = 8). Similarly, the outer portion of the molecular layer had significantly ( P < 0.001) brighter staining in the dilute-lethal sample ( dilute-lethal = 2,820 ± 536, n = 42 vs. heterozygous = 1,974 ± 381, n = 30). This suggests that SV2 accumulates to abnormal levels in dilute-lethal granule cell axons and presynaptic terminals, similar to the peripheral accumulation in cultured SCG neurons. Third, the terminal area and density of synaptic vesicles in synapses from heterozygous and dilute-lethal mouse littermates were compared in two different presynaptic terminals. The presynaptic terminals of P12 Purkinje cells did not show significant differences ( P > 0.5) in terminal cross-sectional area (heterozygous area = 1.1 ± 0.24 μm 2 , n = 6, dilute-lethal area = 1.3 ± 0.67 μm 2 , n = 5) or synaptic vesicle density (heterozygous = 259 ± 89 vesicles/μm 2 , dilute-lethal = 310 ± 114 vesicles/μm 2 ). In contrast, the presynaptic terminals of granule cells appeared to have a much larger average terminal cross-sectional area in dilute-lethal mice at all ages that were inspected (P10, P12, P14, P15, P18) . At P18, the difference was more than fivefold (heterozygous area = 0.76 ± 0.36 μm 2 , n = 21 vs. dilute-lethal area = 4.36 ± 3.6 μm 2 , n = 12, difference was significant, P < 0.001). Synaptic vesicle density was somewhat lower in these enlarged dilute-lethal terminals (P18 heterozygous = 25 ± 8 vesicle/μm 2 , vs. P18 dilute-lethal = 18 ± 12 vesicles/μm 2 , P = 0.05). At P10 the difference in presynaptic terminal area was not as great (heterozygous area = 0.41 ± 0.26 μm 2 , n = 37 vs. dilute-lethal area = 0.96 ± 0.61 μm 2 , n = 22), but the difference was still significant ( P < 0.001). P10 is before the onset of seizures in dilute-lethal mice (our unpublished observations), indicating that the increase in presynaptic terminal area is not a secondary effect of the seizures. The greater area indicates that the presynaptic terminal volume of granule cells will be increased in dilute-lethal mice. Because of the increased volume, the total number of synaptic vesicles in these terminals will be greater. Finally, the amounts of SV2 label in granule cell presynaptic terminals of dilute-lethal and heterozygous mice were compared using immunoelectron microscopy. Presynaptic terminals of labeled P15 dilute-lethal granule cells had a significantly greater number of synaptic vesicles per terminal profile compared with heterozygous littermates as a result of the increased terminal area ( Table ). However, the number of anti–SV2-labeled synaptic vesicles (and gold particles) per terminal profile were the same . In contrast, dilute-lethal presynaptic terminals had slightly more than twice as many large vesicles (>80-nm diameter) per terminal profile that were labeled with the anti–SV2 antibody. In addition, dilute-lethal mice had more anti–SV2 label of large vesicles in parallel fibers away from synaptic sites. This suggests that the increased SV2 staining in the molecular layer of the dilute-lethal cerebellum observed by fluorescence microscopy is at least partially a result of the increased accumulation of large SV2 positive vesicles in or near presynaptic terminals. Two different observations support the conclusion that the microinjected myosin Va-Cy3 antibody labels organelles by binding to their surfaces. First, small spots partially colocalized, and very bright spots showed 100% colocalization, with antibody staining for SV2, an integral membrane protein that is a common marker for synaptic vesicles or their precursors . Second, ultrastructural observations on photoconverted cells that showed small fluorescent spots represented individual organelles surrounded by DAB reaction product. Large fluorescent spots represented antibody-induced organelle aggregates. In nonphotoconverted cells labeled with colloidal gold particles, individual organelle-like structures were associated with gold label on their surfaces. Thus, the accumulated evidence is most compatible with the external binding and concentration-dependent aggregation of vesicular structures by the microinjected myosin Va-Cy3–conjugated antibodies. The presence of individual labeled organelles within neurites suggests that many of the moving spots observed in time-lapse recordings represent individual vesicles. The size of the individual labeled organelles found within neurites was highly variable, but in most cases they were much larger (>80 nm) than synaptic vesicles. It has been reported that some synaptic vesicle proteins are axonally transported in pleomorphic vesicles . This observation, in combination with the data indicating an almost 50% colocalization with the synaptic vesicle protein SV2, suggests that many of the myosin Va–associated vesicles that move in axons may represent synaptic vesicle precursors. It has also been reported that SV2 is axonally transported in separate organelle compartments from other synaptic vesicle components, including synaptophysin . The difference in myosin Va–associated organelle colocalization with SV2 and synaptophysin in neurites is consistent with that observation. The maximum and average rates of movement of both myosin Va-Cy3 antibody and GFP-myosin Va-t–labeled organelles are similar, suggesting that they act as good markers for myosin Va organelle dynamics. The general characteristics of the movement were also the same. Both were bi-directional and saltatory. Movement of GFP-myosin Va-t–labeled organelles in neurons from dilute-lethal animals was also similar to that observed in normal cells, although a bias towards anterograde movement was observed. Furthermore, in untreated cells from both normal and dilute-lethal mice, the maximum rates of movement exceed that observed with purified myosin Va–associated vesicles . Thus, motors other than myosin Va must be responsible for the rapid movements observed. Likely candidates are microtubule-based motors. Consistent with that possibility, the maximum rates of movement are the same as that observed for organelles known to be transported by kinesins . The maximum rate of myosin Va–associated organelle movement is significantly decreased when normal cells are grown in the presence of nocodazole to prevent microtubule polymerization, indicating that microtubules probably represent the transport tracks responsible for the most rapid movements. Treatment with nocodazole to effectively eliminate microtubules does not stop directed movement. Presumably, F-actin acts as transport tracks under these conditions and the organelles are able to move at reduced rates using myosin motors. In contrast to normal cells, movements of GFP-myosin Va-t are absent in dilute-lethal neurons grown under conditions to eliminate most microtubules. This indicates that myosin Va activity is required for movement of these organelles when microtubules are disrupted. This is the first evidence available to directly support myosin Va–mediated organelle movements in intact neurons. It is consistent with recent observations on melanocytes where myosin Va appears to mediate movement of melanosomes in the absence of microtubules . Furthermore, it supports the conclusion that individual organelles contain both actin- and microtubule-based motors. While the results only require that both types of motors be present on the same organelle, they are also consistent with the possibility that a single motor complex with dual functions may exist. Neurons from dilute-lethal mice do not show abnormal accumulations of myosin Va–associated organelles in cell bodies similar to the perinuclear accumulation of melanosomes in melanocytes . The lack of accumulation is likely to be a result of the anterograde bias associated with transport of myosin Va–associated organelles in dilute-lethal neurons. The bias could be a result of a change in the balance of retrograde and anterograde microtubule-based motors associated with these organelles. Alternatively, there may be a change in the efficiency of access to microtubule tracks in peripheral regions that contain lower microtubule densities or increased numbers of microtubule ends. In support of the latter possibility , dilute-lethal neurons showed increased accumulations of SV2 label in varicosities, axon branches, and axon terminations. These regions also show staining for tyrosinated tubulin and are near regions that stain for F-actin. Tyrosinated tubulin is associated with portions of microtubules that contain new polymer or, in other words, are close to dynamic plus ends . This suggests that myosin Va–associated organelles may become stranded in regions of dilute-lethal neurons that have a high concentration of dynamic microtubule plus ends. If this is the case, then it suggests that myosin Va motor activity may facilitate the ability of organelles that have traveled off the ends of microtubules, or are released as a result of depolymerization, to return to these tracks for transport. This is consistent with the reduced percentage of myosin Va–associated organelles undergoing retrograde transport in axons of dilute-lethal neurons. Myosin Va may also mediate entry of organelles into the nearby actin-rich regions for short range transport or tethering. The peripheral accumulation of myosin Va–associated organelles in dilute-lethal neurons is the opposite of that observed in melanocytes. However, the apparent incompatibility with the capture model presented for melanocytes may be primarily a result of differences in the net balance of anterograde and retrograde transport rather than an incompatibility with a myosin Va-dependent tethering. Dendritic spines of Purkinje cells from P21 mice (a strain obtained from Jackson ImmunoResearch Laboratories, Inc.) have been reported to lack smooth endoplasmic reticulum . We have observed a similar defect in Purkinje cells from dilute-lethal (dl-20J/dl-20J) pups at all ages studied (P10–P21). It is not clear if this abnormality fits with a tethering role because the mechanism of spine formation remains controversial . If myosin Va activity is necessary only for local movement or processing of certain organelles in regions devoid of microtubules, then the abnormalities observed in dilute-lethal granule cell presynaptic terminals fit with this model. The increased numbers of large SV2-positive vesicles and synaptic vesicles may result from the absence of local transport in dilute-lethal axon terminals. Transport of synaptic vesicle to fusion/release sites (active zones) may be defective or loading of vesicles destined for microtubule-based retrograde transport may be abnormal. Either defect could lead to accumulations of vesicles in terminals. However, presynaptic terminals of Purkinje cells did not show any noticeable defects in structure, area, or synaptic vesicle density at P12. It is unclear why some terminals appear normal and while others are affected. It is especially surprising that Purkinje cell presynaptic terminals do not show defects, since their dendritic spines clearly do . However, one difference between Purkinje cell presynaptic terminals and those of granule cells is the type of neurotransmitter produced and released. Purkinje cells produce an inhibitory neurotransmitter, while granule cells produce an excitatory neurotransmitter. Possibly there are neuron-specific mechanisms of transport within terminals for different types of synapses. Alternatively, detection of presynaptic structural changes may be activity dependent. Depending on the upstream abnormalities, the output activity (and hence synaptic vesicle turnover) from Purkinje cells may be reduced. | Study | biomedical | en | 0.999999 |
10477759 | The fly stock that contains the marked third chromosome mwh jv ca , and the balancer stock TM6B, h Hu D e ca , were provided by L. Crosby (Harvard University). The stock Gl 1 Sb/TM3 Ser p p e was provided by Dr. Douglas Kankel (Yale University). All third chromosome deficiencies were obtained from the Bloomington Drosophila Stock Center (Indiana University). Df(3R)C4/p p Dp, Sb (89E; 90A) is a third chromosome deficiency that removes the sko locus. The ru h st p p ss e s stock was obtained from the Bowling Green Stock Center (Bowling Green State University), and was recently isogenized in this laboratory for a lethal-free third chromosome. The P element insertion line EP(3)3715 was obtained from the Berkeley Drosophila Genome Project. Oregon R flies were used in all cases for wild-type controls. Flies were raised on standard yeast-cornmeal-agar medium at 25°C. EMS mutagenesis was carried out as described previously . Male flies of the genotype mwh jv ca were starved for ∼1.5 h, fed with 25 mM EMS in 1% sucrose overnight, and mass mated with Gl 1 Sb/TM3 Ser p p e virgins. F 1 progeny of the genotype mwh jv ca / Gl 1 Sb were originally screened for modification (enhancement or suppression) of the rough eye phenotype caused by the dominant Gl 1 mutation. One of the third chromosome suppressors of the Gl 1 rough eye phenotype exhibited female sterility when homozygous. Genetic mapping was conducted by meiotic recombination with a third chromosome containing multiple genetic markers ru h st p p ss e s . The results revealed that two distinct mutations were separately responsible for the suppression of Gl 1 and the female sterility. A recombinant chromosome that carried only the female sterile mutation, designated shi kong ( sko ), was analyzed in the studies described here. The P element in the stock w; EP(3)3715/TM6B, Tb contains a marker that changes the eye color from white to orange. Excision events were scored by loss of the eye color marker. P transposase was introduced by crossing w; EP(3)3715/TM6B, Tb flies with Df(3)/TM3, Sb, Δ2-3 . Individual progeny males of genotype EP(3)3715/TM3, Sb, Δ2-3 were then mated with virgin w; sko/TM6B, D females. Single white-eyed males of genotype ΔP/TM6B, D were mated again with w; sko/TM6B, D virgins. Female progeny of genotype ΔP/sko were tested for sterility. Stocks were established for lines that failed to complement sko , using sibling flies of the genotype ΔP/TM6B, D . Actin-associated proteins were isolated by affinity chromatography, separated by SDS-PAGE, and used as antigens for antibody generation in mice as previously published . Mouse antibody no. 4 described in was used to screen a Lambda Zap expression library constructed from Drosophila ovary poly(A) + RNA. The library was screened as described with minor modifications. The screen produced two unrelated cDNA clones, one of which is 3.2 kb in length and encodes the Drosophila homologue of ABP280 or nonmuscle filamin used in this work. The 7.5-kb cDNA clone GH12209 was obtained from the Berkeley Drosophila Genome Project via Research Genetics. Sequence was obtained using T7, SP6, and custom primers, with an ABI377 sequencer. The entire sequence was manually proofread. Portions were read on only one strand, but all base calls were unambiguous. The nucleotide and protein sequence was analyzed using the UWGCG programs and the MacVector Sequence Analysis Software package (Oxford Molecular Group). Genomic DNA for Southern blots was prepared from adults as previously described . 5 μg of DNA were digested with restriction enzymes, fractioned on a 1% agarose gel, and transferred to Zeta-Probe nylon membrane (BioRad Laboratories) by standard methods. Total RNA used for Northern blot experiments was isolated as described previously . RNA was fractionated on 0.75% agarose formaldehyde gels and transferred to Zeta-Probe membrane. DNA probes were labeled with [ 32 P]dATP (Amersham) using random hexamer primers (Amersham Pharmacia Biotech) according to methods described by Vogelstein and Gillespie 1979 . Prehybridization and hybridization of DNA and RNA blots were carried out using standard methods. Genomic clones were isolated by screening a cosmid library made from iso-1 fly DNA with a fragment derived from the 3.2-kb filamin cDNA. Two overlapping cosmid clones were used to characterize the transcription unit of the filamin gene. DNA labeling and hybridization methods for library screening were those used for Southern and Northern blots. A filamin-6xHis fusion protein was generated using PCR and the 3.2-kb filamin cDNA as a template. The two primers used to amplify a cDNA fragment that encodes the last 90 residues in the filamin protein were primer 1: 5′-GCGCGCGTCGACGCCAGCAAGGTGGTGTCCAA-3′ and primer 2: 5′-CGCGCGAAGCTTCTTGCATACACGTACACATC-3′. Vent DNA polymerase (NEB) was used for the PCR reactions. The 6xHis-tagged cDNA was cloned into the vector PQE9 (Qiaexpress vectors; Qiagen) using a HindIII and a SalI site added during the PCR reaction. The 6xHis fusion protein was produced in E . coli and purified on a Ni-NTA resin column according to the manufacturer's instructions (Qiagen). The filamin antiserum, 4-3D, was raised in rabbits against the purified bacterial fusion protein, and was blot affinity-purified against the His-tagged fusion protein. Ovaries were dissected, fixed, and processed for immunocytochemistry as described in McGrail et al. 1995 . For staining with propidium iodide or Oligreen (Molecular Probes, Inc.), ovaries were first treated with 20 μg/ml RNase in PBS for 1.5 h at 37°C followed by a one hour wash in PBT (PBS supplemented with 0.1% Triton X-100). Ovaries were incubated in 0.1 μg/ml propidium iodide or Oligreen for 30 min at room temperature. To visualize F-actin, ovaries were stained with phalloidin (Sigma Chemical Co.) diluted 1:1,000 for 30 min at room temperature. The anti-phosphotyrosine monoclonal antibody PY20 was purchased from ICN Biochemical Inc. and used in 1:10 dilution. The anti-anillin antibody is a rabbit polyclonal antibody and was used at a final concentration of 5 μg/ml. Both anti-kelch and anti-hts are monoclonal antibodies kindly provided by Lynn Cooley (Yale University) and were used at a dilution of 1:10. The anti-hts antibody used here recognizes only the isoform found in the ring canals . For immunocytochemistry the anti-filamin (4-3D) antiserum was used at a dilution of 1:1. The secondary antibodies used in the immunofluorescence experiments were either FITC-conjugated or Texas red–conjugated goat anti–rabbit (or mouse). In all cases, the secondary antibodies were preabsorbed against embryos and were diluted 1:100. Images were collected by using an MRC-1024 confocal imaging system (BioRad Laboratories). Details on the procedures for fixation, embedding, sectioning, and staining for electron microscopy are published in Tilney et al. 1996 . A female sterile mutation, shi kong ( sko ; “out of control” in Chinese), was generated by standard EMS mutagenesis. Homozygous sko mutant flies are viable, but females do not lay eggs. Mutant ovaries show an apparent arrest in oocyte growth at stage 10b and no mature egg chambers are observed. Further inspection of sko mutant egg chambers reveals several defects in cellular morphology and membrane integrity. In situ staining of DNA reveals the disruption of the normal spatial organization of germline nuclei in sko mutant egg chambers, and in late stages, entire nurse cells and/or nurse cell nuclei are sometimes seen to protrude into the posterior compartment normally occupied only by the growing oocyte . Although follicle cells migrate to form a columnar epithelium surrounding the oocyte in sko mutants, their shape is frequently more elongated than in wild-type egg chambers. The subsequent centripetal movement of follicle cells along the anterior margin of the oocyte is also abnormal in many mutant egg chambers. Our analysis of sko egg chambers stained with rhodamine-labeled phalloidin reveals an abnormal organization of actin filaments. In early egg chambers (stages 1–6), the level of phalloidin staining in ring canals is frequently reduced, and where visible forms a more diffuse pattern than in wild-type, suggesting that actin microfilaments are less tightly organized within the sko mutant ring canals . In late stage 10 egg chambers, actin is no longer detected in organized ring canal structures, although some aggregated clusters of short actin filaments may represent the remnants of collapsed ring canals . The disruption of actin organization in sko egg chambers is not restricted to the microfilaments within ring canals. In early egg chambers, the phalloidin staining pattern suggests that the subcortical network of actin filaments is anomalous along the nurse cell plasma membranes . In late stage 10 egg chambers, the radial arrays of cytoplasmic actin microfilaments that extend from nurse cell membranes toward the centrally located nuclei are also disrupted. Instead, the cytoplasmic actin filaments observed are short, disorganized, and often clumped in small aggregates . Because actin organization is defective in sko mutant ring canals, we have examined other known ring canal components to further characterize the role of the sko gene product. Using an antibody against anillin, we compared patterns of anillin localization in wild-type and sko ovaries . In wild-type egg chambers, anillin localization at ring canals is observed early in region 1 of the germarium and is easily detected through stage 6, with diminished levels still visible through stage 10. In sko ovaries, anillin is present in the stabilized cleavage furrow and persists at ring canals in a pattern similar to wild-type. Moreover, sko egg chambers contain the typical 16 cells, including 15 nurse cells and an oocyte. These results indicate that cytokinesis and initial ring canal formation are not defective. However, in contrast to wild-type, the anillin rings in sko mutant egg chambers appear reduced in size. Similarly, the recruitment of the phosphotyrosine protein(s) to ring canals in the sko mutant egg chambers appears normal during early stages. Beyond stage 3, while we can detect the phosphotyrosine antigen in ring structures, we also frequently detect small cytoplasmic foci, or particles, some of which are also recognized by rhodamine-phalloidin labeling . These foci may represent disrupted or collapsed ring canals. Hts protein is also present in the early ring canals of sko egg chambers, but by stage 4, the localization of hts is often abnormal and only a portion of the ring is labeled. The disruption of hts becomes progressively worse in later stage egg chambers . We did detect actin colocalized with hts protein in some ring canals; however, in most cases the ring canals were devoid of actin filaments. We did not observe concentration of kelch protein in sko mutant ring canals at any stage of oogenesis. Our results indicate that the sko gene product is not required for the initial recruitment of components of the outer rim of the ring canal. Rather, sko is required for proper assembly of the inner rim and/or its stability and growth during development. Thin sections through wild-type and sko ring canals at stage 3 and 4 are consistent with what we have described by light microscopy and expand our resolution of the ring canal structures. As noted in Tilney et al. 1996 , in vertical section the plasma membrane lining the inner margin of each side of the ring canal appears as a T-shaped junction limiting adjacent nurse cells . Attached to these T-shaped extensions of plasma membrane in both wild-type and sko ring canals is a layer of dense material. Inside this material in the wild-type ring canal is a thick layer of actin filaments that is organized circumferentially as a purse string . This layer of filaments is missing altogether in the sko mutants or is reduced to a tiny fraction of the number of filaments seen at this same stage in wild-type. Note that in the sko mutants, the T-shaped extensions are formed with their associated dense material, even though actin filaments are sparse or missing altogether. Since anillin is present through stage 6 in sko mutants, it seems reasonable that anillin may be one (possibly major) component of this dense material lining the cytoplasmic surface of the canal. In thin sections through stages 5 to 6 sko egg chambers, we also observe a number of changes that occur in the association of the plasma membranes that separate adjacent nurse cell cytoplasms. We see breaks or disruptions in the plasma membrane that allow the cytoplasm of adjacent cells to merge in these regions . At these break points the plasma membrane is vesiculated. We also find that many of the ring canals are altered in stages 5 and 6: the normally T-shaped membrane junction appears folded back upon itself . Furthermore, the ring canal as depicted in Fig. 3 e and as seen in other micrographs is frequently incomplete. In these sections we only find half a ring canal; the other half is missing, a situation that we interpret as the breakdown of the ring canal. Overall, the number of actin filaments lining the inner aspect of the ring in stage 2 chambers is initially sparse, and later can completely disappear before membrane disruption and ring canal loss. To address the impact of sko on intercellular transport and cytoplasmic localization we examined the localization of RNAs and proteins known to be required for the proper development of the oocyte . In previous work, we showed that cytoplasmic dynein accumulates within the presumptive oocyte, beginning very early in the germarium and continuing through later stages . This pattern of dynein accumulation is also observed in sko mutant egg chambers, suggesting that the early slow phase of cytoplasmic transport from the nurse cell compartment to the oocyte before stage 9 is not disrupted. By stages 9 to 10 in wild-type egg chambers, dynein becomes concentrated within the oocyte at the posterior pole. In sko egg chambers, this posterior concentration of dynein in the oocyte also appears qualitatively normal. Similar to dynein, we find that the posterior accumulation of staufen protein and oskar mRNA in the oocyte does not depend on sko function. Furthermore, despite the distorted margin at the anterior of the oocyte, we still observe the typical anterior concentrations of both bicoid and gurken mRNAs in sko egg chambers. Meiotic recombination experiments showed that sko is located on the right arm of the third chromosome in the genetic interval between the recessive markers st and e . Deletion mapping studies further defined the position of the sko mutation to the region between 89E7 and 90A. During the characterization of the sko phenotype, we localized the Drosophila homologue of the human actin-binding protein α-filamin to the 89F polytene region using in situ hybridization with a fly filamin cDNA. This 3.2-kb cDNA clone, which encodes a portion of Drosophila filamin, was previously isolated from an ovary expression library using a polyclonal antiserum raised against partially purified fly actin-binding protein . The cDNA represents the 3′ end of the filamin transcript, including a 513-bp 3′ UTR with a polyadenylation tail. It predicts a polypeptide that is homologous (51% identity) to the COOH-terminal domains of human α-filamin. Genomic Southern blot analysis indicates that this filamin cDNA is derived from a single copy gene in Drosophila . However, on RNA blots the probe detects two transcripts, 3 and 7.5 kb, which are differentially expressed . The 7.5-kb transcript is expressed in ovaries and embryos, while the 3-kb transcript is expressed in embryos, but not ovaries. We subsequently identified and sequenced an expressed sequence tag (EST) cDNA clone , derived from adult head mRNA, that potentially encodes the full-length Drosophila filamin protein. The 7,536-bp cDNA contains the original 3′ cDNA sequence and includes a polyadenylation tail. An uninterrupted open reading frame begins from the first nucleotide of the cDNA; however, the first ATG codon is located 401 bp downstream. Although the flanking sequence of this ATG differs from the consensus sequence for translation initiation in Drosophila , conceptual translation from this initial ATG codon predicts a 2343 residue polypeptide with a deduced mass (∼250 kD) and NH 2 -terminal sequence that are highly similar to human filamins . Amino acid sequence comparisons indicate that the Drosophila filamin is ∼50% identical and ∼60% similar to human and chicken filamins along the entire length of the protein. Although we have not unequivocally defined the 5′ end of the transcript, the data suggest that the fly EST cDNA clone represents a full-length copy of the identified 7.5-kb filamin transcript. To characterize the genomic region of the Drosophila filamin gene, we used the filamin cDNA to isolate genomic clones from a cosmid library. A restriction map of the genomic region that contains the filamin gene is depicted in Fig. 7 . Restriction fragments from across the region were subcloned, partially sequenced, and used in RNA blot experiments to define the filamin transcription unit. Both the 3- and 7.5-kb transcripts are detected with a genomic clone that contains the 3′ end of the gene , while only the 7.5-kb transcript is recognized by the more 5′ genomic probes b and c. Probe c contains the 5′ end of the EST cDNA sequence. The genomic probe d fails to detect either the 7.5- or the 3-kb transcript, but does identify a neighboring transcript ∼4 kb upstream. These results allow us to define the Drosophila filamin gene within a ∼30-kb genomic region. However, our experiments have not determined the complete intron/exon organization of the gene. Since the 3-kb transcript is detected only by probes derived from the 3′ end of the filamin transcription unit, it may be expressed from an internal promotor, or as an alternatively spliced product that eliminates 5′ exons. Further sequence analysis of the 5′ end of this transcript will be required to distinguish between these possibilities. Significantly, the 3-kb transcript lacks the 5′ sequence that encodes the highly conserved actin-binding domain; it may encode a filamin isoform that provides a regulatory function. Inspection of the 89F polytene region also identified the mutant cheerio , which exhibits defects in ring canal assembly similar to the sko mutation . The analysis of mutant egg chambers from cheerio mutants showed that the cheerio gene product is required for the incorporation of inner ring canal components and the growth of the ring canal, but the product of the cheerio gene is not known. To test whether cheerio and sko are mutations within the same gene, we conducted a complementation test between cher 1 and sko . We report that the two mutations fail to complement one another; sko/cher 1 females are sterile and exhibit defects in ring canal assembly and egg chamber morphology. To investigate whether the sko mutation represents an allele of Drosophila filamin , we looked at the expression of filamin protein(s) in wild-type and sko mutant flies. A polyclonal antiserum, 4-3D, was raised against a bacterial fusion protein expressing the COOH-terminal 90 residues of filamin. Extracts from whole flies and tissues of sko and cher 1 mutants, as well as from wild-type flies, were analyzed by immunoblotting. As shown in Fig. 8 a, in wild-type ovary extracts the antibody predominantly reacts with a large ∼250-kD polypeptide. The size of this polypeptide correlates well with the single 7.5-kb filamin transcript expressed in ovary RNA, and is comparable in size to human α-filamin. Significantly, the 250-kD filamin polypeptide is not detected in ovaries derived from sterile sko / sko females, nor in homozygous cher 1 ovaries. A less abundant polypeptide of ∼140 kD is detected by the 4-3D antiserum in wild-type ovary extracts, and is not eliminated in the sko or cheerio mutant backgrounds. These results demonstrate that the sko and cher 1 mutations disrupt the expression of a 250-kD filamin product in ovaries. The loss of the filamin polypeptide coincides with the disruption of actin organization and female sterility. In embryo and adult extracts, the major polypeptide detected by the 4-3D filamin antiserum migrates at ∼ 97 kD. A product of this mobility could be encoded by the smaller 3-kb filamin transcript, and like the transcript, the 97-kD polypeptide is found in embryos and adults, but not ovaries. The 250-kD polypeptide is also present in embryos and adults, but at relatively lower levels compared with ovary extracts. Both the 250- and 97-kD polypeptides are absent from immunoblots of adult extracts prepared from the sko/sko mutant background. By comparison, in cher 1 /cher 1 adult extracts the 250-kD polypeptide is missing, but the 97-kD polypeptide is retained. These results provide further evidence that the sko mutation identifies the filamin gene. Our data do not eliminate the possibility that the sko mutation encodes a truncated polypeptide that is not detected by the filamin antibody. The ∼ 140-kD protein detected in ovaries is also detected in adult extracts, but again is not eliminated by the sko or cher 1 mutations. Currently, we can not eliminate the possibility that the 140-kD species is a bona fide product of the filamin gene. Alternatively, it may represent a polypeptide that cross-reacts with the filamin antiserum. Regardless, our results suggest that the sko and cher 1 mutations alter the expression of the 250- and 97-kD filamin polypeptides. The sko phenotype predicts that the 250-kD filamin actin-binding protein might function in ovarian ring canal growth. As shown in Fig. 8 b, the 4-3D filamin antiserum decorates the ring canals in wild-type germline cysts. Filamin is concentrated on ring canals at early stages (regions 2–3 in germarium) and persists through late stages (stage 10b). In the sko mutant egg chambers, filamin is not detected in ring canal structures. This result is consistent with a model in which filamin acts to tether actin microfilaments within the ring canal to the cell membrane. Moreover, the loss of filamin staining and actin organization in ring canals of sko egg chambers supports the interpretation that sko encodes filamin. To confirm that the sko phenotype is caused by a mutation in the filamin gene, we have generated deletions in the gene by imprecise excision of a P-element inserted 5′ to the filamin gene. The P-element insertion line EP(3)3715 was identified from collections in the Drosophila Genome Project. Plasmid rescue and sequence analysis of the genomic sequence flanking the insertion showed that the P-element is inserted 78 bp 5′ to the first nucleotide of the EST cDNA sequence . We determined that the P-insertion itself does not disrupt sko function. However, we took advantage of its position to generate deletions in the filamin gene, by mobilizing the P-element insert in the presence of Δ2-3 P transposase. P - excision lines ( Δ ) were collected and scored for fertility in transheterozygous combination with the sko mutation ( sko/Δ ). Two excision lines, EPSΔ12.1 and EPSΔ5 , fail to complement the sko mutation, resulting in female sterility. Genomic DNA blot analysis confirmed that both lines contain deletions in the filamin gene. The deletion line EPSΔ12.1 removes the entire filamin transcription unit. The EPSΔ5 excision line contains a small deletion at the 5′ end of the transcription unit. The positions of the deletions generated by the P-element excisions are shown in Fig. 7 . As a further verification that the disruption of filamin function accounts for the sko female sterile phenotype, we examined egg chambers in ovaries derived from sko/EPSΔ5 and sko/EPSΔ12.1 heterozygotes. The defects observed are similar to those found in sko/sko ovaries. Most characteristically, actin organization in egg chambers is disrupted and ring canal growth is reduced. In late stage egg chambers from sko/EPSΔ5 and sko/EPSΔ12.1 females, we see few intact ring canals. Our results show that deletions that disrupt the filamin transcription unit are allelic to the sko mutation. We conclude that female sterility and disruption of actin organization in the sko mutant egg chambers results from the loss of filamin function. Our results provide a characterization of the structure and function of Drosophila filamin. We present four lines of evidence that together demonstrate that the female sterile sko mutation represents a lesion within the filamin gene. First, in situ hybridization of a filamin cDNA to polytene chromosomes positions the filamin gene in a genomic region that was shown by deletion mapping experiments to contain the sko mutation. Second, antibodies that were raised against the COOH-terminal domain of the filamin protein detect two major filamin polypeptides. Both these polypeptides are lost in the sko mutants. The 250-kD filamin polypeptide is also eliminated by the cher 1 mutation, which is allelic to sko . Moreover, the antibody detects filamin within ring canal structures of wild-type, but not sko mutant egg chambers. Third, deletions (Δ ) in the filamin gene were generated by P-element excision. These mutations fail to complement the sko mutation and exhibit female sterility, similar to females homozygous for the sko mutation. Finally, the disruption of actin organization is similar in egg chambers derived from either sko/sko or sko/Δ females. Thus P-element excisions known to disrupt the filamin gene are allelic to the sko mutation and, as would be expected, produce a similar phenotype. Given these results, we refer to the sko mutation as the filamin sko allele. Previous structural and functional information on the properties of filamins, together with the comparative cytology of mutant and wild-type egg chambers, suggest several functions for Drosophila filamin in oogenesis. Filamins are a family of actin cross-linking proteins that have been identified in multiple tissues and cell types from several organisms . Our analysis of the Drosophila filamin sequence predicts a polypeptide that shares the main structural elements present in other filamins, as defined by the extensive characterization of human α-filamin . Overall there is ∼50% amino acid identity with the predicted human , and chicken filamin sequences. The characteristic signatures of an actin-binding domain are present within an NH 2 -terminal region that is ∼70% identical to the corresponding region in the human α-filamin sequence . The human sequence predicts an elongated central rod domain comprising 24 repeats of ∼96 amino acids each, interrupted in two locations by insertions of 20–40 residues that are thought to provide flexible hinge regions within the rod-like backbone. A series of repeats also constitute the central domain of the fly polypeptide, and sequence alignments indicate that the relative positions of both hinge regions are also conserved. However, a striking distinction is the deletion of repeats 6–9 in the rod domain of the fly filamin. The remaining repeats show 34–52% identity with the corresponding human domains. In addition to promoting dimerization between filamin monomers, the COOH terminus of human filamin interacts with a number of membrane proteins, including presenilin , β1 and β2 integrin , and tissue factor . These interactions are thought to regulate the linkage between the actin cytoskeleton and the plasma membrane. Evidence for conservation of these functions in the COOH-terminal domain of Drosophila filamin was recently provided by a two-hybrid screen for proteins that interact with the toll receptor . The toll transmembrane receptor binds the extracellular ligand, Spatzle, to trigger a signaling pathway that includes the downstream cytoplasmic effectors tube, pelle, cactus, and dorsal. Clones encoding the COOH terminus of filamin , plus a variable number of repeats, were identified by their interaction with toll. The COOH-terminal filamin fragment was judged to be competent to dimerize, since coexpression of both the LexA DNA binding and activation domains as separate fusions to filamin resulted in elevated reporter expression. How the actin cytoskeleton functions in the toll pathway, and how the dimerization of filamin and its interaction with toll are regulated, will be important to investigate further. Our results identify filamin as a new component of ring canals. Antibodies raised against a filamin fusion protein detect filamin within the ring canal and mutations in the filamin gene disrupt ring canal structure and function. Filamin can provide several functions missing from the previously known complement of ring canal proteins. Most significantly, filamin can cross-link antiparallel actin filaments , as well as link such cross-linked filaments to the plasma membrane . A simple model for filamin function is that the COOH-terminal tail of filamin anchors the dimer to the plasma membrane in the outer rim of the ring canal. The distal, NH 2 -terminal actin-binding sites would act to recruit and/or stabilize actin filaments in the ring canals. This model is consistent with the early arrival of filamin at the ring canal, and with the loss of filamin and actin from the ring canal in the filamin sko mutant background. We have shown that in the presence of the sko mutation, the 4-3D antiserum does not detect filamin in situ in ring canals or on immunoblots of ovary extracts. Since this filamin antiserum is directed against the COOH terminus, which participates in membrane association, our results suggest that if a filamin product is encoded by the sko mutant, it lacks the COOH terminus and therefore cannot attach to the membrane or recruit actin filaments to the ring canals. Filamin is required for the maturation of the ring canal and the normal assembly of the inner rim components. Cytokinesis, stabilization of arrested cleavage furrows, and the initial construction of ring canals proceed normally in the sko mutant as judged by proper cell numbers in sko egg chambers and the early staining pattern of anillin and at least one phosphotyrosine protein. These outer rim components are thought to fortify the initial intercellular bridges and are present before the assembly of the inner rim components including hts, actin filaments, and kelch protein . Only partial staining of the hts protein and actin filaments were obtained in sko mutants even at early stages. In addition, no accumulation of kelch protein in ring canal structures was detected. Despite the disruption of inner rim assembly, anillin organization in the outer rim of ring canals was apparent up until stages 5–6. However, the rings were reduced in size and later disintegrated or collapsed. This disruption may result from the inability of the ring canal to fully expand. How does filamin act to promote ring canal expansion? Before expansion, overlapping actin filaments of opposite polarity extend circumferentially around the ring perimeter with no apparent seam. The sliding of actin filaments of opposite polarities is proposed to account for expansion of the ring canal . One explanation for the defect in ring canal growth associated with filamin mutations is that actin filaments fail to be recruited to the ring canal or are present in insufficient numbers to support continued sliding during expansion. This interpretation is consistent with partial growth at early stages and the disruption or collapse of ring canals at later stages. During ring canal expansion, the homogeneous monolayer of parallel actin filaments is transformed into a branching network of overlapping actin filament bundles . The known property of filamins to cross-link actin filaments at high angles may also serve to establish the spacing between this network of actin bundles and act to stabilize the growth of the ring canal. Mutations in three other genes, Tec29 , Src64 , and cheerio , affect the growth of ring canals. The tyrosine kinases Tec29 and Src64 have recently been shown to be essential for ring canal growth during oogenesis . Mutations in either of these genes result in defective transfer of cytoplasm from the nurse cells to the oocytes and eliminate the staining of ring canals by phosphotyrosine antibodies. Furthermore, antibodies to Tec29 show that the encoded kinase is localized to the ring canal, suggesting that Tec29 protein could provide at least one of the major phosphotyrosine antigens present in ring canals. Src64 protein does not itself accumulate in ring canals, but is required for the accumulation of Tec29. Although expansion of the ring canal is blocked in the Tec29 mutant, the incorporation of actin, hts, and kelch appears normal and suggests a parallel pathway that regulates ring expansion . Filamin is a phosphoprotein and its stability and association with actin may be mediated by its state of phosphorylation . In this regard, it will be of interest to test whether filamin is a substrate of the Tec29 kinase. A third gene, cheerio , was previously identified by a female sterile mutation that exhibits defects in the assembly and the expansion of ring canals . The phenotypes of mutations in cheerio are similar to the sko mutation and our complementation analysis has shown the both are alleles of the filamin gene. Based on preliminary examinations, it has been reported that the cher 1 mutation does not eliminate the localization of Tec29 . This result is consistent with the proposed model that Tec29 and Src64 act in an independent pathway that regulates the growth, but not the assembly of ring canal components . Defects in the morphology of cells within the nurse cell compartment, as well as the disruption of nurse cell membrane integrity, suggest that filamin function may not be specific to ring canals. Most notably, the protrusion of whole nurse cells or nurse cell nuclei into the oocyte compartment suggests that filamin acts in sites of intercellular adhesion where the cytoskeletons of adjacent cells are linked together. Electron microscopy substantiates the compromised integrity of membranes and indicates that this defect precedes the later disruption of the ring canal structures. While ring canal structures can be observed at stages when breaks in nurse cell membranes are already apparent, the inner rim of such ring canals is often devoid of actin filaments. Our observations suggest that filamin is required for cross-linking actin filaments to the membrane throughout the cortical cytoplasm of nurse cells, as well as in ring canal structures. In this regard, the role of cortical actin filaments in cell adhesion and the maintenance of cell shape and membrane integrity in the Drosophila egg chamber has been previously demonstrated . For example, mutations in armadillo , a fly homologue of β-catenin, disrupt all three types of actin filaments and exhibit disruptions in cell arrangements and membrane integrity within egg chambers . The rapid phase of cytoplasmic transport that results in a rapid increase in oocyte size in stages 10–11 depends on the contraction of the cortical actin network and presumably its linkage to the nurse cell membrane . The disruption of the membrane linkage and/or organization of the cortical actin network may also contribute to the arrested growth of oocytes in sko egg chambers. Our interpretation that filamin function is required outside the ring canal is consistent with the functions described for filamins in other cell types. Filamin is known to increase the rigidity of actin networks in vitro, and in vivo filamin increases the elastic modulus of the cell cortex . Moreover, filamin cross-linking of cortical actin to integrin receptors can mediate the protective responses of cells subjected to mechanical stress . Cells deficient for filamin show an increase in permeability of the calcium channel and elevated cell death . Interestingly, mutations in the Drosophila dcp-1 caspase , a critical component of the apoptotic pathway, disrupt actin organization, block cytoplasmic transfer, and partially inhibit nurse cell death . Perhaps the regulation of filamin cross-links mediates changes in the mechanical strength of the nurse cell membranes during contraction of the cortical actin network and the subsequent onset of nurse cell death. In addition to its role in mechanoprotection, the requirement for filamin function in cell movement is well established. Tumor cell lines derived from human malignant melanomas that lack filamin (ABP280) show impaired locomotion . These cells exhibit extensive blebbing of the cell membrane, but fail to spread and extend lamellae. Moreover, there is evidence that filamin participates in the formation of filopodia in migrating cells as a downstream target of Cdc42-activated RalA. Activated RalA may act to elevate the local concentration of filamin and direct the formation of actin bundles in filopodial extensions . The developmental significance for filamin function in cell movement was recently revealed in a study that shows loss-of-function mutations in human filamin prevent migration of cerebral cortical neurons . These mutations give rise to the human disorder periventricular heterotopia (PH). The X-linked PH, or filamin , mutations lead to epilepsy and vascular disorders in females and embryonic lethality in males. While the filamin sko mutation is not recessive lethal, our studies do not rule out additional functions for filamin during embryogenesis or in adult animals. Indeed, we do find that filamin transcripts and protein products are expressed in embryos and adults. It will be important in future work to establish the functional significance of this expression. | Study | biomedical | en | 0.999997 |
10477760 | The karst stocks mwh ve kst 1 e/TM6B , mwh ve kst 2 e/TM6B , mwh kst 14 . 1 red e/TM6B , mwh Df(3L)1226 red e/TM6B, and kst 01318 /TM6B have been previously described . The recombinant stocks mwh kst 1-1151 FRT80B/TM6B and mwh kst 2-2119 FRT80B red/TM6B were derived from the above stocks by recombination with the rucuca chromosome to remove ancillary lethals and to w ; FRT80B for other purposes. The slbo 1 /CyO stock was a gift from Dr. D. Montell (Johns Hopkins University) and was introduced into the karst mutant background by standard genetic methods. To detect β H , we used serum #243 at 1/200. To detect α-spectrin, we used ascites fluid #N3 at a dilution of 1/5,000 . To detect β-spectrin, we used rabbit serum #89 at a dilution of 1/200. To detect D E-cadherin, we used the monoclonal antibody #DCAD2 at a dilution of 1/20. To detect myosin IB, we used affinity-purified rabbit antibodies at a dilution of 1/200. To detect Notch, we used the monoclonal antibody 9C6 at 1/200. To detect cytoplasmic myosin II, we used the rabbit serum #656 at 1/200. To detect Fasciclin III, we used the monoclonal antibody 7G10 at 1/10. FITC, Cy3 or Cy5 conjugated, and affinity-purified secondary antibodies used were all made in goat and were obtained from Jackson ImmunoResearch Laboratories, Inc. These antibodies were rehydrated according to the manufacturer's instructions and used at dilutions of 1/100. Alexa 488 conjugated secondary antibody was obtained from Molecular Probes and used at a dilution of 1/200. 2–4-d old females fed with yeast paste at 25°C were dissected and their ovaries were teased apart into individual ovarioles in PBS (130 mM NaCl, 7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 , pH 7) using a tungsten needle. The tissue was fixed in buffer B for 15–20 min at room temperature. After a 30-min wash in PBT (10 mM Na 2 HPO 4 /NaH 2 PO 4 , pH7.4, 175 mM NaCl, 0.1% Triton X-100), samples were blocked in PBT-NGS (PBT, 5% normal goat serum). The samples were then incubated in PBT-NGS containing the primary antibody for 4–5 h at room temperature or overnight at 4°C. After one wash in PBT and one wash in PBT-NGS for 30 min each, the samples were incubated in PBT-NGS with the appropriate secondary antibody for 2–3 h at room temperature or overnight at 4°C. After two washes in PBS, the stained ovaries were equilibrated and mounted in mounting medium (100 mM Tris-Cl, pH 8.5, 80% glycerol, 2% n -propyl gallate). To costain for F-actin, samples were further incubated for 30 min at room temperature in 165 nM FITC- or TRITC-phalloidin (Molecular Probes) in the first wash after secondary antibody incubation. Nuclei were visualized by staining with 5 μg/ml propidium iodide in PBT for 20 min at room temperature . Ovaries were dissected in PBS and fixed in 1% glutaraldehyde in PBS for 20 min at room temperature. After three to four washes in PBS, the tissue was incubated in prewarmed reaction solution (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1 mM MgCl 2 , 3 mM K 3 Fe(CN) 6 , 3 mM K 4 Fe(CN) 6 , 0.1% X-Gal) for at least 30 min at 37°C. The samples were extensively washed in PBS and mounted in mounting medium. Imaging of immunofluorescently stained ovaries was done using an MRC1024 confocal microscope (Bio-Rad Laboratories). Imaging of ovaries stained for β-galactosidase activity was done on a BX50 microscope (Olympus Corp.) equipped with a Dage/MTI CCD72T camera and DSP2000 signal processor, and were imported directly into a Power Macintosh 8100/80AV (Apple Computer) using a DT2255 frame grabber (Data Translations) controlled by the public domain program NIH Image (v1.61 available on the internet at http://rsb.info.nih.gov/nih-image). Images were contrast stretched as appropriate using Adobe Photoshop v4.0 (Adobe Systems, Inc.) and the figures assembled and annotated in Adobe Illustrator v6.0. Images of sagittal optical sections of 81 wild-type and 318 karst mutant egg chambers at stage 9 or 10A (a comprehensive combination of kst 1 , kst 2 , kst 14 . 1 , and kst 01318 alleles over one another and over Df(3L)1226 ), that had been stained for α-spectrin, D E-cadherin, actin, or Notch, were acquired. The distances described in text and Fig. 2 were then measured and analyzed using Excel 98 (Microsoft Corp.) and/or Deltagraph (DeltaPoint, Inc.). To measure the follicle cell apical surfaces, we stained wild-type and kst 01318 egg chambers for D E-cadherin to outline the apical contours. To ensure that only cells being viewed en face were measured, the following procedure was used. The z-axis motor on the confocal was stepped gradually out from the sagittal plane of the oocyte towards the apical domain of the upper follicle cell monolayer until the oocyte had just disappeared. Measurement of the apical surface areas was done using NIH Image software. Specifically, we used the Threshold option to isolate the cell outlines, after which the Measure option was used to quantify the number of pixels per apical surface per cell. Any breaks in the D E-cadherin staining, particularly in karst mutant egg chambers, were manually closed with straight, one pixel-wide lines before area measurement. The position of the center of each cell measured relative to the nurse cell/oocyte boundary was also recorded for each cell. Oogenesis in flies takes place in ovaries formed of 12–16 ovarioles, each of which consists of an anterior structure called the germarium and several egg chambers sequentially ordered with regard to their developmental stage . The germarium is comprised of three zones . In zone 1, two germline stem cells divide asymmetrically to give rise to a cystoblast and a stem cell. The cystoblast then divides synchronously four times to produce 16 cell cysts interconnected by ring canals as a result of incomplete cytokinetic events. In zone 2, 16 cell cysts become surrounded by a pool of follicle cells produced by asymmetric division of two to three somatic stem cells. By this stage, 1 of the 16 germ cells has adopted an oocyte fate and becomes located at the posterior of the cyst. The remaining 15 nurse cells undergo polytenization. In zone 3, fully formed stage 1 egg chambers begin to emerge from the germarium, bounded by a well polarized follicular epithelium . Egg chambers will continue to grow and increase in size up to stage 9, while the follicle cell monolayer accommodates this growth by a series of cell divisions. At the onset of stage 9, after all divisions have ceased, the majority of the follicle cell monolayer undergoes a concerted migration towards the posterior of the egg chamber onto the oocyte membrane. Those follicle cells that are left behind become squamous and continue to reside on the nurse cells. During this morphogenetic event, the migratory follicle cells undergo a change in cell shape from cuboidal to columnar, a process that is instrumental in accommodating them on the growing oocyte membrane. Concomitantly, a group of 6–10 anterior follicle cells called border cells round up and plunge in between the nurse cells to reach the anterior of the oocyte . After the follicle cell monolayer and the border cells have reached the anterior of the oocyte, a specialized subset of follicle cells called centripetal cells migrate along the nurse cell–oocyte interface such that the follicle cells now completely surround the oocyte. Communication between nurse cells and the oocyte is maintained via ring canals that remain open until the nurse cells dump their cytoplasmic contents into the oocyte at stage 11. Although two partial descriptions of the distribution of β H during oogenesis have been published by other groups , no complete description exists. We therefore began by fully characterizing the distribution of β H in developing wild-type ovarioles . β H expression in the germline is low to undetectable in the most anterior region of the germarium, and first appears in zone 2 . In zone 3, fully formed stage 1 egg chambers emerge, β H is found uniformly along the nurse cell and oocyte membranes . Later, in mid-oogenesis, β H is slightly enriched on the outer edge of the ring canals . In the soma, β H is strongly expressed at the very anterior tip of the germarium in the terminal filament and the cap cells that contact the germline stem cells . In close proximity to the 16 cell cysts in region 2, we detect high levels of β H expression in the vicinity of the somatic stem cells and in their progeny, the follicle cells . As individual cysts become enveloped in follicle cells, β H is slightly enriched in the cells that move in to segregate adjacent cysts . β H continues to be strongly expressed here as these become the stalk cells that separate successive egg chambers along the ovariole . β H is apically polarized in the follicle cells . β H is downregulated in the migrating border cells at stage 9 , but is again expressed on the apical surface of these cells when they begin to secrete the micropyle after stage 10 . At stage 10, β H is part of a prominent terminal web-like structure at the apical ends of the follicle cells that are secreting egg components . This structure appears to be anchored in the ZA by fine fibers of staining around its edge. β H is also expressed on the apical surfaces of the follicle cells secreting the dorsal appendages and the chorion (data not shown). β H colocalizes with α-spectrin in the germline and soma at all these locations ; however, the latter is more widespread presumably through its association with the conventional β isoform. In mid–stage 9 egg chambers, the follicle cell monolayer and the border cells migrate in a concerted fashion . However, in 73% of karst egg chambers the border cells migrate ahead of the follicle cell monolayer . The degree to which the border cells migrate ahead of the follicle cells during stage 9 is quite variable in keeping with the variable expressivity of the karst mutation . We occasionally find egg chambers where the border cells are retarded relative to the follicle cells; however, this extreme situation probably arises from a combination of weak expression of the follicle cell phenotype combined with strong expression of the border cell phenotype (see Discussion). The lack of coordination in cell migration makes it difficult to assess the progression of each chamber through stage 9. We therefore resorted to a morphometric approach. The four distances indicated in Fig. 2 B were measured in sagittal optical sections of 81 wild-type and 318 karst mutant egg chambers. Since the nurse cells do not grow or shrink at this time, all these measurements were normalized against the anterior-posterior length of this cell cluster. Pairwise comparisons of the parameters (FC/NC), (BC/NC), and [oocyte/(oocyte+NC)] reveals the following defects in karst mutant egg chamber morphogenesis. (a) Most karst border cell clusters are migrating ahead of the follicle cell monolayer during stage 9 . This could arise either due to faster border cell migration or slower follicle cell migration. (b) Most karst mutant oocytes occupy a larger portion of the egg chamber than in the wild-type during follicle cell migration, while the oocyte exhibits no significant overgrowth at the completion of migration . This suggests that karst follicle cells are delayed in their migration relative to growth of the oocyte, or may respond more slowly to oocyte growth. (c) Similarly, most karst mutant oocytes occupy a larger portion of the egg chamber than in wild-type during border cell migration . This effect is not as strong as for the follicle cells, consistent with the observation that the border cells generally migrate ahead of the follicle cells in karst mutant egg chambers, but it does suggest that there is a slight delay in border cell migration. The most parsimonious model accounting for these data suggests that the karst mutation causes a significant disruption of follicle migration onto the oocyte membrane and a slight delay in border cell delamination or migration through the nurse cells. Consistent with the hypothesis that the primary morphogenetic defect lies in follicle cell migration, some follicle cells in karst mutant egg chambers often remain in contact with the nurse cell membranes at stage 10A. These follicle cells still attempt to make the appropriate adhesive contacts with the oocyte membrane , pulling the oocyte membrane towards them and grossly distorting the nurse cells/oocyte interface . In most cases, the subsequent inward migration of the follicle cell layer at stage 10B proceeds along the nurse cell/oocyte interface in a relatively normal fashion. However, in rare, extreme cases, the centripetally migrating cells penetrate between nearby nurse cell membranes and cause one or more nurse cells to become included within the egg along with the oocyte . The failure of karst follicle cells to complete their migration onto the oocyte by the onset of stage 10B implies that the total apical surface area of the epithelium is greater than that of the oocyte membrane. Moreover, karst mutant follicle cells often appear to have a more cuboidal shape than in the wild-type . Since there is no over-proliferation in the mutant monolayer (data not shown), this cannot arise due to an increase in cell number. However, an inability of karst follicle cells to properly change their cell shape or constrain their apical surface area at the appropriate size would explain this observation. We therefore compared the apical surface area of wild-type and mutant follicle cells during monolayer migration. This analysis reveals a sharp decrease in the apical surface area of the wild-type follicle cells as they approach and migrate onto the oocyte . In contrast, the majority of karst mutant follicle cells fail to apically constrict . The mean apical surface area of the mutant follicle cells is almost twice that of the wild-type . Moreover, comparison of the apical surface areas of mutant follicle cells in chambers during migration with those where migration has been completed reveals a slight increase ( Table ; P < 0.001). This suggests that, in addition to the constriction defect, the monolayer cannot withstand the forces exerted by the growing oocyte. Examination of the follicle cell apices stained for D E-cadherin also reveals conspicuous disruptions in the staining pattern of D E-cadherin in karst mutant egg chambers . In the mildest cases, this staining is missing at three- or four-cell vertices, but we also see large breaks in the normally continuous belt of staining in more extreme cases. These observations are consistent with the hypothesis that the absence of β H weakens the ZA, and that it breaks up as the apices attempt to constrict or accommodate the growth of the oocyte. However, the apicolateral polarization of the ZA is largely unaffected . The border cell cluster delaminates from the follicle cell epithelium and migrates between the nurse cells to the anterior of the oocyte during stage 9. In ∼10% of karst mutant egg chambers, we observe migratory cells that are well separated from, or trail behind, the main border cell cluster . The trailing cells upregulate α-spectrin and D E-cadherin (data not shown) in a manner that resembles wild-type clusters, suggesting that they are true border cells. To confirm that these cells have a border cell fate, we looked for the expression of the border cell marker slow border cells ( slbo ) by introducing the enhancer trapped LacZ gene associated with the slbo 1 P-element allele into the karst mutant background and staining for β-galactosidase activity. All of the migratory cells in slbo 1 /+; kst egg chambers express β-galactosidase, although the intensity of staining exhibited by the separated cells is often lower than the main border cell cluster . None of the trailing cells express Fasciclin III, indicating that they do not contain polar cells , and thus do not represent second, independently organized, migratory clusters. Furthermore, the total number of β-galactosidase–positive cells ranges from 7 to 11, very close to the normal range in border cell number in wild-type chambers . Together, these data suggest that all of the migratory cells are bona fide border cells and that the normal number of border cells is specified in karst mutants, but that the cluster is unable to remain together as a unit. β H is no longer detectable at the apical domain of the follicle cells in any allelic combination of karst alleles that we have examined (data not shown). The localization of β H to the apical domain has been previously shown to be dependent on α-spectrin . To see if α-spectrin is dependent on β H for its localization to the apical domain, and to confirm that no apical spectrin function remains in karst mutants, we examined the distribution of α-spectrin in karst follicle cells. While the lateral α-spectrin distribution is unaffected by this mutation, apical α-spectrin is no longer detectable by immunofluorescence . This indicates that the stable recruitment of α-spectrin to the apical domain is dependent on β H , and that there is thus a mutual interdependence between α-spectrin and β H . This further suggests that αβ H -spectrin is recruited to the apical domain as a heterodimer or tetramer, or that following separate recruitment only the dimers or tetramers remain stably associated with the apical domain. Spectrin associated with E-cadherin has been implicated in the apicobasal cell polarization pathway , and fly α-spectrin mutations cause a breakdown in monolayer polarity including the loss of apical β H . However, karst mutants form a follicle epithelium that appears to have a well polarized morphology. To further verify this observation, we examined the distribution of the apical markers Notch, myosin II, and unconventional myosin IB , as well as the apical concentration of actin in the follicle cell brush border. These and similar experiments in mutant eye and wing imaginal discs (data not shown) indicate that the distribution of several apical markers is not conspicuously affected by the lack of β H . Given the significant similarity between β H and β-spectrin , it is also possible that polarity is retained in karst mutant epithelia because of functional redundancy between these two proteins. Moreover, the karst mutant phenotype exhibits variable expressivity despite genetic evidence that we have null alleles , and this might also result from functional redundancy. Such redundancy would predict that conventional β-spectrin would be recruited to the apical domain in the absence of β H . However, this is not the case , confirming that β H is unnecessary for apicobasal polarity and indicating that we must look elsewhere for the source of variability in the karst phenotype. β Heavy -spectrin is a member of the spectrin family of proteins that have been implicated in cadherin-mediated cell polarization , and is associated with the ZA . In this paper, we have characterized the effects of loss of function karst mutations (which eliminate β H ) on fly oogenesis. These mutations cause defects in the migration of the follicle cell monolayer and in border cell delamination, but do not compromise the apicobasal polarity of its constituent cells. The defect in monolayer migration is characterized by a failure of the follicle cells to constrict their apical surfaces and by visible breaks in the ZA. We present here the first complete description of the distribution of β H during Drosophila oogenesis . β H is strongly expressed in the terminal filaments and cap cells that sit adjacent to the germline stem cells at the anterior end of the germarium. In the germline, it is first seen on the membrane in region 2, in the 8–16-cell cysts. β H is not obviously polarized at any of these locations, and remains uniformly distributed in the nurse cells and oocyte. In the soma, β H is prominently expressed in the vicinity of the somatic stem cells and their derivative, the follicle cell epithelium, where it is restricted to the apical domain. These results corroborate the previous partial descriptions of the distribution of β H during oogenesis . We have also found that β H is prominently expressed in the stalk cells, at a number of sites of high secretory activity late in oogenesis, and is downregulated in the border cells during their migration. The downregulation of β H in the anterior region of the germarium would suggest that β H has no role in germ cell division and/or oocyte specification within the germline. However, β H is strongly expressed in the terminal filament and cap cells that sit adjacent to the germline stem cells and early cystoblasts, the cellular activities of which are believed to regulate germline stem cell division and polarization . β H could thus interact with one or more components in the terminal filament and cap cells to generate signals that affect polarity or proliferation in the germline. β H is part of a prominent terminal web subtending the follicle cell brush border that forms as they migrate onto the oocyte at stage 9 and begin to secrete yolk protein. β H is also expressed in the border cells once these cells begin to secrete the micropyle and in the follicle cells that are secreting chorion to form the dorsal appendages. The prominence of β H in all these locations of high secretory activity suggests that there may be a role for the apical membrane skeleton in the targeting or delivery of secretory vesicles even though it is not required for overall apicobasal polarity (see below). A similar role has been proposed for the gut-specific terminal web β-spectrin, TW260, in the chicken . In karst mutant ovaries, the primary defect in follicle cell morphogenesis is a failure of this monolayer to complete migration onto the oocyte membrane by the end of stage 9 . Specifically, these cells fail to undergo the normal apical contraction associated with the development of a more columnar shape upon contacting the oocyte membrane . Furthermore, staining for the ZA marker D E-cadherin revealed conspicuous breaks in the normally continuous belt of staining for this protein around the follicle cell apices . Apical contraction is a well established process for the generation of form in an epithelium; however, the exact mechanism by which cells achieve this phenomenon is still unclear. β H is associated with two actin-rich structures at the apical pole, the ZA and the terminal web subtending the microvillar brush border. F-actin at the ZA lies in a circumferential band of microfilaments of mixed orientation that can be induced to undergo a purse string–like contraction in vitro that is mediated by myosin II . β H may be necessary for contraction of this bundle. Spectrin cross-linking of F-actin can stimulate myosin ATPase and is necessary for cortical contraction of sea urchin eggs . The distribution of myosin II was not conspicuously affected in karst mutant follicle monolayers (data not shown); however, β H may be necessary for the correct organization of the contractile actin bundle through its ability to cross-link F-actin, or for the attachment of the bundle to the membrane or the ZA. Alternatively, β H may play a structural role in the follicle cell terminal web . This is a region of dense actin cross-linking between bundles of microfilaments that support the overlying microvilli and is integrated into the circumferential F-actin bundle and the ZA at its margins. This structure also contains myosin II , but is not contractile. However, a decrease in apical surface area presumably requires remodeling of the terminal web to produce a corresponding decrease in the size of this network or an increase in microvillar density. Neither a terminal web nor β H is required to produce microvilli . However, β H in the terminal web might help stabilize the contractile process. The apical domain of all of the follicle cells must apically constrict, presumably generating significant tension in the adhesive network that binds the epithelium together. In this particular case, the terminal web may be an essential structural component of a supracellular actin network. The lack of cross-linking by β H in the terminal web might thus result in excessive strain on the ZA and its ultimate breakage. The fact that follicle cell migration does initiate and proceed to some extent in karst mutants indicates that not all motile forces are eliminated by this mutation. This may simply mean that not all intercellular tension generated by apical constriction is eliminated by the karst mutation. However, another possibility is that apical constriction is not the only force-generating mechanism responsible for monolayer migration. Indeed, the observation that hypomorphic mutations in the regulatory light chain of cytoplasmic myosin II do not prevent the migration of the follicle cell monolayer does indicate this. At least two other forces should be considered. First, the oocyte is growing through the uptake of hemolymph and yolk during stage 9, and this inflation may contribute to migration and to tension at the ZA. Second, the follicle cells make specific adhesive contact with the oocyte membrane and this might produce localized tension as each follicle cell first contacts, and then moves onto the oocyte. The failure of karst follicle cells to complete their migration onto the oocyte by the onset of stage 10B leads to aberrant centripetal cell migration in a small number of egg chambers. In such cases, the inwardly migrating cells find their way in between nurse cell membranes enclosing one of the latter along with the oocyte. Given the high frequency of defective migration, it is perhaps surprising that this latter defect is relatively rare and that oogenesis can often proceed to completion in the absence of β H . We suspect that the ability of the centripetally migrating cells to seek out the nurse cell/oocyte interface by specific adhesion to the oocyte membrane results in a substantial compensation for the migration defect that is generated by the karst mutation. Thus, the inclusion of a nurse cell with the oocyte after centripetal cell migration is infrequent and a relatively normal egg results. Moreover, after migration is completed, the oocyte continues to grow as dumping occurs and the egg matures, during which time the follicle cell apices must again grow and presumably any slack in the karst monolayer is taken up. This would explain why karst mutant females are not completely sterile and do lay some fertilized eggs. The border cells delaminate from the follicular epithelium at the onset of stage 9 and migrate between the nurse cells to the anterior of the oocyte . As part of this developmental program, we have shown that they downregulate β H . While there is no detectable β H at any of the border cell–border cell interfaces, it is difficult to say with certainty that there is no β H in a normal cluster. This is because these cells do not fully depolarize , and the residual apical surface is closely juxtaposed to the surrounding nurse cell membranes that contain abundant β H protein. Migration normally occurs as a tight cluster of 6–10 cells; however, in karst mutant egg chambers, one or more border cells are separated from the main cluster . The presence of β H at two or possibly three locations during normal border cell delamination and migration suggests a number of possible roles for this protein in border cell morphogenesis. Its presence at the nurse cell membranes on which the border cells migrate could contribute to the rigidity of this substratum or to inter-nurse cell adhesion. In this context, we note the similarity between the patchy distribution of β H along the border cell migration route and that of D E-cadherin, a molecule required for migration . The loss of either of these functions might impact border cell migration. β H might also be present on the outermost apical surface of the migrating border cell cluster. Here it could contribute to the adhesion between the border cells and the nurse cells. Loss of this function might cause the border cell cluster to have difficulty attaching to the nurse cells during delamination and the subsequent migration. Finally, β H is present in the nonmigratory follicle cells that surround the delaminating cluster, where it might play a role in border cell delamination. This latter hypothesis is the most likely explanation for the border cell phenotype. If the absence of β H either on the apical border cell surface or on the nurse cell surface was responsible for the breakup of the cluster, we would expect to see either a more dramatic or a preferential breakup of clusters late in migration. However, there is no indication that the number of separated cells increases with the distance migrated and we see many examples of early break up. The normal boundary between the border cells that will delaminate and the surrounding follicle cells is marked by a dramatic decrease in the level of β H in the border cells. In addition, β H is also prominent at the follicle cell/border cell membrane interface during delamination. This boundary must have at least two properties. It must serve to allow the border cells to detach and it must allow the follicle cell monolayer to reseal the gap left by the departing cluster. We hypothesize that the presence of β H in the surrounding follicle cells is part of a differential adhesion system that causes the surrounding, nonmigratory follicle cells to seek out one another to reseal the gap and in doing so to sacrifice contact with the border cells. Elimination of β H in the nonmigrating cells would affect the precise physical boundary between groups of cells with different adhesive properties preventing this rearrangement of cell contacts, and thus proper detachment of the border cells. The precise role of β H in generating this boundary remains open. β H is localized in part at the ZA and its presence or absence could be responsible for modulating D E-cadherin–based adhesion. Such differential adhesion is clearly part of the mechanism by which the oocyte positions itself relative to the overlying follicle cells . However, such sharp transitions in cell fate can be generated by lateral inhibition mechanisms , and it is also possible that β H is responsible for stabilizing some of the signaling molecules involved in such processes during border cell specification. The initial development of the follicular epithelium is essentially unaffected by the loss of either α-spectrin or β H (this paper), suggesting that the spectrin membrane skeleton is not essential for the establishment of polarity in this particular case. We have also demonstrated that there is no conspicuous breakdown of apicobasal polarity in the absence of the apical SBMS later in oogenesis . This contrasts with the phenotype of α-spectrin mutants in Drosophila , in which a loss of cell polarity and breakdown of the follicular epithelium is seen. Together, these two results indicate that the loss of apicobasal polarity in the α-spectrin mutants reflects a requirement for the basolateral SBMS specifically, or is a synergistic consequence of losing both the apical and basolateral membrane skeletons. Resolution of this ambiguity awaits further characterization of β-spectrin mutants. Although overall apicobasal polarity remains intact in karst mutants, it remains possible that specific proteins that are dependent on binding to the apical SBMS for delivery and/or retention in the apical membrane are depolarized by the absence of (αβ Η ) 2 . In keeping with the observation that different spectrin isoforms do not generally colocalize , β H and β-spectrin are found in mutually exclusive domains in epithelial tissues . β-spectrin is recruited to the membrane by proteins that bind to the adapter protein ankyrin . However, β H lacks an ankyrin binding site and is not recruited in this manner . Integral membrane proteins that bind to ankyrin thus provide a polarizing influence that can specifically establish a basolateral membrane skeleton. In this paper, we have shown that in the absence of β H , β-spectrin does not become recruited to the apical surface. This indicates that β H is not excluding β-spectrin from potential binding sites in this domain and that β H must therefore be specifically recruited to this domain. This result is also significant because it implies that the variable expressivity associated with the karst phenotype does not arise through redundancy of function between β H and β-spectrin. Current models for the origins of epithelial polarity suggest that spectrin plays a key role in establishing and/or maintaining the apicobasal axis . This model is largely based on the behavior of molecules involved in establishing the basolateral domain. The combined observations on the α-spectrin and karst mutant phenotypes suggests that the basolateral membrane skeleton may be playing such a role; however, our results indicate that apical spectrin [i.e., (αβ Η ) 2 ] does not. It remains unclear what the precise mechanism is by which apical spectrin acts during morphogenetic events. β H exhibits a close colocalization with the ZA, and its levels at this location are regulated in concert with D E-cadherin . Furthermore, we have observed mild disruptions of the ZA in eye/antennal imaginal discs (Zarnescu, D.C., and G.H. Thomas, unpublished observations) that are similar to the effects reported in this paper. None of our results to date reveal how closely (αβ Η ) 2 is associated with the ZA; however, the contrasting behavior of the apical and basolateral SBMS during the emergence of apicobasal polarity combined with the phenotypic data presented in this paper strongly suggest that these two cytoskeletal structures have somewhat distinct rather than identical roles in their respective domains, at least when it comes to the generation and/or maintenance of apicobasal polarity. The results presented in this paper add to a growing body of evidence that apical spectrin is essential for epithelial morphogenesis. Moreover, we show that an apical SBMS is not required for establishing or maintaining apicobasal polarity, as seems to be the case for the basolateral SBMS. It is unknown at present whether or not β H or any other β-spectrin plays a similar role in morphogenesis in vertebrates. The observations that β H is evolutionarily old , that there is a homologue in C. elegans encoded by the sma1 gene , and that the chicken brush border protein TW260 has a similar size and contour length to β H , all strongly suggest that there is a vertebrate homologue of β H that has yet to be cloned. The karst phenotype bears close resemblance to the phenotype exhibited by C . elegans embryos mutant at the sma1 locus that encodes the worm homologue of β H . In sma1 mutants, the worms fail to elongate at the wild-type rate during embryogenesis, resulting in a smaller embryo. Moreover, this phenotype seems to arise from a defective contraction of the embryonic epithelium . Thus, while the geometry of this developmental event is quite different from follicle cell migration in the fly, β H appears to be playing a similar role in the two organisms. In vertebrates, the apical accumulation of spectrin has been correlated with the initiation of neurulation in mice , and, in the sea urchin embryo, an apical accumulation of α-spectrin is associated with the involution of tissues during embryogenesis . This suggests that apical spectrin has a general role in the morphogenesis of epithelia mediated by apical contraction. | Study | biomedical | en | 0.999994 |
10477761 | Males of Ascaris suum were collected from the intestines of infected hogs at the Lowell Packing Company. Worms were transported to the laboratory in phosphate-buffered saline containing 10 mM NaHCO 3 , pH 7, at 37°C, and maintained in this buffer for up to 1 d. Spermatids were isolated by draining the contents of the seminal vesicle into HKB buffer (50 mM Hepes, 65 mM KCl, and 10 mM NaHCO 3 , pH 7.1). The spherical spermatids were then treated with vas deferens extract, prepared according to Sepsenwol et al. 1986 , to initiate lamellipodium formation and completion of development into motile spermatozoa. Activated sperm were pipetted into chambers formed by mounting a 24 × 60-mm glass coverslip onto a glass slide with two strips of hematoseal tube sealant. All glassware was washed in ethanol before use. Sperm were examined under a 100× differential interference contrast (DIC) oil immersion Zeiss plan/neofluar objective (1.3 N.A.) on a Zeiss Axiovert 35 microscope. Preparations were maintained at 37°C using an airstream incubator (Nicholson Precision Instruments). When desired, the solution bathing the cells was changed by pipetting 5–7 chamber volumes (1 chamber volume = 350–400 μl) of the new solution at one side of the chamber, using a tissue wick to draw the fluid into the chamber. All images of cells were obtained using a charged-coupled device (CCD) camera (Model TI-24A; Nippon Electronics Corp.), digitized, and processed by background subtraction and contrast enhancement using Image-1AT software and hardware (Universal Imaging), and recorded on a super VHS VCR . Video images were imported into Adobe Photoshop, processed, and printed on a Codonics printer. Rates of cell movement and related parameters were measured using Image 1AT subroutines. The dynamics of the cytoskeleton of Ascaris sperm can be observed directly in crawling cells using DIC microscopy and indicate that both MSP assembly and disassembly are important in their amoeboid motility. The MSP-based cytoskeleton in these cells form fiber complexes, each comprised of a meshwork of MSP filaments, that can be observed in living cells . Distinctive features, such as branches in the fiber complexes, can be followed and show that the cytoskeleton treadmills as sperm locomote . When sperm crawl, filaments assemble into fiber complexes along the leading edge and flow retrograde to the base of the lamellipodium, where they disassemble. The rates of cytoskeletal assembly and disassembly are balanced and are coupled to the pace of locomotion. Thus, as illustrated by analysis of morphological markers in the cell shown in Fig. 1 , the lamellipodium maintains its shape over time while the cytoskeleton flows rearward with respect to the cell, but does not move, or moves very slowly, with respect to the substrate. This pattern of lamellipodial dynamics has also been measured by computer-assisted microscopy . For example, difference pictures comparing the shape of the lamellipodium of crawling sperm at two-second intervals showed that the area of the zone of expansion at the leading margin was similar to the area lost due to retraction at the base of the lamellipodium. This study also showed that the average instantaneous velocity of sperm crawling on glass was 30 μm/min, but that the cells surged forward about every 0.35 min, increasing their velocity by an average of 47%. The cells maintained their lamellipodial shape during these surges, indicating that cytoskeletal assembly and disassembly remain balanced even when crawling speed changes . The behavior of crawling sperm that have become tethered to the glass substrate at their cell body indicated that lamellipodial extension is not sufficient for cell locomotion. In these cells, the lamellipodium continued to exert force against the cell body sufficient to distort its shape from hemispherical to elongate, but there was no stretching of the lamellipodium . In tethered cells, the leading edge underwent cycles of extension and retraction with no net advance, and yet cytoskeletal flow continued. In some cells, the lamellipodium lost its attachment and the entire cell was pulled toward the tether site. Others, like the sperm shown in Fig. 2 , broke the tether, after which the cell body recoiled toward the lamellipodium and regained its hemispherical shape as protrusion of the leading edge and locomotion resumed. By contrast, there was no shortening of the lamellipodium when the cell body recoiled. These observations show that the cell body does not follow passively behind the motile lamellipodium but, instead, that tension within the cytoskeleton pulls the cell body forward. Moreover, the force pulling against the cell body appears to be generated at the base of the lamellipodium. If that tension was produced at the leading edge by the force that drives protrusion, then the entire cell would stretch when tethered at the rear and recoil when the tether was broken. To probe the mechanism of cell body retraction and its role in sperm locomotion, we used pH to uncouple the cytoskeletal assembly and disassembly that occur at opposite ends of the lamellipodium. The MSP cytoskeleton is sensitive to intracellular pH . We found that treating sperm with HKB buffer containing 20 mM sodium acetate (HKB-acetate ) at different pH values caused cytoskeletal assembly and protrusion of the leading edge to either slow dramatically or stop entirely, while cytoskeletal disassembly and retraction of the cell body continued unaltered. The cell shown in Fig. 3 , for example, was crawling at 15 μm/min until it was perfused with HKB-acetate, pH 6.75. This treatment caused protrusion of the leading edge to slow to <3 μm/min. However, retraction of the cell body continued at 15 μm/min for a further 30 s, then stopped. This continuing retraction of the cell body appeared to be correlated with localized disassembly of the fiber complexes near the cell body because the distance between the cell body–lamellipodium junction and distinctive morphological features of the fiber complexes decreased as the cell body moved forward. Moreover, the cell body appeared to move forward due to shortening of the lamellipodium rather than rolling forward over the rear of the lamellipodium. If the cell body was rolling, the organelles within it should also roll, but this was not observed. Instead, the organelles maintained their position in the cell body as it retracted . Perfusing moving cells with HKB-acetate buffer at pH 6.35 for 5–10 s stopped both lamellipodial protrusion and cell body retraction and also arrested filament assembly at the leading edge . However, in these cells the fiber complexes of the cytoskeleton continued to disassemble at the base of the lamellipodium. Remarkably, the tips of the fiber complexes pulled away from the plasma membrane at the leading edge of the lamellipodium and the entire array of fiber complexes then progressed retrograde in concert towards the cell body. As this process continued, the fiber complexes became progressively shorter and the gap between their tips and the leading margin of the lamellipodium widened, so that after 30–60 s, the cytoskeleton was disassembled completely. The rate at which fiber complexes moved rearward ranged from 10–28 μm/min (mean, 18 ± 6 μm/min; n = 11). In general, the rate at which the fiber complexes receded was similar to the rate of forward movement of the cell body before acid treatment. Inspection of the morphology of the cytoskeleton during this retrograde recession allowed us to establish the site of fiber complex disassembly during HKB-acetate buffer treatment. Thus, if cytoskeletal disassembly was due primarily to filament depolymerization throughout the fiber complexes, we would have expected to observe changes in the morphology and optical density of fiber complexes. However, neither property changed markedly. Instead, characteristic features such as branches in the receding fiber complexes moved rearward and remained visible until they reached the site of disassembly at the base of the lamellipodium. Moreover, the optical density of the fiber complexes remained essentially constant as they receded. Treatment of sperm with HKB-acetate at pH 5.5 caused the entire MSP cytoskeleton to disassemble rapidly and the lamellipodium to round up . This effect was completely reversible. The pattern by which the cytoskeleton was rebuilt and locomotion resumed showed that cytoskeletal assembly and disassembly produce independent forces and that both are required for locomotion. When cells treated with pH 5.5 buffer were washed with HKB buffer without acetate, fiber complexes began to form around the periphery of the lamellipodium . This localized assembly resulted in formation of protrusions from the cell surface, but the cell body remained stationary. As these new fiber complexes continued to elongate, those emanating from the side of the lamellipodium reached the cell body and began depolymerization and treadmilling. In the cell shown in Fig. 5 , the fiber complexes on the right side of the lamellipodium reached the cell body before those from the other side. When these fiber complexes began to treadmill, the cell body was pulled to that side . Soon, additional fiber complexes growing from the right side of the lamellipodium reached the cell body and the entire cell began to move to the right before the fiber complexes from the other side were completely rebuilt . This asymmetry in cytoskeletal reconstruction, with treadmilling resuming earlier on one side of the lamellipodium than the other, resulted in a 60° change in the direction of locomotion, compared with that observed before acid treatment. The behavior of this unusual cell emphasized that movement, first of the cell body and then the whole cell, was determined by the location where cytoskeletal depolymerization resumed along the cell body–lamellipodium junction. In most cells recovering from this acid treatment, cytoskeletal reconstruction was symmetric and the directions of movement before and after treatment were similar. In each, however, movement of the cell body did not occur until the onset of depolymerization of the reconstructed fiber complexes. Manipulation of intracellular pH allowed us to uncouple cytoskeletal assembly from disassembly and thereby examine the separate roles of these processes in locomotion. We sought to determine if other factors are involved in locomotion by identifying a method for keeping the cytoskeleton intact, but blocking both polymerization and depolymerization. Previously, we had shown that antiphosphotyrosine antibodies stained the leading edge of the pseudopod preferentially and so we treated sperm with 30 μM phenylarsine oxide (PAO), a protein tyrosine phosphatase inhibitor. In PAO-treated cells, the fiber complexes remained clearly visible, but we were unable to detect either cytoskeletal flow or locomotion . However, the effect of the drug was completely reversible; cytoskeletal treadmilling and locomotion resumed within 10 s after washing the cells with a PAO antagonist, dimercaptopropanol, at 5 mM in HKB buffer. Thus, without the forces associated with cytoskeletal polymerization and depolymerization, sperm exhibit no detectable motility. Ascaris sperm offer a powerful experimental system for assessing the roles of cytoskeletal polymerization and depolymerization in amoeboid cell motility because their cytoskeletal dynamics can be observed directly using DIC microscopy. When the cells crawl, MSP polymerization and depolymerization take place simultaneously and at the same rate, but at separate locations: polymerization is localized primarily to the leading edge of the lamellipodium, whereas depolymerization takes place primarily at its base adjacent to the cell body. Previously, we demonstrated that local polymerization and bundling of MSP filaments can move membranes . In sperm, as in actin-based cells , this polymerization-derived force appears to mediate protrusion of the leading edge. By using pH to uncouple lamellipodial extension from cell body retraction, we have demonstrated here that tension associated with cytoskeletal disassembly pulls the cell body forward and that this force is required for locomotion. The deformation of sperm that become tethered to the substrate during locomotion indicated that the cell body was pulled forward by tension generated within the cytoskeleton itself. Polymerization of MSP at the leading edge is unlikely to be the source of this force directly because by pushing against the membrane, the fiber complexes would be placed under compression rather than tension. In principle, pushing the elongating fiber complexes against the leading edge could generate sufficient tension in the plasma membrane or cortical cytoskeleton to drag the cell body forward. However, if this were the case, the tension should cause the entire cell to stretch, not just the cell body. We also would have expected to observe movement of the cell body in cells recovering from treatment at pH 5.5 when polymerization was occurring, but depolymerization was not. Crucially, neither membrane nor cortical cytoskeletal tension can account for the rearward movement of the detached fiber complexes observed at pH 6.35 . Therefore, our results indicate that cell body retraction is mediated directly by tension in the cytoskeleton rather than by an indirect mechanism, such as release of tension in the cortical cytoskeleton or the plasma membrane at a rate controlled by fiber complex depolymerization. The results obtained by varying pH showed that this cytoskeletal tension was still produced when polymerization, and thus protrusion, was inhibited, but cytoskeletal depolymerization was not. Thus, the force that generates the tension in the cytoskeleton to pull the cell body forward must be produced by another mechanism than that used to push the leading edge forward. It may seem paradoxical to propose that the cytoskeleton is simultaneously under tension and compression, with polymerization-induced compression in the cytoskeleton pushing the leading edge forward while tension in the distal portion pulls the cell body. However, these two processes are separated spatially and, because the fiber complexes are coupled to the substrate through adhesions under the lamellipodium, they are also separated mechanically. Thus, these contacts can adsorb the opposing forces, thereby allowing the extension of the leading edge and retraction of the cell body to occur simultaneously. Our observations indicate that tension in the sperm cytoskeleton is generated locally at the base of the lamellipodium next to the cell body. The behavior of tethered sperm is consistent with this interpretation, as is the difference in the effects of treatment of sperm with pH 6.75 and 6.35 buffers. At the higher pH, polymerization and lamellipodial extension were greatly reduced, but depolymerization at the base of the lamellipodium continued and the cell body was pulled forward as the fiber complexes shortened . At pH 6.35, the cytoskeleton detached from the plasma membrane and under these conditions, rather than the cell body moving forward, the cytoskeleton was pulled rearward . Thus, in both cases, movement was toward the base of the lamellipodium, indicating that this is where tension is generated. The motile behavior of cells recovering from treatment at pH 5.5 shows that the cytoskeletal tension associated with depolymerization is required for sperm locomotion. During recovery, polymerization and depolymerization were uncoupled until the newly assembled fiber complexes reached the cell body. During this stage, when polymerization was active but depolymerization was inactive, the cell was capable only of protrusion. However, when depolymerization began at the base of the lamellipodium the cell body started to move. As shown in Fig. 5 , this movement required depolymerization of only a few fiber complexes and their position determined the direction of cell body movement. The involvement of a force specific for retraction has also been demonstrated in actin-based crawling cells, although, in these cells, there is not the direct link between retrograde flow and retraction seen in Ascaris sperm. For example, in Aplysia neuronal growth cones, treatment with cytochalasin D stops actin assembly along the leading margin, but the existing actin cytoskeleton continues to flow rearward as it is disassembled at the base of the growth cone . The behavior of the actin cytoskeleton in growth cones treated in this way parallels the recession of the MSP cytoskeleton in sperm incubated in pH 6.35 buffer. In fish epithelial keratocytes, forward movement of the cell body can be uncoupled from protrusion of the leading edge by treatment with cytochalasin B, demonstrating that forward movement of the cell body is not directly dependent on polymerization at the leading edge . Thus, these cells exhibit the same pattern of cell body retraction as Ascaris sperm treated with pH 6.75 buffer. The force that generates tension in the MSP cytoskeleton and the forward movement of the cell body could, in principle, be produced at the cell body either by a motor-driven contraction followed by depolymerization or alternatively by the depolymerization of the fiber complexes themselves. In actin-based cells, the force mediating retraction has been thought to involve primarily an actomyosin-based contraction . Although the morphological data we have presented here do not allow us to exclude completely the possibility that a contractile mechanism produces cell body retraction in Ascaris sperm, no MSP-based motor protein has been identified and we argue, based on several lines of evidence, that the tension in the MSP cytoskeleton that drags the cell body forward is more likely generated by local depolymerization than by molecular motors. In addition to the coupling of cell body retraction to localized cytoskeletal disassembly that occurs in crawling sperm, retraction (or its equivalent) is correlated both spatially and temporally with depolymerization under several different conditions, including selective stretching of the cell body in tethered sperm , retraction of the cell body in concert with the depolymerizing fiber complexes in cells treated with pH 6.75 buffer , recession of the entire cytoskeleton toward the site of depolymerization in pH 6.35 treated sperm , and simultaneous resumption of retraction and fiber complex disassembly in cells recovering from HKB-acetate at pH 5.5 . Conversely, when cytoskeletal depolymerization was blocked by PAO treatment , retraction was also inhibited, although the cytoskeleton remained intact to support any contractile activity. Moreover, in addition to the failure to identify any MSP-based motor proteins, structural studies have shown that the helices from which the filaments of the MSP cytoskeleton are constructed lack the structural polarity, characteristic of actin filaments and microtubules, that allows motor proteins to function. Depolymerization-associated tension in the MSP cytoskeleton may be analogous to the tension generated by microtubule depolymerization . For example, depolymerization of kinetochore microtubules at the kinetochore is associated with anaphase chromosome movement , and depolymerization of the plus-end of cytoplasmic microtubules has been shown to be able to move both membranes and vesicles in Xenopus laevis egg extracts . In the case of anaphase chromosome movement, microtubule depolymerization is thought to be coupled mechanically to the kinetochore through the binding of proteins of the kinesin family to the microtubule . However, these proteins are thought to function primarily to hold the microtubule (and so couple its length change mechanically to the kinetochore) rather than move the microtubule directly. Although we have not been able to identify an MSP-binding protein that could couple MSP depolymerization to the cell body in an analogous manner, such a mechanism would not be inconsistent with the apparent lack of polarity of MSP filaments because simply holding, rather than moving, does not require filament polarity a priori. Alternatively, it could be that depolymerization of the MSP-based cytoskeleton gel in the vicinity of the cell body generates contraction . Clearly, further work at the molecular level will be needed to distinguish between these possibilities. However, although the results obtained with Ascaris sperm do not rule out a contribution by motor proteins to retraction in actin-based amoeboid motility, they do show that it would, in principle, be possible both to extend the lamellipodium and retract the cell body by modulating the polymerization state of the cytoskeleton alone and certainly raise the possibility that such a mechanism may contribute to locomotion in at least some of these systems. | Study | biomedical | en | 0.999997 |
10477762 | NG108-15 cells (gifts of Drs. David Julius and Neil Smalheiser, University of California San Francisco) were cultured in GIBCO BRL DME H21 cell culture media supplemented with 10% FCS, penicillin/streptomycin, and 1× H.A.T. at 37°C and 5% CO 2 . 4–5 d before microinjection, the media was supplemented with 1 mM dibutyryl cyclic AMP, an agent that induces formation of axons and growth cones in these cells . In preparation for microinjection, cells were briefly trypsinized (0.05% in 1 mM EDTA) and replated on a 25-mm-circular glass coverslip attached with silicone grease to the bottom of a 35-mm polystyrene tissue culture dish in which we had drilled a hole. These coverslips were precoated with poly- d -lysine and coated with matrigel (a mixture of extracellular matrix proteins, primarily collagen and laminin) just before plating cells, essentially as described . Cells were transferred to coverslips 24–48 h before microinjection and incubated in 1 ml of media without phenol red. 30 min before microinjection, the media was supplemented with 25 mM sodium-Hepes, pH 7.4. On the microscope, the culture dishes were placed in a water-heated machine chamber maintained at 38°C. The temperature near the cells was close to 30°C because of loss of heat through contact with the microscope objective. For photoactivation, we labeled rabbit muscle actin on its reactive thiol with the caged rhodamine derivative α-carboxy-dimethoxy-C2CQRd-IA (caged Q-rhodamine) 1 as described . This actin derivative has been shown to form filaments in vitro and to localize to actin-containing structures . Caged Q-rhodamine is the rhodamine derived from 7-hydroxy-quinoline, caged as a bis-carbamate with two α-carboxy-dimethoxy-nitrobenzyl groups. None of the other caged fluorochromes we have worked with were satisfactory for actin labeling because of either poor photostability of the final fluorochrome (fluorescein and resorufin derivatives) or slow uncaging (caged rhodamines without α-carboxy substitution on the nitrobenzyl caging group). Caged Q-rhodamine actin was stored at 4–5 mg/ml in G buffer (5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl 2 , 1 mM ATP, 5 mM glutathione) in 5-μl aliquots at −80°C. For microinjection, it was diluted twofold with a solution of Cy5-conjugated dextran in G buffer. Injection was verified, and cells found after stage movement, using the Cy5 signal. Gerrard Marriot (Max Planck Institute, Martinsried, Germany) provided us with a plasmid encoding Dictyostelium discoideum actin fused to GFP . Cells were transfected using a standard calcium phosphate method . A Zeiss Axiovert inverted microscope with a bottom port was modified for photoactivation and photobleaching experiments. A mercury arc lamp illuminating a slit in a conjugate focal plane was used to generate a bar of 360-nm light for photoactivation experiments. An argon ion laser focused through a cylindrical lens was used to generate a bar of 488-nm light for GFP photobleaching experiments. Descriptions of our photoactivation microscopes have been published elsewhere . Phase and fluorescence images were acquired through a 100× objective to cooled CCD cameras from Princeton Instruments (http://www.prinst. com). For photoactivation experiments, we used a Kodak KAF1400 chip with 6.9-μm pixels cooled to −40°C. For phase images, we used 2 × 2 binning and for fluorescence images, 4 × 4 binning. For photobleaching experiments, we used a Sony 768x512 interline chip with 8.3-μm pixels cooled to –10°C. Phase images were collected without binning, and fluorescence images were collected with 2 × 2 binning. In both cases, we typically used 200–400-ms exposures with 100 W Hg illumination and standard fluorescence filters. Images were acquired with Princeton Instrument's WinView software with additional home-written software to control image acquisition. Delay between phase and fluorescence images was typically <2 s. Negligible changes occur in NG108 filopodia over this time-scale, so these image pairs effectively represent a single time point for our purposes. Locations of filopodium tips and photo-marks were determined by measuring the position of each with respect to the substrate along the axis of the filopodium. In practice, a single reference line was overlaid on the phase image of each filopodium, and tip distances were calculated with respect to the end of the line for each time point in the sequence. Filopodia were sometimes observed to pivot about an axis located in the growth cone body, as previously reported . Only filopodia that rotated <15° were included in our study. Generally, filopodia that exhibited minor pivoting behavior rotated smoothly from one position to another. Reference lines were drawn to bisect the angle of filopodium rotation and intersect with the axis of rotation. The position of the tip was projected via a perpendicular onto the reference line. Subsequently, an identical reference line was generated in the fluorescence channel, and similar measurements of the mark were taken. When we generated distance versus time plots of this data, all measurements were normalized with respect to the position or length recorded at t = 0, the time the mark was made. Fig. 1A and Fig. B , shows a typical growth cone photoactivation experiment. A differentiated NG108 mouse neuroblastoma cell was microinjected with actin derivatized with caged Q-rhodamine. 30 min after microinjection, the rhodamine actin had incorporated into existing cytoskeletal structures and a fluorescent mark was made on the actin filaments in a growth cone near the tips of filopodia by brief irradiation with a bar of 360-nm light. This allowed us to track the movement of a population of actin filaments in single filopodia over time . The photoactivation mark made at the tip of a filopodium maintained a constant intensity as it moved backward, consistent with tip assembly followed by retrograde flow. Fig. 1B and Fig. C , shows a typical growth cone photobleaching experiment. NG108 cells were transfected with a plasmid expressing a GFP-actin chimera that has been shown to incorporate into the actin cytoskeleton . Photobleaching also allowed us to track movement of a population of actin filaments in single filopodia over time . Marks made using photoactivation and photobleaching techniques were qualitatively and quantitatively similar in terms of their direction and rate of movement. In both cases, behavior of marked filopodia, as observed by phase-contrast microscopy, was similar to that of filopodia in untreated cells as well as unmarked filopodia in the same cell. Thus, both techniques appear to provide a nonperturbing probe of cytoskeletal dynamics. It is difficult to conclusively rule out the possibility that photodamage influenced our results, but two arguments suggest it was not a significant problem. First, the behavior of marked and unmarked filopodia by phase-contrast imaging is similar; second, the behavior of Q-rhodamine activation marks and GFP-actin bleach marks is similar since the photochemistry is different for these two probes. Photodamage can occur with long 360-nm irradiations. Increasing the duration of the 360-nm photoactivation pulse 10-fold over our experimental protocol caused freezing or breaking of filopodia, but we never observed such behavior in the experiments reported here. 490-nm illumination of GFP-actin appeared to cause very little perturbation, even when the fluorochrome was significantly bleached. Where our results or conclusions apply equally to photoactivation or photobleaching experiments, we use the term mark to refer to both types of mark interchangeably. Photobleaching experiments were technically easier since they did not require microinjection, whereas photoactivation experiments allowed us to track movements of actin filaments from filopodium tips deep into the growth cone as well as estimate the rate of actin filament turnover. Marks made at or near filopodium tips moved backward at rates ranging from −3.3 to 0 μm/min; measurements of mark and tip movement are made with respect to the direction of filopodium extension, so positive values indicate forward movement and negative values indicate backward (retrograde) movement. We never observed forward movement of marks made at filopodium tips ( n = 75 sequences). Also, we never observed splitting or broadening of marks. These data suggest that the actin filaments in filopodium bundles under our conditions assemble at the tip and flow rearward as a single unit, with extension or retraction resulting from the difference between the assembly and flow rates. If a small fraction of actin filaments moved at a different speed, they would have to have comprised <15% of total actin, or else not have exchanged with injected or expressed actin over the time course of our experiments, to have avoided detection. From images of the type shown in Fig. 1 we determined two parameters: cytoskeleton assembly rate, measured as the change in distance between the filopodium tip and the mark as a function of time; and retrograde flow rate, determined as the change in position of the mark with respect to the substrate as a function of time, measured along the vector defined by the filopodium. Measuring retrograde flow with respect to the substrate restricted us to measurements in filopodia whose angle with respect to the substrate did not change greatly during the time course of observation. This was the case for most of the filopodia in our images. Small angular changes were corrected as described in Materials and Methods. We first sought to determine how cytoskeleton dynamics changed in filopodia whose extension rate changed. Of the 75 filopodia observed in photo-marking experiments, 11 extending filopodia switched to either stationary ( n = 3) or retracting ( n = 8), 2 stationary filopodia switched to either extending ( n = 1) or retracting ( n = 1), and 6 retracting filopodia switched to extending ( n = 4) or stationary ( n = 2). For each of these, we asked how the rates of cytoskeleton assembly or retrograde flow changed from one phase of movement to another. Fig. 2 A shows an example in which a filopodium switched from extension to retraction. In Fig. 2 B, we plot the changing distance of the mark (black squares) and filopodium tip (solid line) with respect to substrate as a function of time. From this graph, we see that the switch in filopodium tip movement is accompanied by an abrupt change in the cytoskeleton assembly rate. This type of regulation, where a change from extension to retraction or vice versa was caused primarily by a change in assembly rate while the flow rate remained constant, was observed most frequently (14/19). We also found a smaller number of examples in which changes in retrograde flow alone ( n = 2), or both parameters ( n = 3) contributed to a switch in filopodium tip movement. In Fig. 3 A, a filopodium changes from a stationary to retracting movement. Here, switches in movement of the tip coincide primarily with changes in the rate of retrograde flow (dark lines). The plot in Fig. 3 B shows that the rate of cytoskeleton assembly (white circles) remains relatively constant over the course of the experiment. In Fig. 4 , assembly and flow rates changed in equal and opposite directions, resulting in little change in the rate of filopodium movement. These results show that assembly and retrograde flow rates can vary independently of each other. The results presented above demonstrate that cytoskeleton assembly and retrograde flow rates can vary within a single filopodium over time, and that the two parameters can be regulated independently. However, the cases where the filopodium tip switched from one consistent direction of movement to another during the experiment represent only a subset (19/75) of the filopodia studied. In most cases, the filopodium under observation extended or retracted continuously, though the rate might fluctuate. To summarize the contribution of assembly and flow rates to the direction and rate of filopodium tip movement for all our experiments, we grouped all our data by filopodium tip velocity and calculated average assembly and flow rates for each group . An approximately linear relationship can be discerned between average actin assembly rate and tip velocity. In contrast, average flow rate was relatively constant for different tip velocities. The only exception was a significant change in flow rate to more negative values in rapidly retracting filopodia. Thus, on average for the data collected in this study, most of the variations in filopodium motility could be attributed to differences in the rates of actin cytoskeleton assembly while the flow rate was relatively constant. We occasionally observed small decreases in the distance between the mark and the filopodium tip for rapidly retracting filopodia, as is apparent from the left in Fig. 5 . This may indicate the occurrence of actin cytoskeleton disassembly at filopodium tips, or throughout the structure, in rapidly retracting filopodia. However, the average cytoskeleton assembly rate in rapidly retracting filopodia was not significantly different from zero, and individual sequences were ambiguous with respect to the question of tip disassembly because of limitations of spatial or temporal resolution. Growth cone turning requires differential regulation of motile or adhesive activities across a single growth cone, and it is well known that individual filopodia can behave differently within a single growth cones . We sought to determine whether cytoskeleton assembly and retrograde flow could be regulated differently within a single growth cone. Fig. 6 A shows a growth cone in which several filopodia were marked simultaneously. Visual inspection of this sequence shows that retrograde flow rates vary between different filopodium bundles; marks on filopodia at the lower edge of the growth cone have moved a shorter distance than the marks above them . Similarly, the change in distance between mark and tip varies between different filopodia, indicating different rates of cytoskeleton assembly; compare marks on filopodia 8 and 13 at t = 0 and 4 min. We determined assembly and flow rates for 13 filopodia in this growth cone, labeled in Fig. 6 A, t = 0, right column. The bar chart in Fig. 6 B compares cytoskeleton assembly and retrograde flow rates for these filopodia. Cytoskeleton assembly rate varied between 0.34 and 1.77 μm/min (average = 1.0 μm/min). Retrograde flow rates varied between −0.08 and −0.90 μm/min (average = 0.63 μm/min). We performed a similar analysis on six other cells in which we had created marks on multiple filopodia, and each of these showed variable flow and assembly rates across the growth cone. One striking feature of our data that is readily apparent in Fig. 6 A is the long lifetime of photoactivation marks in filopodia. The remarkable stability of filaments in these bundles allowed us to observe the rearward transport of filaments deep into the growth cone body where they coalesced with filaments from other bundles . We estimated the half-life of actin filaments generated in filopodia to be at least 25 min in our experiments. This contrasts sharply with actin filaments in lamellipodia of other cell types that have been reported to display a half-life on the order of 0.5–3 min using rhodamine bleaching and resorufin photoactivation . To assay turnover in lamellipodia with the same probe we used for filopodia, we marked caged Q-rhodamine actin in fan-shaped lamellipodial of our cells . Lamellipodia were identified by their lack of filopodia, and active ruffling behavior in phase-contrast. Such lamellipodia occur frequently in NG108 cells under our conditions, and we often observed spontaneous transitions between filopodial and lamellipodial growth cones morphologies. Rapid actin filament turnover, with a half-life in the range of 1–3 min, was observed in lamellipodia ( n = 3). Similar results were obtained using GFP-actin photobleaching. Indeed, photobleached marks were very difficult to follow in lamellipodia because of rapid turnover (not shown). Thus, the stability of the filopodium bundles in the growth cones we imaged is due to cellular factors that stabilize actin filaments and not to an artifact of the new probe used in this study. Our results support a model for filopodium dynamics in which cytoskeleton assembly at the tip and retrograde flow are regulated independently, with filopodium extension/retraction resulting from the difference between the two rates. Within the error of our measurements, we found that marks in filopodia always moved rearward or were static, and that they remained of constant length and constant or slowly diminishing intensity. Thus, we strongly disfavor models for filopodium extension involving forward transport or outward telescoping of filaments and models for filopodium retraction involving inward telescoping. Our data are ambiguous on models for retraction involving cytoskeleton disassembly at tips and/or crumbling of the whole filament bundle. Most retraction in our study is accounted for by the action of retrograde flow with zero or slow assembly, but not disassembly, at the tip. Systematic studies of actin dynamics in rapidly retracting filopodia, for example in growth cones responding to collapsing factors , are needed to test whether tip disassembly and/or crumbling are important physiological retraction mechanisms. We observed instances in which changes in the extension rate of an individual filopodium were governed by changes in either assembly or flow rates and some cases where both parameters changed concurrently. However on average , we found that regulation of assembly was the dominant influence on filopodium extension rate in our study. It is not clear whether flow or assembly regulation will dominate during pathfinding in embryos. Most likely both will be important, with their relative contribution varying according to the specific situation. Although assembly and flow can be regulated independently, they may be coupled under conditions where the filopodium maintains constant length . Many of the filopodia in our data set were stationary with assembly exactly balancing flow . Such coupling most likely results from unknown molecular mechanisms that make flow rate limiting for assembly or vice versa. Cytoskeleton assembly and retrograde flow represent distinct molecular processes, and, thus, are likely to be regulated by distinct mechanisms. Independent regulation of assembly and flow allows for considerable flexibility in control of growth cone behavior by signaling and adhesion pathways. What are the likely mechanisms for regulating the two processes? Cytoskeleton assembly is thought to be driven by actin polymerization, though whether polymerization itself generates the force to extend the membrane forward is controversial . Cytoskeleton assembly is, thus, likely to be regulated by molecules that affect the rate of actin polymerization, such as barbed end cappers, polymerization factors, or actin monomer sequestering agents . We think that regulators of nucleation and pointed end dynamics, notably the arp2/3 complex, are less likely to be involved (discussed below). Since actin polymerization occurs right at the tip of the filopodium, the signaling pathways regulating polymerization may also be localized to the tip. One intriguing piece of evidence supporting this view is the finding that stationary and extending filopodia are differentiated by the presence of a phosphotyrosine epitope at their tips . Flow is thought to be generated by myosin activity , though in other cell types flowlike behavior may not be myosin driven and in general flow is poorly understood. Flow is known to be regulated by adhesion molecules that couple the cytoskeleton to the substrate , and it is also likely that the motors that cause flow can be regulated directly. There is now great interest in the mechanisms by which signaling pathways influence growth cone motility. Our experiments suggest that different pathways may control cytoskeleton assembly and retrograde flow. Obvious candidates for assembly regulation are the small GTPases cdc42 and rac since these two molecules are known to regulate actin polymerization in leading edge structures and cdc42 is known to induce de novo filopodia formation via NWASP . NWASP is thought to function at least in part by activating Arp2/3 complex , which in turn nucleates actin filaments and stabilizes pointed ends . Since actin filaments in filopodia are thought to be relatively long and do not turn over rapidly (as shown here), we suspect that continued assembly at filopodium tips is regulated by control of elongation rather than nucleation. Thus, we favor a model in which arp2/3 functions in initiating new filopodia, a process recently described in detail in growth cones , and not in regulating extension. Continuous action of arp2/3 is likely to be of more importance in lamellipodia, where filaments turn over rapidly and must be re-nucleated, than in filopodia. Factors that regulate actin filament elongation, such as capping factors , and bundling/stability, such as filamin , may regulate cytoskeleton assembly at filopodium tips. Candidates for flow regulation include cell adhesion molecules and the small GTPase rho since the latter is thought to regulate myosin II activity in growth cones . Growth cone turning is likely to be a complex process involving microtubule and membrane dynamics as well as actin dynamics, and it is likely that there are multiple ways of making a turn. Our data allow limited new conclusions about the role of actin dynamics in filopodia in turning growth cones. Differential extension/retraction of individual filopodia within a single growth is known to play a key role in turning . We can add that differential extension/retraction can be caused by differential regulation of both cytoskeleton assembly and flow by mechanisms that can act autonomously in single filopodia. Assembly regulation in particular seems to vary widely between filopodia within a single growth cone . Since assembly occurs right at the tip, the signals that regulate it autonomously in individual filopodia also may well be generated at or near the filopodium tip. Control of assembly at the tip will influence the stability and mechanics of the whole filopodium, and can, thus, transmit a mechanical signal back to the growth cone. During axonal pathfinding, a single filopodium whose tip contacts a distant guidance cue can induce turning of the whole growth cone . Dissection of the molecular pathways that can act autonomously within a single filopodium in response to guidance cues to govern cytoskeleton assembly rate at the tip, presumably through regulating actin polymerization onto existing barbed ends, should be informative about physiological guidance mechanisms. | Study | biomedical | en | 0.999998 |
10477763 | The expression plasmid pUCCAGGS containing either full-length DOCK180, myc-tagged c-CrkII, c-CrkII cDNA with a mutated amino-terminal SH3 domain (tryptophan 109 to cysteine), or Crk with a mutated SH2 domain (arginine 38 to valine) have been previously described . The pEBG expression plasmid containing glutathione- S -transferase–tagged or untagged wild-type p130CAS or CAS with an in frame deletion of its substrate domain (CAS-SD, amino acids 213–514) have been previously described . Myc-tagged dominant negative Rac 1 (N17) and mutationally activated MEK in pcDNA3 has been previously described . PD98059 (2-[2′-amino-3′methoxyphenyl]-oxanaphthalen-4-one; Calbiochem-Novobiochem). Anti-myosin antibody that recognizes the myosin IIB isoform present in COS7 cells was a gift from Dr. Robert Adelstein (Molecular Cardiology, National Lung Heart and Lung Institute, National Institutes of Health, Bethesda, MD). The phosphoERK antibody was from Promega. The anti-Rac, DOCK180, and myc antibodies were from Santa Cruz Biotechnology. FG-C are human pancreatic carcinoma cells stably overexpressing c-CrkII as previously described . Transient transfection of COS cells and Transwell migration assays were performed as previously described . In brief, COS-7 cells were cotransfected with lipofectamine and expression vectors containing cDNAs encoding wild-type and/or mutant forms of MEK, Crk or CAS, Rac, DOCK180, along with a reporter construct encoding β-galactosidase (pCMV5-β-gal) or green fluorescent protein (pEGFP-C1; Clontech). Control cells were mock-transfected with the appropriate amount of the empty expression vectors along with the β-gal reporter. Cells were allowed to incorporate the cDNA constructs for 6–8 h, washed, and then allowed to incubate for 40 h which provides optimal transient expression in these cells. COS cells were then prepared for haptotaxis cell migration using X-gal as a substrate and analyzed for expression of specific proteins by immunoprecipitation and immunoblotting as previously described . Transfection efficiency and cell adhesion of these cells to purified extracellular matrix proteins were monitored as described below. The migration of FG cells, metabolic labeling of transfected cells with [ 32 P]orthophosphate, immunoprecipitation of myosin light chains, SDS-PAGE, and autoradiography were performed as previously described . Controls for transfection efficiency and cell adhesion to ECM proteins were performed as previously described . In brief, an aliquot of cells from the migration experiments above was allowed to attach to culture dishes coated with purified ECM proteins. The dishes were washed and adherent cells transfected with the β-gal reporter gene were detected using X-gal as a substrate according to the manufacturer's recommendation (Promega). In typical transfection experiments with these cells, we obtain 70–75% efficiency, as determined by counting the number of β-gal positive cells relative to the total number of cells attached per field (200×). It is important to note that in an individual experiment, transfection efficiently varies <10%. The efficiency and adhesion control assures that changes observed in cell migration is not simply the result of differences in transfection efficiency or expression of the β-gal reporter gene or differences in the ability of transfected cells to attach to the ECM. COS-7 cells were cultured on glass coverslips and then transfected as described above along with a reporter construct encoding either GFP or β-gal. Cells were serum-starved for 24 h then treated with or without insulin (10 μg/ml) or IGF-1 (20 ng/ml) for 15 min. In some cases, MEK or mock-transfected cells were exposed to 50 μM PD98059 for 2 h before being treated with insulin or IGF-1 as described above. To visualize F-actin containing membrane ruffles, cells were rinsed in PBS then fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with rhodamine-conjugated phalloidin. Cells with prominent actin-rich membrane ruffles were quantified blindly by two independent investigators and represents the average number of transfected cells (i.e., GFP cells) with ruffles of eight high powered fields (400×). To observe F-actin filaments and myosin IIB colocalization in cells, we used a procedure that preserves the association of these proteins in whole cells. In brief, cells were extracted with 0.1% Triton X-100 detergent, fixed in 4% paraformaldehyde, then stained with rhodamine-conjugate phalloidin and rabbit anti-myosin IIB, followed by FITC-conjugated secondary antibodies as previously described . In some cases, cells transfected with the β-gal reporter construct were detected by staining with mouse anti-β-gal and donkey anti-Cy5-conjugated secondary antibodies along with three color cell fluorescence and a laser confocal microscope . Myosin content per cell was obtained by determining the fluorescence intensity (sum of total green pixels × intensity) in the green/FITC channel per cell area (μm 2 ) using Adobe Photoshop and IPLab Spectrum P computer software. Cell contraction of a three-dimensional collagen matrix was performed as previously described . In brief, 1 × 10 6 cells/ml collagen were cultured for 2 d in DME containing 10% FBS. Cells were serum-starved for 4 h then exposed for 60 min to serum-free culture media containing PD98059 (50 μM), M7 (1 μM; [(5-iodonaphthalene- 1 -sulfonyl)homopiperazine, HCL; Calbiochem-Novabiochem], or BDM (10 mM; butanedione monoxime; Sigma Chemical Co.). To initiate contraction, collagen gels were mechanically released from the culture dish in the continued presence of the inhibitors. The change in diameter in millimeters was measured with a ruler at various times after release. In some cases, cells were washed to remove the inhibitors then cultured an additional 24 h in drug-free media before initiation of contraction as described above. Recently, we reported that CAS/Crk coupling as well as ERK activation facilitate cell migration . Each of these events were shown to be required for cell migration, yet it remains unclear whether these signals represent the same or parallel pathways involved in regulation of this process. To address this issue, cells were transfected with CAS lacking its substrate domain (CAS-SD) or Crk with a mutated SH2 domain (CRK-SH2), either of which are capable of preventing CAS/Crk coupling and downstream signaling events . Cells containing these cDNAs were treated with the cytokine insulin and examined for migration as well as ERK activity. Expression of either CAS-SD or Crk-SH2 blocked insulin-induced cell migration, yet had no effect on ERK activity in these cells . Similar findings were observed in cells expressing Crk with a mutated amino-terminal SH3 domain that retains its ability to couple to CAS, but is unable to link to downstream effector molecules such as DOCK180 or C3G . These findings reveal that disruption of CAS/Crk coupling or its binding to downstream effectors can suppress cell migration without influencing ERK activity. To determine whether ERK signaling represents an independent pathway necessary for cell migration, cells were induced to migrate with insulin and then exposed to the compound PD98059 that blocks ERK kinase (MEK), and thereby prevents ERK activation . These cells were then examined for cell migration, ERK activity, and formation of CAS/Crk complexes. While the MEK inhibitor prevented insulin-induced cell migration and ERK1/ERK2 activation, it did not impact CAS tyrosine phosphorylation or the formation of CAS/Crk complexes in these cells . Similar findings were observed when cells were stimulated to migrate with either EGF or IGF-1 (data not shown). Thus, CAS/Crk coupling and ERK activation appear to represent components of distinct signaling pathways necessary for cytokine-induced cell migration. To investigate directly whether formation of a CAS/Crk complex could activate ERK, serum-starved COS-7 cells were transiently transfected with vectors encoding CAS and Crk or mutationally activated MEK. These cells were then examined for ERK activity and migration. Expression of MEK in these cells promoted a four- to fivefold increase in cell migration and significantly increased ERK activity compared with mock-transfected control cells . However, while CAS/Crk transfected cells showed a fourfold increase in cell migration, there was no change in ERK activity . Similar findings were obtained in FG carcinoma cells stably transfected with c-Crk (FG-C). These cells also showed significantly enhanced migration, yet ERK activity was the same as control cells . Together, these findings indicate that CAS/Crk-induced cell migration does not result from increased ERK activity. However, since ERK signaling appeared to be a separate event necessary for cell migration, we investigated whether ERK activity was also necessary for CAS/Crk-induced cell movement. To investigate this possibility, FG-C and COS-7 cells transfected with CAS and Crk were exposed to PD98059 and analyzed for their ability to migrate on ECM proteins. In this case, PD98059 blocked cell migration induced by CAS/Crk without affecting the formation of CAS/Crk complexes in these cells (data not shown). Conversely, MEK-induced cell migration was blocked by expression of CAS-SD or dominant negative RacN17 without impacting ERK activity (data not shown). Thus, CAS/Crk/Rac signaling and ERK activation appear to be separate biochemical pathways necessary for cell migration. Activation of cell migration is characterized by the assembly of actin into membrane ruffles as well as cell contraction . To explain how the coupling of CAS/Crk and activation of ERK might influence the migration machinery, cells expressing CAS-SD were stimulated with insulin and then examined for actin-containing membrane ruffles. Exposure of mock-transfected control cells to insulin induced prominent membrane ruffles rich in F-actin . Approximately 18% of the control cell population showed membrane ruffling before stimulation with insulin and this was increased to 80% after cells were exposed to this cytokine . Importantly, expression of CAS-SD in these cells completely blocked the insulin-induced membrane ruffling response . As expected, cells within the field of view not transfected with CAS-SD showed prominent membrane ruffles . Expression of Crk-SH2 in cells also blocked insulin-induced membrane ruffles (data not shown). Recently, it was reported that CAS/Crk coupling facilitates Rac activity which can promote membrane ruffling . Therefore, we determined whether CAS/Crk-induced membrane ruffles required Rac activity. Cells were transfected with CAS and Crk along with a dominant negative form of Rac (RacN17) and then examined for F-actin containing membrane ruffles. Expression of RacN17 in these cells blocked CAS/Crk-induced ruffles . RacN17 also blocked cell migration without impacting ERK activity (data not shown). Thus, CAS/Crk coupling promotes membrane ruffles that depend on Rac, but not on ERK activity. Crk is known to bind to a number of downstream effector molecules via its SH3 domain, including c-Abl, SOS, C3G, Eps15, and DOCK180 . Among these Crk-binding proteins, DOCK180 has been associated with Rac activation . Moreover, in Caenorhabditis elegans and Drosophila melanogaster , the DOCK180 homologue ced-5 and mbc, respectively, control cell migration events associated with development . Therefore, we were prompted to investigate the role of DOCK180 in cell migration. As shown in Fig. 4D and Fig. E , expression of DOCK180 was able to potentiate CAS/Crk-induced cell migration in a Rac-dependent manner, yet it had no effect on ERK activity in these cells. Together these findings suggest that CAS/Crk in conjunction with DOCK180 can form a signaling module involved in Rac-mediated membrane ruffling and cell movement that is independent of ERK activation. To investigate the role of ERK-dependent signaling in actin assembly, cells exposed to the cytokine insulin were treated in the presence or absence of the MEK inhibitor, PD98059. This inhibitor, which blocks cell migration, but not adhesion or spreading on collagen or vitronectin substrates , failed to disrupt membrane ruffling in response to insulin . In fact, 70% of cells exposed to PD98059 and insulin showed membrane ruffling, compared with 80% of cells exposed to insulin alone . Together these findings indicate that CAS/Crk coupling is necessary for membrane ruffling, whereas ERK activity is not. Cell migration also involves myosin light chain phosphorylation leading to actin-myosin association and cell contraction. While ERK can phosphorylate MLCK leading to increased MLC phosphorylation, it is not yet known if this event promotes assembly of a functional actin-myosin motor capable of generating force necessary for cell contraction . This, and the fact that ERK did not influence membrane ruffling, yet appeared critical for cell migration, prompted us to examine its role in actin-myosin assembly and contractile function. We also investigated the role of CAS and Crk in this process since it is not known if these proteins regulate actin-myosin activity independent of their ability to organize actin into membrane ruffles. Cells were stimulated with insulin or transfected with mutationally activated MEK and examined for phosphorylation of MLC, which facilitates actin-myosin binding and motor activity . These cells were also examined for actin-myosin colocalization by immunofluorescent staining and laser confocal microscopy. Cells exposed to insulin or those expressing MEK+ showed increased phosphate incorporation into MLC compared with unstimulated or mock-transfected control cells . Exposure to insulin or transfection with MEK also promoted increased actin-myosin colocalization compared with control cells . In fact, the amount of insoluble myosin associated with these cells after extraction in detergent was increased by two- to threefold . Importantly, PD98059 blocked insulin and MEK-induced MLC phosphorylation as well as actin-myosin colocalization, indicating that ERK activity was required for this response . In contrast, transfection of cells with CAS-SD, which blocks membrane ruffling , failed to block MLC phosphorylation and actin-myosin colocalization in response to insulin or expression of MEK+ . Therefore, ERK activation selectively promotes MLC phosphorylation and actin-myosin colocalization, whereas CAS/Crk coupling facilitates membrane ruffling. While cells require actin-myosin motor function for motility, these same events generate mechanical force necessary for wound contraction . Therefore, we investigated the role of ERK and CAS/Crk coupling in cell-mediated contraction of the extracellular matrix. Cells were transfected with CAS-SD or exposed to PD98059 and then examined for their ability to contract a three-dimensional collagen matrix. Exposure of cells to the MEK inhibitor significantly reduced the rate of cell contraction . In fact, the time necessary to achieve half-maximal contraction was increased from 16 min in control cells to 36 min in cells exposed to the MEK inhibitor. Similar findings were obtained when cells were transfected with the ERK phosphatase MKP-2 which blocks ERK activity (data not shown). The time for half-maximal contraction was also significantly increased in cells exposed to the MLCK inhibitor M7 or in cells transfected with a dominant negative form of MLCK (MLCK−) , which prevents ERK-induced cell migration and myosin light chain phosphorylation . In this case, half-maximal contraction time was increased to 76 and 48 min in M7 and MLCK− cells, respectively, whereas control cells responded within 16 min. The difference in cell contraction between M7 and those transfected with MLCK− is likely due to the fact that ∼75% of transfected cells express the MLCK− construct, whereas 100% of the cells are exposed to M7. In support of these results, cells exposed to BDM, a general inhibitor of myosin ATPase activity , showed minimal contraction indicating that myosin plays a central role in this process . In contrast, contraction was not altered in cells transfected with CAS-SD which blocks membrane ruffling, but not actin-myosin colocalization . Similar findings were observed when cells were transfected with Crk-SH2 (data not shown). Importantly, the inhibition of cell contraction induced by these compounds was not due to nonspecific cell toxicity since their effect was reversed when they were removed from the culture media . Together, these findings support the notion that ERK activation represents a distinct signaling pathway involved in the regulation of actin-myosin assembly and cell contraction, whereas CAS/Crk coupling represents an independent pathway that regulates actin membrane ruffling in migratory cells. However, while inhibition of ERK or MLCK activity decreased cell contraction, it did not completely block this event, suggesting that additional signals may exist to regulate MLC phosphorylation and actin-myosin contraction. Consistent with this possibility, Rho kinase is known to promote MLC phosphorylation independent of MLCK activity . ERK activation and the molecular coupling of CAS and Crk are initiated upon cell adhesion to ECM proteins and/or exposure to various growth factors. Our findings that formation of a CAS/Crk complex and activation of the GTPase Rac are necessary for membrane ruffling, whereas ERK activity is involved in actin-myosin contraction, suggest that these signaling events regulate specific components of the migration/contraction machinery. During wound healing, the actin-myosin motor generates contractile force necessary for both cell migration as well as contraction of fibrin or collagen matrices. These cellular events appear to be independently regulated of CAS/Crk and Rac-associated events, while ERK regulates force generation and cell contraction. Several lines of evidence suggest that CAS/Crk and ERK activation operate as components of separate signaling pathways necessary for cell migration. First, dominant negative forms of CAS and Crk that prevent CAS/Crk coupling blocked cytokine-induced cell migration without impacting ERK activation. Furthermore, inhibition of ERK activation prevented cytokine-induced cell migration without impacting CAS tyrosine phosphorylation or the formation of a CAS/Crk complex. Second, while formation of a CAS/Crk complex was sufficient to induce cell migration in serum-starved cells, it failed to promote ERK activation, indicating that this migration response was not the result of increased ERK activity. However, ERK activity was found to be a separate response necessary for CAS/Crk-induced cell migration since blocking endogenous ERK activity with PD98059 prevented CAS/Crk induced cell migration, but had no effect on the coupling of these proteins. In addition, we have observed that expression of mutationally activated MEK in cells, while sufficient to induce ERK activation and cell migration, did not impact CAS/Crk coupling (data not shown). That dominant negative forms of CAS and Crk were able to block MEK-induced cell migration, but not ERK activation, provided additional evidence that CAS/Crk and ERK are separate events required for cell migration. Finally, CAS/Crk and ERK represent distinct signals capable of regulating membrane ruffling and contraction in migratory cells. That Ras effector mutants deficient in their ability to facilitate ERK activity retain the capacity to promote membrane ruffling further support this notion . Our findings that CAS/Crk coupling was sufficient to induce membrane ruffles suggests that at least one important consequence of the molecular coupling of these proteins in cells is to facilitate Rac activation and/or its localization to the cell membrane. In fact, recent evidence indicates that CAS/Crk coupling in cells can potentiate Rac activity and cell spreading . This was found to depend on the recently described Rac-activating protein DOCK180, which binds to the amino-terminal SH3 domain of Crk . Our findings indicate that DOCK180 can potentiate CAS/Crk-induced cell migration, an event that depends on Rac activity. Together, these findings suggest that DOCK180 is an important downstream mediator in the CAS/Crk motility response. Interestingly, cellular expression of farnesylated, but not wild-type DOCK180, induced cell spreading and ruffling , suggesting that DOCK180 requires localization to the cell surface to fully activate Rac and the associated changes in the actin cytoskeleton. During cell migration, CAS/Crk may serve to target DOCK180 to the membrane where it can interact with Rac. Indeed, CAS/Crk and Rac are known to localize to membrane ruffles of migratory cells . Alternatively, the association of DOCK180 with CAS/Crk complexes may regulate signaling from integrin adhesion receptors as recently suggested . In either case, this appears to be independent of ERK signaling as cells expressing CAS/Crk/DOCK180 complexes showed enhanced cell migration without significant changes in ERK activity . The ability of CAS/Crk complexes to couple to DOCK180 may have important implications for cell migration associated with development and cell metastasis. The DOCK180 homologues, mbc and ced-5, isolated from Drosophila melanogaster and Caenorhabditis elegans , respectively, play a role in cell motility associated with the development of these organisms . In contrast, CAS/Crk coupling may contribute to the migratory/invasive behavior of tumor cells through its ability to couple to the Rac and PI3 kinase signaling pathway . Indeed, carcinoma cells that showed increased invasive and metastatic potential in vivo were found to have increased CAS/Crk complexes compared with non-metastatic cells . While it is not yet clear how ERK is regulated in migratory cells, our findings indicate that CAS and Crk do not play a central role in this signaling cascade. Recent evidence suggests that several signals exist to regulate ERK activity independent of CAS and Crk . It is known that Src can phosphorylate FAK at tyrosine 925 leading to a Grb2/SOS association and direct ERK activation . Furthermore, the FAK-related tyrosine kinase Pyk2 directly couples integrin signals to ERK activation independent of CAS/Crk coupling . Protein kinase C and Grb2 binding to Shc provide additional pathways capable of regulating ERK activity in response to integrin events . Alternatively, CAS-dependent mechanisms may exist to link integrin and cytokine receptors to the RAS/ERK pathway. For example, Nck couples to SOS as well as tyrosine phosphorylated CAS. This could serve as an alternate pathway to facilitate a low level of ERK activity in some cells . However, it is not yet known if formation of a CAS/Nck complex is necessary for ERK activation or cell migration. Assembly of an actin-myosin motor unit is critical for cell-mediated contraction of the ECM as well as cell movement, suggesting that these processes may be related. In fact, contraction of a collagen matrix involves a rapid smooth muscle–like contraction that is associated with increased ERK activity and myosin light chain phosphorylation . Migratory cells also assemble actin-myosin motors and exert force on the ECM . This is thought to generate the force necessary for the rapid retraction of the tail region that is known to occur in migratory cells. Previous work has shown that ERK can directly phosphorylate and, thereby, activate MLCK leading to MLC phosphorylation . In this report, we have extended these findings by showing that ERK activation can promote assembly of a functional actin-myosin motor unit capable of promoting cell contraction. Based on these findings, we propose that during cell migration ERK facilitates MLCK activity and MLC phosphorylation leading to the assembly of actin-myosin motors, an event necessary for cell contraction, but not membrane ruffling. On the other hand, CAS/Crk coupling independently regulates Rac activity and membrane ruffling in migratory cells. It is likely that additional signals operate to control cytoskeletal changes involved in cell movement. In fact, Rho modulates cell migration through its ability to inactivate myosin phosphatase leading to increased myosin light chain phosphorylation and cell contractility . Furthermore, v-Crk can regulate Rho activity, suggesting that in some cells Crk may be able to facilitate myosin contractility . p21-activated kinase (Pak1) also regulates MLC phosphorylation and cell motility in fibroblasts , and Ras/ERK regulates integrin affinity and modulates adhesive contacts with the ECM, which is important for cell migration . Our findings that assembly of a CAS/Crk complex and Rac activation are necessary for membrane ruffling, whereas ERK activity facilitates actin-myosin contraction, indicate that these signals regulate specific components of the migration machinery. These findings provide molecular insight as to how cellular recognition of growth factors and adhesive proteins regulate the process of cell movement during development, wound healing, and inflammation, as well as tumor cell dissemination. | Study | biomedical | en | 0.999996 |
10477764 | A mouse brain cDNA library (Stratagene) was screened with a probe that was derived from the rat l-afadin cDNA . 32 positive clones were subcloned into the pBluescript II vector and sequenced. A cDNA fragment containing the NH 2 -terminal open reading frame of the mouse afadin cDNA was used to isolate genomic clones from a 129SVJ mouse genomic DNA library (Stratagene). Overlapping genomic clones were obtained and mapped with respect to the mouse afadin cDNA sequence. A SacI-SphI genomic fragment (4.6 kb) 5′ to the afadin exon 2 encoding amino acids (aa) 36–100 was blunt-ended and inserted into the SmaI site of pBluescript neo/DT-A, which contained neomycin-resistance and diphtheria toxin A genes under the control of the MC1 promoter . The XbaI genomic fragment (5.3 kb) 3′ to exon 2 was then inserted into the EcoRV site of pBluescript neo/DT-A containing the 5′ genomic fragment. The ∼1.0 kb SphI-XbaI fragment was targeted for disruption and replaced by the neomycin-resistant gene cassette . This fragment began at the SphI site within exon 2 and ended in the following intron, and contained the coding sequence of aa 85–100. In the targeting vector, a stop codon resided 36 bp 3′ from the encoded aa 84. For Southern blot analysis, a 0.9-kb SacI-HindIII fragment and a 1.0-kb XbaI fragment were used as 5′ and 3′ probes, respectively. 129/Sv RW4 ES cells (Genome Systems Inc.) were cultured on STO feeder cells in high-glucose DME supplemented with 20% FCS, 0.1 mM 2-mercaptoethanol (Sigma Chemical Co.), 1,000 U/ml leukemia inhibitory factor (Amrad Co.), 0.1 mM nonessential aa (GIBCO BRL), 3 mM adenosine, 3 mM cytosine, 3 mM guanosine, 3 mM uridine, and 1 mM thymidine (Sigma Chemical Co.) . The targeting vector (50 μg) was linearized by NotI digestion and electroporated into ES cells using an Electro Cell Manipulator 600 (BTX) set at 270 V and 500 μF . ES cells were plated onto G418-resistant STO feeder cells in normal growth medium for 48 h, followed by selection with 175 μg/ml G418. G418-resistant STO feeder cells were prepared as described . After 7–10 d, G418-resistant colonies were picked up and their DNAs were isolated for Southern blot analysis . The colonies were screened by cleaving genomic DNA (15 μg) with HindIII and probing the Southern blot with the 5′ probe . The HindIII-digested genomic DNA was also similarly analyzed with the 3′ probe. Among the 120 G418-resistant ES clones, 14 clones underwent homologous recombination, which was confirmed by Southern blot analysis. Three different ES clones with the targeted mutation of the afadin gene were used to generate chimeric mice by injection of C57BL/6 blastocysts with 10–20 ES cells . The blastocysts were implanted into pseudopregnant MCH foster mothers. Chimeric mice were mated with BDF1 mice. Offsprings with agouti coat color were tested for the presence of the targeted afadin allele by Southern blot analysis. Heterozygous mice were interbred, and the resulting mice were genotyped. Genomic DNA from ES cells was prepared as described and analyzed by Southern blot analysis as described above. The mice were genotyped by Southern blot analysis and/or PCR. Two sets of primers were used in the PCR reactions. One set of primers corresponded to the neomycin-resistance gene: 5′-GGGCGCCCGGTTCTTTTTGTC-3′ and 5′-GCCATGATGGATACTTTCTCG-3′. The other set of primers corresponded to exon 2 of the afadin gene: 5′-TTCTAGGATTTGGAGTTTCAT-3′ and 5′-GGTCAGGACACAGTCTTCACT-3′ . Tail or yolk sac DNA was used for the PCR reactions . In the targeting vector for generation of afadin +/− ES cells, the G418-resistance gene was replaced by the puromycin-resistance gene . The resulting targeting vector was linearized by NotI digestion and electroporated into afadin +/− ES cells as described above. ES cells were then subjected to selection with 1.0 μg/ml puromycin (Sigma Chemical Co.) and analyzed by Southern blotting as described above. Among the 120 puromycin-resistant ES clones, 9 clones underwent homologous recombination, which was confirmed by Southern blot analysis. They were further analyzed by reverse transcription PCR using one set of primers corresponding to aa 187–316 of mouse afadin: 5′-CTCAAGGGGATGACAGTGAG-3′ and 5′-TCCTTAGCACCTCTCTCATC-3′. ES cells (6 × 10 6 ) were cultured without feeder cells on gelatin-coated 10-cm dishes for 3 d in the normal growth medium described above. Embryoid body (EB) formation was initiated by hanging drops of 1,000 cells in 20 μl of DME-supplemented 10% FCS in the absence of leukemia inhibitory factor . After 2 d, formed EBs were transferred to 10-cm bacteriological dishes and allowed to grow in suspension culture in DME supplemented with 10% FCS. The media were changed every other day. Rabbit polyclonal and mouse monoclonal anti–l-afadin antibodies were prepared as described . A rabbit polyclonal antibody recognizing both l- and s-afadins (rat, aa 577–592) was prepared as described . A mouse mAb recognizing both l- and s-afadins was purchased from Transduction Laboratories. Mouse anti–ZO-1 and antioccludin mAbs were kindly supplied by Drs. Sh. Tsukita, M. Itoh, and M. Furuse (Kyoto University, Kyoto, Japan). Mouse and rat (ECCD2) anti–E-cadherin mAbs were purchased from Transduction Laboratories and Takara Shuzo, Co., Ltd., respectively. Mouse antivinculin and anti–β-catenin mAbs were purchased from Sigma Chemical Co. and Zymed Laboratories Inc., respectively. Rat anti-PDGF receptor α (PDGFRα) and anti-fetal liver kinase 1 (Flk1) mAbs were prepared as described . Embryos dissected from their deciduae were fixed with 2% paraformaldehyde in PBS for 1 h, followed by extensive washing with PBS. The embryos were then suspended in 20% sucrose for 4 h, replaced with a solution (1:1 of 20% sucrose/OCT compound; Sakura Finetechnical Co., Ltd.), frozen in OCT compound, and sectioned on a cryostat at a thickness of 10 μm. Sections were mounted on glass slides, air-dried, and blocked in PSS (5% skim milk and 0.005% saponin in 0.1 M phosphate buffer, pH 7.5). The samples were then incubated with primary antibodies in PSS at 4°C overnight. They were washed with PSS and then incubated with secondary antibodies in PSS at 4°C for 2 h. After being washed with PSS, the samples were embedded and viewed with a confocal imaging system . EBs were also similarly fixed, frozen in OCT compound, and sectioned. Alternatively, EBs were directly frozen in OCT compound and sectioned on the cryostat. Sections were mounted on glass slides, air-dried, fixed with 95% ethanol at 4°C for 30 min, and then fixed with 100% acetone at room temperature for 1 min. They were processed as described above. Whole-mount immunohistostaining was performed as described with slight modifications. In brief, embryos dissected from their deciduae were washed with 0.1 M phosphate buffer and fixed with 2% paraformaldehyde in PBS at 4°C for 2 h to overnight according to the size of samples. The fixed samples were then bleached in a solution (methanol/30% H 2 O 2 of 4:1). For staining, the rehydrated samples were first blocked in PBSMT (1% skim milk and 0.2% Triton X-100 in PBS), incubated with the anti–l-afadin polyclonal antibody in PBSMT at 4°C overnight, and washed extensively with PBSMT. The sample was then incubated with an HRP-conjugated antibody at 4°C overnight. After extensive washing, the samples were soaked in a solution of metal-enhanced DAB substrate kit (Pierce). The enzymatic reaction was allowed to proceed until the desired color intensity was reached, and the samples were washed with PBST. Staining with hematoxylin and eosin was performed as described . In brief, embryos dissected their deciduae and EBs were fixed with 2% paraformaldehyde in PBS at 4°C overnight, washed with PBS at 4°C overnight, dehydrated in graded alcohols, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin. Whole-mount in situ hybridization with the antisense RNA of Brachyury ( T ) as a probe was performed as described with slight modifications. In brief, the RNA probe was labeled with digoxigenin-UTP by RNA in vitro transcription according to the manufacturer's protocols (Boehringer Mannheim). The cDNA of T was kindly supplied by Dr. S. Takada (Kyoto University, Kyoto, Japan). The samples were incubated with an antidigoxigenin antibody conjugated to alkaline phosphatase, and the color was developed in NBT/BCIP solution (Boehringer Mannheim). The samples were frozen in OCT compound, sectioned as described above, and counterstained with neutral red. Protein concentrations were determined with BSA as a reference protein . SDS-PAGE was done as described . We first examined expression and localization of l-afadin during mouse early embryogenesis. At embryonic day 6.5 (E6.5), embryos developed to egg cylinders containing embryonic and extraembryonic regions and proamniotic cavities. The embryonic ectoderm was composed of high columnar epithelial cells surrounded by the visceral endoderm. Yolk sac and ectoplacental cone were clearly observed in this stage. Immunofluorescence microscopy of E6.5 embryos revealed that l-afadin was localized at the most apical regions of cell–cell adhesion sites, called the junctional complex regions, of the entire embryonic ectoderm, whereas the signals of F-actin were observed along the entire cell surface . l-Afadin was hardly detected in the extraembryonic regions such as the visceral endoderm. At E7.0, embryos underwent primitive streak formation and mesoderm generation. Gastrulation began by the recruitment of embryonic ectodermal cells to the primitive streak, followed by exfoliation of cells from the primitive streak. Whole-mount immunohistochemistry revealed marked expression of l-afadin in the primitive streak and the migrating paraxial mesoderm . At E7.5, l-afadin was highly concentrated at the junctional complex regions in the primitive streak region (neuroepithelium) and the neural fold/groove region, but it was hardly detected in other areas of the ectoderm . It remains to be clarified whether l-afadin is downregulated or whether the proportion of ectodermal cells with low expression of l-afadin increases. By E8.5, normal embryos completed gastrulation and began organogenesis. The primitive streak regressed and newly organized tissues developed. High expression of l-afadin was detected in the tail bud, somites, and the paraxial mesoderm, which is being reorganized to form somites, neural tube, and intraembryonic coelomic cavity/pericardio-peritoneal canal that gives rise to pleura and pericardium . l-Afadin was highly concentrated at the junctional complex regions in neural tube, somites, and pericardio-peritoneal canal . These results indicate that l-afadin is highly expressed in a restricted set of epithelial structures and highly concentrated at their junctional complex regions. To determine the function of l-afadin in these epithelial structures, the mouse afadin gene was disrupted by homologous recombination. A targeting vector was designed so as to delete the exon 2 . The linearized targeting vector was introduced into ES cells and subjected to selection using G418. To screen for homologous recombination events, genomic DNAs from G418-resistant clones were subjected to Southern blot analysis with a 5′ probe. The wild-type afadin allele displayed a 13.9-kb band on Southern blotting of HindIII-digested DNA, whereas the disrupted locus showed a 5.0-kb band . Corrected targeting was confirmed by Southern blot analysis with a 3′ probe. The wild-type afadin allele displayed a 13.9-kb band, whereas the disrupted locus showed a 7.8-kb band. Three different ES clones (A46, A59, and A97) with the targeted allele were separately injected into host blastocysts, and the blastocysts were transferred to the uteri of pseudopregnant female mice. Germline transmission of the targeted allele was achieved with all ES clones. Inheritance of the targeted allele was determined by Southern blot analysis of the genomic DNA isolated from tail biopsies . The heterozygous (afadin +/− ) mice appeared normal compared with the wild-type littermates. The afadin +/− mice were intercrossed and genotypes of the progeny were determined by Southern blot or PCR analysis using tail DNAs . No homozygous (afadin −/− ) mice were detected among 82 progeny analyzed. These results indicate that deficiency of afadin causes embryonic lethality. Embryos were isolated at various stages of gestation and their genotypes were determined ( Table ). Distribution of each genotype examined at E7.5–E9.5 followed the Mendelian law, whereas no homozygous embryos were detected from E10.5. At E6.5, gross morphological analysis did not distinguish the homozygous embryos from the wild-type and heterozygous littermates (data not shown), indicating that implantation and egg cylinder formation occur normally in the absence of afadin. To examine the presence of residual maternal afadin, which may affect implantation and egg cylinder formation in the homozygous embryos, preimplantation embryos at E3.5 (early blastocysts) were cultured and their levels of l-afadin were determined by immunofluorescence microscopy. Of 20 embryos examined, 16 showed weak but significant staining, whereas the remaining did not show any signal (data not shown). It is most likely that the latter embryos are homozygous and that there is no residual maternal afadin in the homozygous embryos. In contrast to embryos at E6.5, it was easy to distinguish the homozygous embryos from the wild-type and heterozygous littermates in embryos at E7.5–E9.5. Compared with wild-type embryos, the architecture of the homozygous embryos was apparently distorted and reduced in size . Of note, however, is that their extraembryonic regions, including ectoplacental cone and yolk sac, developed normally, indicating that the anomalies are basically restricted to embryos proper. Whereas the anterior–posterior distinction could easily be made by gross appearance, and some vascular system and blood were detectable, the homozygous embryos were always flat and short, and no landmark tissues such as heart developed at this stage. The anomalies specific for these embryos proper in afadin −/− embryos are consistent with the expression patterns of l-afadin. In wild-type embryos at E7.5, delamination of mesodermal cells from the primitive streak was undertaken in a strictly polarized manner, thereby recruiting mesodermal cells into the space between the embryonic ectoderm and the visceral endoderm . Of importance is that the integrity of epithelial structures of the ectoderm, including the primitive streak and neural groove, is maintained even under the stimulation to induce delamination. In afadin −/− embryos at E7.5, generation of mesodermal cells at this space did occur, indicating that mesoderm induction itself occurs normally . However, these mutant embryos had the wider space between the ectoderm and the endoderm, where more mesodermal cells were observed compared with wild-type embryos. Ectodermal cells of afadin −/− embryos appeared flat or cuboid, and their polarized epithelial structures appeared to be entirely impaired . At the region corresponding to neural fold/groove (from the anterior region to the distal region), the ectoderm became multilayered and appeared as a cell mass . At the posterior region corresponding to the primitive streak, the ectoderm was invaginated toward the amniotic side . The invaginated ectoderm often ran along with the ectoderm of the lateral region, resulting in the appearance of two layers. The space between the two layers corresponded to the amniotic cavity, which was compressed to an inverted U-shape. Cells were detected at the space surrounded by the invaginated ectoderm. Furthermore, in afadin −/− embryos, amniotic and exocoelomic cavities did not develop normally and formation of allantois was not observed . In contrast to the severe defects of the embryonic ectoderm, a single-layered epithelial structure in the endoderm remained intact . Hence, all of these phenotypes may be an outcome of abnormal progression of amniotic and chorionic membrane formation from the most proximal region of the primitive streak. In wild-type embryos at E8.5, tissues with epithelial structures, such as neural groove, intraembryonic coelomic cavity, and somites, were clearly evident . In afadin −/− embryos, consistent with histological findings showing generation of mesodermal cells, gross appearance showed that somite-like blocks and some vascular structures were detected (data not shown), although no epithelial structure of somites was established . Formation of neither neural tube nor heart was observed. To further dissect the developmental defects of afadin −/− embryos, we next investigated expression of E-cadherin and mesoderm markers, including T , PDGFRα, and Flk1 at E7.5. Consistent with an earlier observation , T was highly expressed in the primitive streak and its nascent mesoderm in wild-type embryos . In afadin −/− embryos, the T -positive area appeared to be divided into two portions that corresponded to the most posterior regions of the two-layered ectoderm . At the primitive streak stage, E-cadherin is expressed in the entire embryonic ectoderm . PDGFRα is expressed in the paraxial mesoderm , and Flk1 is expressed in the proximal lateral mesoderm and the extraembryonic mesoderm . After completion of exfoliation from the primitive streak, E-cadherin was downregulated, and PDGFRα and Flk1 were expressed in the mesodermal cells . In afadin −/− embryos, E-cadherin–negative and PDGFRα-positive cells were detected at the space between the ectoderm and the endoderm . These cells corresponded to the mesodermal cells of the paraxial region. At the proximal region of the primitive streak, E-cadherin–positive cells were jammed at the space between the ectoderm and the endoderm . The staining for E-cadherin clearly demonstrated that the E-cadherin–positive ectoderm was invaginated from the posterior region toward the amniotic side . The most posterior region of the two-layered ectoderm corresponded to the area positive for T . E-cadherin–negative and PDGFRα-positive cells were detected at the space surrounded by the invaginated ectoderm. These cells appeared to migrate from the primitive streak. At the regions corresponding to neural fold/groove (from the anterior region to the distal region), the multilayered cells were E-cadherin–positive . At the distal region, some cells in the cell mass expressed not only E-cadherin but also PDGFRα . This cell mass was surrounded by a layer of the ectodermal cells that were E-cadherin–positive and PDGFRα-negative. Similar observations were obtained with the double staining for E-cadherin and Flk1, except that Flk1 was not expressed in the cell mass . These observations strongly suggest that the major histological basis of the developmental defects of afadin −/− mice is disorganization of the embryonic ectoderm, and that distorted placement of various cell lineages is the secondary outcome of this disorganization. To investigate whether or not the apparatus for maintaining cell polarity is disturbed in the embryonic ectoderm, we examined the localization of E-cadherin and ZO-1 in afadin −/− embryos at E7.5. At the primitive streak region (neuroepithelium) and the neural fold/groove region in wild-type embryos, E-cadherin was concentrated at the junctional complex regions, although its signals were detected along the lateral membrane . ZO-1 was exclusively localized at the junctional complex regions of the embryonic ectoderm . At the primitive streak region in afadin −/− embryos, E-cadherin hardly showed such an organized concentration as observed in wild-type embryos . In the cell mass from the anterior region to the distal region, E-cadherin was distributed diffusely over the entire cell surface . The localization of ZO-1 was also disturbed in afadin −/− embryos. At the primitive streak region, ZO-1 was mainly localized at the most apical regions, but the signals were also detected in the basal regions . In the cell mass, ZO-1 showed dotty signals in a random manner . These results indicate that deficiency of afadin induces disorganization of cell–cell junctions of the embryonic ectoderm. The results of afadin −/− embryos indicate the following: (a) afadin is not essential for processes earlier than egg cylinder formation; (b) afadin is not required for anterior–posterior body plan placement in egg cylinders; and (c) afadin is expressed specifically in the embryonic ectoderm, particularly the primitive streak region (neuroepithelium) and the neural fold/groove region, and plays an essential role in the junctional organization in the ectoderm during gastrulation. To determine whether or not the defects in afadin −/− embryos can be reproduced in a simpler model system, we took advantage of EB formation of ES cells where development of two-layered epithelial structures and subsequent mesoderm induction from the inner layer are shown to be reproduced in vitro . For this purpose, we first established an afadin −/− ES cell line by introducing another targeting vector harboring puromycin-resistant gene . This targeting vector was introduced into afadin +/− ES cells (clone A46). ES cells were subjected to selection using puromycin. Southern blot analysis showed that three clones (B3, B8, and B103) resistant to puromycin underwent gene conversion, resulting in disruption of both alleles . Western blot analysis using the anti–l-afadin mAb and the mAb recognizing both l- and s-afadins revealed the loss of afadin in afadin −/− ES cells and EBs . Similar results were obtained with the polyclonal antibody recognizing both l- and s-afadins (rat, aa 577–592) (data not shown). Western blot analysis also revealed that l-afadin was a major expressed variant in wild-type ES cells and EBs, and that s-afadin was hardly detected . Reverse transcription PCR analysis using the primer set corresponding to aa 188–316 of mouse afadin showed the loss of the afadin mRNA in afadin −/− ES cells (data not shown). The three independent clones of afadin −/− ES cells showed the same growth rate with undifferentiated morphology as wild-type ES cells did (data not shown). Since these clones showed the same phenotypes as far as examined, the data obtained from clone B3 were represented below. When wild-type ES cells were subjected to suspension culture, cells aggregated to form EBs, some of which eventually develop to a two-layered cystic structure consisting of the outer endodermal layer and the inner high columnar ectodermal layer, although EBs with more complex structures with a large yolk sac–like cyst were often observed . During earlier stages of EB formation, afadin −/− ES cells showed no significant difference (data not shown). Moreover, EBs with a large yolk sac–like cyst were often formed, suggesting that the endodermal components function normally . On the other hand, in EBs with an amniotic cavity–like cyst, many necrotic cells were found in the cavity, while leaving the outer layer intact . Generation of mesodermal cells at the space between the ectodermal and endodermal layers was observed, but the well-organized ectodermal layer did not develop. These results indicate that the ectoderm-specific defects of afadin −/− embryos may be reproduced in the in vitro EB model. Moreover, the presence of cellular components in the EB cavity may reflect the defects in the polarity of the ectodermal layer. To investigate whether deficiency of afadin affects expression of other components of cell–cell junctions, we compared expression levels of E-cadherin, β-catenin, vinculin, ZO-1, and occludin, in wild-type and afadin −/− ES cells during EB formation. We could not detect any significant difference in their expression levels (data not shown). EBs with an amniotic cavity–like cyst were selected and subjected to immunofluorescence microscopy using antibodies against various components of cell–cell junctions. Consistent with the results on embryos, cells in the ectodermal layer, but not in the outer endodermal layer of wild-type EBs expressed l-afadin . However, unlike embryos with the high expression along the anterior–posterior axis, l-afadin was expressed ubiquitously in the ectodermal layer. l-Afadin was concentrated at the junctional complex regions where F-actin was concentrated, although diffuse distribution of F-actin along the entire cell surface was also observed . In afadin −/− EBs, no afadin signal was observed , indicating that the function of the gene is completely disrupted. Although no abnormality was found in the outer endodermal layer, formation of the organized junctional complex was severely inhibited in the ectodermal layer . F-actin showed diffuse distribution along the entire cell surface without any concentration in the ectodermal layer . Compared with the organized concentration of E-cadherin at the junctional complex regions in wild-type EBs , E-cadherin was diffusely distributed over the ectodermal cell surface . Likewise, compared with the organized localization of ZO-1 in wild-type EBs , it was displayed as dotty signals in a random manner in the cell mass . The first implication of this study is that l-afadin is expressed only in a restricted set of epithelial structures during early embryogenesis. This goes against the notion that l-afadin is a constitutive component of cadherin-based cell–cell AJs. In fact, high expression of l-afadin is observed in such regions as the primitive streak region (neuroepithelium), neural fold/groove, and somites, where dynamic tissue rearrangements are ready to occur. On the other hand, only a low level if any of l-afadin expression is detected in other epithelial structures such as the visceral endoderm, which may expand but take a simpler differentiation course. The essentially similar expression patterns are reproduced in an in vitro model system of EB formation, suggesting that its expression is coordinated to each cell specification program. Further studies are required to determine whether l-afadin is expressed inducibly in the regions where new tissues develop from epithelia through dynamic cell rearrangement. In agreement with the expression patterns of l-afadin during gastrulation, afadin −/− embryos display major defects in the embryonic ectoderm. This indicates that no redundancy exists in the function of afadin during gastrulation. Delamination of mesodermal cells does occur. As the cells, which are observed at the space surrounded by the invaginated ectoderm as well as at the space between the ectoderm and the endoderm, are E-cadherin–negative and PDGFRα-positive, mesoderm differentiation can occur normally if placed in the correct environment. This is consistent with the fact that mesoderm-derived structures develop to some extent also in afadin −/− embryos. Thus, mesoderm differentiation per se is not affected by this mutation. The most remarkable phenotype is the invagination of the ectoderm to the amniotic side, which never occurs in normal embryogenesis. This invagination divides the primitive streak into two portions as examined by the staining for T . Furthermore, this mutation produces the cell mass in the region corresponding to neural fold/groove. Some cells in the cell mass express both E-cadherin and PDGFRα, but not Flk1. As such double positive cells are found in stripes of ectodermal cells at the edge of neural groove of prorhombomeres , it is likely that the cell mass represents this subset of the ectoderm. While it is attractive to think that afadin also plays a direct role in organization of epithelial structures such as neural tube, somites, and intraembryonic coelomic cavity/pericardio-peritoneal canal where a high level of l-afadin expression is detected, disorganization of the ectoderm in afadin −/− embryos does not allow investigation of these processes. The design of the targeting vector used in this study raises the possibility that the truncated afadin polypeptide (aa 1–84) is produced both in afadin −/− mice and ES cells and in afadin +/− mice and ES cells. It can not completely be neglected that the truncated afadin polypeptide may contribute to the phenotypes of afadin −/− mice and EBs shown here. However, this possibility is unlikely because any known, functional domain or motif is not included in this polypeptide, and afadin +/− mice and ES cells develop normally. An issue here is closely related to the problem of cell biology, i.e., how epithelial cells maintain the structural integrity with polarity. Our results clearly indicate that structural integrity of epithelia such as the trophoectoderm and the visceral endoderm can be maintained in the absence of afadin. This is more clearly addressed in the development of afadin −/− EBs. Formation of yolk sac–like large cysts in some of afadin −/− EBs indicates that endodermal cells differentiate, proliferate, and maintain the integrity of epithelial structures, which are essential for transporting and sealing fluid in the cyst. Thus, at least during early embryogenesis, afadin plays a key role in actively rearranging epithelia of the embryonic ectoderm, such as the primitive streak and neural fold/groove. As the mesoderm can be produced in afadin −/− embryos, gastrulation itself appears to occur. Instead, we have highlighted processes during formation of the primitive streak and neural fold/groove. Under such a situation, active reorganization of epithelial structures is induced, but the structural integrity of epithelia should be maintained and its basic structures such as polarity have to be preserved, otherwise newly generated tissues cannot be organized. Afadin is indeed a key molecule for these processes. Afadin is a linker of nectin, an Ig-like CAM, with the actin cytoskeleton . Ig-like CAMs are implicated in tissue morphogenesis, particularly cell migration . The result, that epithelial organization as defined by a correct distribution of ZO-1 in the ectoderm is disrupted completely in the absence of afadin, suggests that afadin regulates the functional state of nectin, thereby contributing to the basic organization of cell–cell junctions of the ectoderm. Although further studies are necessary to establish the role of afadin in these processes, this study has highlighted not only the role of afadin but also the importance of epithelial organization of the embryonic ectoderm during gastrulation. Thus, we expect that further studies on the role of afadin and its associated molecules will open a new avenue bridging the border of cell biology and developmental biology. | Study | biomedical | en | 0.999996 |
10477765 | Expression vectors encoding rat MuSK, rat trkC, and a rat MuSK-rat trkC chimera were described previously . In all three vectors, the cDNA is expressed under the control of the Rous Sarcoma virus long terminal repeat. In the chimera, the extracellular domain of MuSK except for the last three amino acids before the predicted transmembrane domain (amino acids 1–492) is followed by 32 amino acids of the extracellular domain plus the complete transmembrane and cytoplasmic domains of rat trkC. In the trkC and MuSK-trkC constructs, a myc tag consisting of three tandem copies of a 12–amino acid myc epitope was fused in-frame at the carboxy-terminal of the coding region. MuSK and the MuSK-trkC chimera are called construct 1 and construct 1T, respectively. Constructs 2–6 are identical to construct 1, and constructs 2T, 5T, 6T, 14T, and 15T are identical to construct 1T except that in each case a fragment of MuSK was deleted. Deletions, constructed by PCR, were as follows: 2 and 2T, amino acids 25–112, inclusive; 3, amino acids 115–204; 4, amino acids 205–299; 5 and 5T, amino acids 299–396; 6 and 6T, amino acids 397–484; 14T, 25–204; and 15T, amino acids 25–299. Constructs 7–13 were derived from a vector in which the rat MuSK cDNA was expressed under the control of late adenovirus promoter, and a Srf I site was engineered in-frame just upstream of the transmembrane domain. Deletions were constructed by PCR, removing successively larger fragments of the ectodomain as follows: 7, amino acids 447–492; 8, amino acids 398–492; 9, amino acids 283–492; 10, 233–492; 11, amino acids 191–492; 12, amino acids 142–492; and 13, amino acids 99–492. In constructs 16–25, the ectodomain of MuSK was intact, but portions of the cytoplasmic domain were mutated or deleted. The mutations, generated by mutagenesis, were as follows: 16, Lys608 was replaced by Ala; 17, Tyr553 was replaced by Phe; 18, Asp545-Arg546-Leu547-His548 and Pro549 were replaced by Leu-Tyr-Leu-Ser-Ser; 19, amino acids 545–549 were replaced by Ile-Pro-Ile-Leu-Glu; 24, Tyr831 was replaced by Phe; and 25, Val866 Gly867 and Val868 were deleted. The deletions, constructed by PCR, were as follows: construct 20, amino acids 515–594, inclusive; 21, amino acids 655–868; 22, amino acids 721–868; and construct 23, amino acids 799–868. The coding sequence of a tyrosine kinase from Torpedo electric organ was inserted in place of rat MuSK in construct 7. In all constructs, ligation junctions were sequenced to confirm correct insertion and maintenance of the reading frame. MuSK +/ − mice were bred with transgenic mice bearing a temperature-sensitive SV40 T antigen under the control of an interferon-inducible promoter . MuSK +/ −, transgene-positive offspring were backcrossed to MuSK +/ − mice and a myogenic line was established from hindlimb muscles of a MuSK −/ − transgene-positive embryonic day 18 pup using the methods detailed by Donoghue et al. 1992 . Cells were cultured as myoblasts at 33°C in DME containing 10% heat-inactivated FCS, glutamine, penicillin, streptomycin, and 20 U/ml γ-interferon (R&D Systems, Inc.). To induce formation of myotubes, cultures were transferred to 37°C and the medium was replaced with DME containing 2% horse serum, glutamine, and antibiotics, but no FCS or interferon. Cells were transfected as myoblasts with Fugene6 reagent (Roche Molecular Biochemical), used according to the manufacturer's instructions. Fusion was initiated 1 d after transfection and cultures were harvested 5–7 d later. Between 5 and 20% of myotubes expressed the exogenous protein, as assessed by immunostaining. For immunoblotting analysis, the cells were cultured on 10-cm plates. In some experiments, a recombinant carboxy-terminal fragment of agrin was added to the cultures for 10 min before harvesting. For immunohistochemistry, cells were cultured on 13-mm-diam glass coverslips. Where specified, the recombinant agrin fragment was added to the cultures for 18 h before harvesting. The quail fibroblast cell line QT-6 was obtained from the American Type Culture Collection and cultured in DME containing 10% FCS, 1% DMSO, penicillin, and streptomycin. Cells were cultured on glass coverslips and transfected using the calcium phosphate method described by Phillips et al. 1991b . Cultured cells were lysed and solubilized in NP-40, the extracts were subjected to immunoprecipitation with antibodies to MuSK and the resulting precipitates were immunoblotted with antibodies to phosphotyrosine (4G10; Upstate Biotechnology Inc.). After detection of the phosphorylated species, the immunoblots were stripped and reprobed with antiserum to the MuSK cytoplasmic domain (see below) to ascertain total MuSK levels. MuSK −/− myotubes were incubated live for 1 h with rhodamine-α-bungarotoxin (rBTX; Molecular Probes, Inc.), washed, and fixed for 20 min at room temperature in 2% paraformaldehyde in PBS. In some experiments, cells were permeabilized by incubation for 10 min in 1% Triton X-100. Nonspecific binding sites were blocked by overnight incubation with 10% FCS at 4°C and then cultures were incubated sequentially with antibodies to MuSK (for 2 h) and fluorescein-conjugated second antibody (for 1 h). AChR clusters were counted with rhodamine illumination using a 40× oil objective. 10 fields were chosen at random on each coverslip, all clusters >3 μm in diameter were counted, and this number was divided by the number of myotubes that crossed the field. QT-6 cells were rinsed in PBS, fixed in 1% paraformaldehyde, 100 mM l -lysine, and 10 mM sodium metaperiodate, rinsed again, and permeabilized with 1% Triton X-100. They were incubated with a mixture of mouse anti-rapsyn and rabbit anti-MuSK, washed, and reincubated with a mixture of fluorescein- and rhodamine-conjugated second antibodies. Rabbit antisera were generated to the cytoplasmic domain and to the ectodomain of rat MuSK, using standard techniques . Antiserum to a soluble fusion between the ectodomain of mouse MuSK and immunoglobulin Fc segment was a gift of M. Ruegg (Basel, Switzerland). When myoblasts fuse to form myotubes, AChR subunit genes are activated and AChRs synthesized. Most of the receptors are diffusely distributed at low density on the myotube surface, but some form high density aggregates, which are readily visualized with the selective ligand rBTX . Addition of a recombinant carboxy-terminal fragment of agrin increased the number of such AChR clusters ∼10-fold . In contrast, myoblasts derived from MuSK −/ − mutant mice form myotubes that are unresponsive to agrin, even though they bear normal levels of diffuse AChRs . Likewise, myotubes of an immortalized myogenic cell line derived from MuSK −/ − muscle did not form AChR clusters, either spontaneously or in response to agrin . These results are consistent with the notion that MuSK is a critical component of the agrin receptor and that even spontaneous AChR clusters are dependent on MuSK. To establish an assay for MuSK function, we transfected MuSK −/ − myoblasts with an expression vector encoding rat MuSK, induced fusion, incubated the myotubes with or without agrin, then stained them with rBTX. MuSK-transfected myotubes formed a small number of clusters in the absence of exogenous agrin, and approximately eightfold more clusters in the presence of agrin . AChR clusters were present only on those myotubes that had been transfected and were expressing MuSK , and over 80% of MuSK-positive myotubes in the agrin-treated cultures bore AChR clusters (data not shown). Thus, spontaneous as well as agrin-induced AChR clustering requires MuSK. Because MuSK −/ − myotubes form AChR clusters in response to other, possibly nonphysiological, clustering agents , we believe that MuSK is not required as a structural component of AChR clusters. Instead, spontaneous clustering is likely to result from a low level of MuSK activation, either by an endogenous ligand or in the absence of ligand. Data that distinguish these alternatives are presented below. To test the specificity of the response to agrin, we introduced two other kinases into MuSK −/ − myotubes: rat trkC and a receptor tyrosine kinase isolated from Torpedo electric organ . TrkC, the receptor for neurotrophin 3 is homologous to MuSK in its cytoplasmic domain, but unrelated in its extracellular domain. In contrast, the Torpedo kinase is homologous to MuSK throughout its length and is selectively expressed in muscle; therefore, it is a plausible MuSK orthologue. Myotubes transfected with trkC formed no AChR clusters either spontaneously or in response to neurotrophin 3, whereas the Torpedo kinase endowed MuSK −/ − myotubes with the ability to form AChR clusters spontaneously and in response to agrin . This result, combined with the inactivity of trkC in this assay, supports the presumption that the Torpedo kinase is orthologous to MuSK; we call it Torpedo MuSK hereafter. Together, these results confirm a specific requirement of MuSK for agrin signaling. More important for the present study, the ability of MuSK to restore agrin sensitivity to MuSK −/ − cells provides an assay system to determine the relationship between MuSK's structure and its function. The extracellular segment of MuSK contains five distinct domains: four immunoglobulin-like domains and a cysteine rich region called a C6 box. All five domains are conserved in sequence and arrangement in rat, mouse, human, Xenopus , chicken, and Torpedo MuSK . To determine which portions of the MuSK ectodomain are required for its activation, we generated the mutants diagrammed in Fig. 2 a. In a first series (constructs 2–6), sequences were deleted that encoded either the first, second, or third immunoglobulin-like domain, the C6 box, or the fourth immunoglobulin-like domain. In a second series (constructs 7–13), nested deletions extended variable distances toward the amino terminus from a common site just upstream of the predicted transmembrane domain. Each mutant, as well as wild-type MuSK (construct 1), was transfected into MuSK −/ − myoblasts, which were fused, treated, and stained as described above. Four principal results were obtained from this series of experiments. First, all of the constructs restored the ability of MuSK −/ − myotubes to form spontaneous AChR clusters . Because the deletions span the entire ectodomain, we conclude that spontaneous clustering reflects ligand-independent activation of MuSK rather than activation by an endogenous ligand. Second, all of the mutants with deletions confined to the carboxy-terminal three fifths of the ectodomain were able to mediate agrin-induced AChR clustering . We did not rigorously control for differences in transfection efficiency, but conclude that differences between any of these mutants and wild-type MuSK were less than twofold. Thus, the third and fourth immunoglobulin domains and the C6 box are dispensable for agrin-mediated AChR clustering. Interestingly, Hesser et al. 1999 recently reported on the occurrence of a naturally occurring splice variant of MuSK that lacks the third immunoglobulin-like repeat, yet retains the ability to induce AChR clustering. Third, mutants with deletions in the amino-terminal two fifths of the ectodomain were markedly impaired in their ability to mediate agrin-induced AChR clustering . This inability did not result from failure of these mutants to reach the cell surface, because staining of nonpermeabilized cells with antibodies to MuSK showed clear immunoreactivity (see below). Thus, sequences in or near the first or second immunoglobulin-like domain interact, directly or indirectly, with agrin. Fourth, whereas mutants lacking sequences within the first immunoglobulin-like domain (constructs 2 and 13) were completely agrin-insensitive, agrin had limited ability to stimulate AChR clustering (∼2.5-fold above spontaneous levels) in myotubes transfected with mutants lacking the second immunoglobulin-like domain . The difference between constructs 2 and 13 on the one hand and constructs 3 and 12 on the other indicates that sequences in or near immunoglobulin-like domain 1 are sufficient to endow MuSK with agrin responsiveness. Previous studies have shown that agrin causes rapid phosphorylation of MuSK, and that this phosphorylation is required for MuSK to mediate agrin's effects . If this is so, MuSK ectodomain mutants able to mediate agrin-induced AChR clustering should exhibit agrin-dependent phosphorylation. To test this prediction, we assayed agrin-dependent phosphorylation of MuSK. Wild-type or mutant MuSK was precipitated from lysates of transfected MuSK −/ − cells with an antibody to an intracellular epitope, which was present in all mutants. Precipitated material was separated by gel electrophoresis, transferred to nitrocellulose, and probed with antiphosphotyrosine antibody. The blots were stripped and reprobed with anti-MuSK, to permit normalization to MuSK protein levels and to confirm the electrophoretic mobility of the mutants. As shown in Fig. 3 , MuSK was undetectable in untransfected MuSK −/ − cells, but readily detectable in transfected cells. Wild-type MuSK was phosphorylated at a low level in the absence of added agrin, and its phosphorylation was increased >10-fold 10 min after the addition of agrin to the cells (lanes 3 and 4). Tyrosine phosphorylation of construct 11 (which lacks immunoglobulin-like domains 3 and 4 and the C6 box yet remains responsive to agrin) was also greatly stimulated by agrin (lanes 5 and 6). In contrast, construct 13, which lacks most of the ectodomain and is agrin unresponsive, was highly phosphorylated in the absence of agrin and showed only slightly increased phosphorylation after treatment with agrin (lanes 7 and 8). This result raises the possibility that the ectodomain of MuSK keeps the cytoplasmic domain inactive in the absence of agrin; ligand binding would alter the conformation of the ectodomain to relieve the inhibition. MuSK and rapsyn cocluster when coexpressed in QT6 fibroblasts . Surprisingly, even though rapsyn is a cytoplasmic protein, it is the ectodomain of MuSK that is essential for this association: chimeras composed of the ectodomain of MuSK and the cytoplasmic domain of trkC cocluster with rapsyn in QT6 cells, whereas chimeras containing the ectodomain of trkC and the cytoplasmic domain of MuSK do not . Therefore, we hypothesized that RATL mediates the association. We asked whether the portions of MuSK required for association with rapsyn were distinguishable from those required for activation by agrin. For this purpose, we modified construct 2, which lacks the first immunoglobulin-like domain and is agrin unresponsive. In construct 2T , the ectodomain from construct 2 was fused to the transmembrane and cytoplasmic domains of trkC, to exclude any contribution of cytoplasmic MuSK sequences to its localization. We used trkC as a negative control and the chimera between wild-type MuSK and trkC (construct 1T) as a positive control. These constructs were transfected into QT-6 cells either alone or with an expression vector encoding rapsyn. 2 d later, cells were fixed, permeabilized, and doubly stained with antibodies specific for rapsyn and MuSK or trkC. Rapsyn formed small aggregates when transfected by itself, whereas construct 1T, construct 2T, and trkC were all diffusely distributed when introduced alone . Cotransfection of rapsyn and trkC had no effect on the distribution of either component, whereas cotransfection of rapsyn with constructs 1T or 2T led to nearly perfect colocalization of the two components in most cells . No differences were detected between constructs 1T and 2T in this assay. Thus, sequences required for activation by agrin are not required for colocalization with rapsyn. Based on these results, we generated and tested constructs in which additional sequences were deleted. Constructs 14T and 15T, in which immunoglobulin-like domains 1 and 2 or 1–3, respectively, were deleted behaved like constructs 1T and 2T in this assay: they were diffusely distributed when introduced on their own, but coclustered with rapsyn when both components were expressed . In contrast, constructs 5T and 6T, which lacked carboxy-terminal portions of the ectodomain (the C6 box and the fourth immunoglobulin-like domain, respectively), were diffusely distributed both in the absence and in the presence of rapsyn . For each construct, similar results were obtained when AChR subunits were cotransfected along with rapsyn. That is, constructs 1T, 2T, 14T, and 15T aggregated with rapsyn and AChRs, whereas 5T and 6T did not (data not shown). Thus, juxtamembranous portions of the MuSK ectodomain are required for its association with rapsyn, presumably via RATL. Together, the results in Fig. 2 and Fig. 4 show that sequences in the MuSK ectodomain required for colocalization with rapsyn are dispensable for activation by agrin. What role might this juxtamembranous region play in muscle cells? One obvious possibility is that it is required for the association of MuSK with AChR rich aggregates. To test this hypothesis, we transfected MuSK −/ − cells with MuSK mutants, and then stained nonpermeabilized cells with anti-MuSK and rBTX to determine whether the MuSK constructs reached the cell surface and whether they colocalized with AChRs. As shown in Fig. 5 , constructs that contained the fourth immunoglobulin-like domain of the MuSK ectodomain (constructs 1, 2, 4, and 5) were concentrated at AChR clusters. In contrast, the two constructs that lacked sequences within the fourth immunoglobulin-like domain (constructs 6 and 7) were present on the myotube surface, but not concentrated at significantly higher levels in AChR rich than in AChR poor areas. Thus, the domain required for MuSK to associate with rapsyn in QT-6 cells is required for association with rapsyn–AChR clusters in myotubes. Two results from this series deserve further comment. First, cells transfected with construct 2 were unresponsive to agrin, but MuSK was concentrated at spontaneous clusters in these cells; likewise MuSK was present at both spontaneous and agrin-induced AChR clusters in cells transfected with constructs 1, 4, and 5. Thus, the association of MuSK with rapsyn in muscle cells does not require that MuSK be activated by agrin. Second, construct 5T, which lacked the C6 box, failed to colocalize with rapsyn and AChRs in QT-6 cells but construct 5 did do so in myotubes. We speculate that deletion of sequences adjacent to the fourth immunoglobulin-like domain prevented that domain from assuming an appropriate conformation in heterologous cells but not in muscle cells. How does agrin-induced phosphorylation of MuSK lead to postsynaptic differentiation? According to a widely accepted model , phosphorylation activates multiple signal transduction pathways that eventually combine to generate the biological response. The signal transduction components are recruited to the kinase by adaptor proteins that bind to conserved sequences. Therefore, one strategy for defining the pathways through which MuSK signals, is to identify critical sites in its cytoplasmic domain. As a first step, we tested a construct in which a critical lysine residue in the consensus ATP binding site was replaced with alanine . Such mutations in other receptor tyrosine kinases abolish tyrosine kinase activity , and we showed previously that this MuSK mutant inhibits AChR clustering when expressed in wild-type myotubes , suggesting that it had reduced activity. Therefore, if MuSK is acting as a receptor tyrosine kinase, construct 16 should be unable to mediate AChR clustering. Alternatively, if MuSK were only a substrate for other kinases, this mutation might not abolish activity. Here, we transfected construct 16 into MuSK −/ − cells, and then assayed for tyrosine phosphorylation of the mutant by immunoblotting and AChR clustering by staining with rBTX. Construct 16 was devoid of detectable phosphotyrosine and was completely unable to induce formation of AChR clusters, either spontaneously or in response to agrin. Inactivity did not reflect poor expression or misrouting because construct 16 was expressed at levels similar to those of wild-type MuSK and reached the cell-surface without impediment (data not shown). These results indicate that the kinase activity of MuSK is essential for its biological activity. Next, we tested the function of the cytoplasmic sequence NPMY, which is perfectly conserved among rat, mouse, human, Xenopus , and Torpedo MuSKs , and which corresponds to a consensus binding site (NPXY) for signaling proteins that contain a PTB domain . We changed the critical tyrosine residue to phenylalanine (construct 17), a mutation known to abolish binding of most PTB domain proteins. This mutation had no detectable effect on the expression level or the ability of MuSK to reach the cell surface . However, myotubes transfected with construct 17 formed neither spontaneous nor agrin-induced AChR clusters . Moreover, construct 17 was not detectably phosphorylated either spontaneously or after addition of agrin . These results suggest that PTB domain–containing proteins are required for activation of MuSK's kinase activity. Muscle fibers express at least two PTB domain proteins, insulin receptor substrate 1 (IRS-1) and shc . To determine whether either of these was activated by MuSK, we incubated wild-type myotubes with or without agrin, immunoprecipitated IRS-1 or shc, and probed immunoblots with antiphosphotyrosine. Neither protein was detectably phosphorylated in response to agrin treatment (data not shown). These results raised the possibility that MuSK associates with PTB proteins other than IRS-1 or shc. In support of this possibility, MuSK lacks residues upstream of NPXY that favor interaction with IRS-1 or shc. Although all PTB domains recognize the NPXY motif, residues amino-terminal of this tetrapeptide dictate binding specificity. A hydrophobic residue such as I or L is present five residues upstream of Y in sites that bind shc, and L is present eight residues upstream of Y in sites that bind IRS-1 . In rat, mouse, human, Xenopus , and Torpedo MuSKs, the fifth and eighth residues are H and D, respectively, so the extended sequence (DRLHPNPMY) would not be expected to bind either shc or IRS-1. To assess the importance of the sequence upstream of NPMY in MuSK (DRLHP), we altered it either to LYLSS (construct 18), which is found upstream of NPXY in the IRS-1 binding site of the insulin receptor, or to IPILE (construct 19), which is upstream of NPXY in the shc binding site of trkB and trkC. Both of these constructs mediated agrin-dependent AChR clustering, but at lower levels than wild-type MuSK . In addition, construct 18 was poorly phosphorylated in response to agrin . The fact that residues adjacent to the NPXY motif modulate MuSK activity supports the idea that PTB domain proteins are involved in MuSK signaling. However, the finding that optimizing the MuSK cytoplasmic domain to bind shc or IRS-1 decreased the ability of MuSK to induce AChR clustering suggests that wild-type MuSK interacts with a PTB domain protein that is neither shc nor IRS1. The essential ATP- and PTB domain–binding sites of MuSK are within the amino-terminal third of its cytoplasmic domain. To ask whether the carboxy-terminal portion of the cytoplasmic domain also contained essential sites, we deleted the last 214, 148, or 70 amino acids . All three of these constructs were completely inactive in the presence or absence of agrin (data not shown), suggesting that sites in the carboxy-terminal fifth of the cytoplasmic domain were essential for activity. Examination of the sequence revealed two consensus binding sites in this region. The first was YXXM, which is the consensus binding site for p85, the regulatory subunit of phosphatidylinositol-3-kinase. This enzyme mediates signal transduction by several tyrosine kinases, including the trk kinases which, as noted above, are related to MuSK . The second was VXV at the carboxy terminus, which resembles the consensus binding site for PDZ domain proteins. Proteins in this family that have been implicated in the aggregation of numerous membrane proteins include components of the postsynaptic membrane such as glutamate receptors and ephrins . Moreover, using the yeast two-hybrid system, we have identified PDZ domain proteins that bind to MuSK, and shown that deletion of its carboxy-terminal three residues abolishes the ability of MuSK to bind these proteins (Torres, R., E.D. Apel, H. Zhou, D.J. Glass, J.R. Sanes, and G.D. Yancopoulos, unpublished observations). We changed the essential tyrosine of the putative p85 binding site to phenylalanine in construct 24, and deleted the carboxy-terminal three resides (VGV) in construct 25. Surprisingly, both constructs were equivalent to wild-type MuSK in their abilities to mediate spontaneous and agrin-induced AChR clustering . Thus, both the p85- and the PDZ domain–binding sites are dispensable for MuSK function in cultured myotubes. In view of the similarity of MuSK to trk, it is interesting that the p85-binding domain in trk is dispensable for at least some aspects of neurotrophin signaling . In light of the known role of PDZ domain proteins in aggregation of postsynaptic components, we also asked whether construct 25 was impaired in its ability to associate with AChR–rapsyn clusters. The association of this mutant with AChRs and rapsyn in myotubes was indistinguishable from that of wild-type MuSK . Numerous polypeptides promote growth or differentiation by binding to the ectodomain of receptor tyrosine kinases. This binding induces dimerization or conformational changes that lead to autophosphorylation. Once phosphorylated, the cytoplasmic domain recruits and/or phosphorylates additional components that transduce the biological signal . In keeping with this model, agrin interacts with and activates MuSK, and MuSK phosphorylation is required for AChR clustering. However, agrin and MuSK are unusual among growth factor/receptor tyrosine kinase pairs in at least two respects. First, agrin does not bind MuSK directly, but requires an accessory component, MASC . Second, the ectodomain serves not only to activate MuSK but also to recruit AChR–rapsyn aggregates, via another accessory component, RATL . Here, we have mapped the domains of the MuSK ectodomain that mediate interactions with agrin–MASC and rapsyn–RATL. In addition, we have taken the first steps toward mapping the cytoplasmic domains through which MuSK transduces its signals. In initial studies, we characterized a MuSK −/ − myogenic cell line and showed that introduction of rat MuSK or a related Torpedo kinase rescued the ability of MuSK-deficient myotubes to form AChR clusters, both spontaneously and in response to agrin. Although these experiments were designed to establish an assay for structure–function analysis, they led to three novel conclusions. First, the Torpedo receptor kinase first described by Jennings et al. 1993 and later shown to be homologous to MuSK is likely to be a true orthologue of MuSK. Second, the requirement for MuSK is limited to muscle cells. Results in vivo had not excluded the possibility that expression of MuSK was also required at earlier stages of development or in other tissues to generate agrin-sensitive myotubes, but analysis of rescued MuSK −/ − cells demonstrates that this is not the case. Third, formation of spontaneous as well as agrin-induced AChR clusters requires MuSK. In principle, MuSK might be activated at low level in a ligand-independent fashion, or muscles might produce an endogenous ligand. We favor the hypothesis that activation is ligand-independent, based on our inability to block spontaneous clustering by deletion of any portion of the ectodomain. Using MuSK −/ − cells as an assay system, we found that the amino-terminal two fifths of the ectodomain is both necessary and sufficient to confer agrin sensitivity on MuSK: deletions within this region compromised agrin sensitivity, whereas constructs lacking the remainder of the ectodomain were fully agrin-sensitive. The possibility that these sequences are required for basal MuSK function was excluded by the observation that a construct lacking them restored the ability of MuSK −/ − cells to form spontaneous AChR clusters. The critical region includes two immunoglobulin-like domains, which are known to mediate ligand binding in numerous cell surface receptors. Thus, these domains may interact with agrin, MASC, or both. Further analysis suggests that the region of the first immunoglobulin-like domain is more critical than that of the second immunoglobulin-like domain for interactions with agrin. Deletion of sequences within the first immunoglobulin-like domain completely abolished agrin-sensitivity (constructs 2 and 13), whereas deletion of the second immunoglobulin-like domain left MuSK with significant albeit impaired agrin sensitivity (constructs 3 and 12). One interpretation of these results is that only sequences in or near the first immunoglobulin-like domain is required for agrin responsiveness, but its conformation is altered by mutations in neighboring areas. Alternatively, the second immunoglobulin-like domain may contain sites that stabilize or enhance interactions of MuSK with agrin–MASC. We cannot yet distinguish between these alternatives. Next, we attempted to determine which portions of MuSK are necessary for it to associate with rapsyn. Although rapsyn is entirely intracellular, we showed previously that MuSK–rapsyn interactions require the MuSK ectodomain, implying the existence of a transmembrane linker, RATL . Studies in both fibroblasts and MuSK −/ − myotubes suggest that the juxtamembranous portion of the ectodomain is both necessary and sufficient for the association of MuSK and rapsyn–RATL: MuSK fails to coaggregate with rapsyn in its absence, whereas deletion of more amino-terminal sequences does not impair MuSK–rapsyn coaggregation. The critical region includes the fourth immunoglobulin-like domain and a unique juxtamembranous segment, either or both of which might be involved in MuSK–rapsyn interactions; additional constructs will be needed to distinguish these possibilities. The observation that agrin induced AChR clustering in constructs lacking the juxtamembranous domain showed that MuSK–rapsyn/RATL interactions are dispensable for at least some aspects of MuSK function. What roles might such interactions play? We showed previously that MuSK-dependent phosphorylation of AChRs is reduced in rapsyn-deficient cells, which is consistent with the idea that rapsyn and RATL bring AChRs into proximity with MuSK . One possibility is that this interaction is more important in vivo than in the transfected MuSK −/ − cells, which express MuSK at higher than normal levels. Alternatively, the requirement of rapsyn for AChR phosphorylation may reflect a role of rapsyn (which is present in MuSK −/ − cells) but not of MuSK–rapsyn interactions. In either case, it is important to note that AChR phosphorylation may not be required for AChR clustering . Another possibility, which we favor, is that MuSK–rapsyn interactions (and possibly AChR phosphorylation) may be important for the localization of AChR clusters to synaptic sites but not for their formation per se. Although it is natural to interpret our results on colocalization as reflecting recruitment of MuSK to an AChR–rapsyn cluster, the relevant interaction in vivo may be one that recruits AChR and rapsyn to a subsynaptic concentration of MuSK. This point is crucial in evaluating the model proposed below. In a final series of studies, we initiated an analysis of the MuSK cytoplasmic domain. Two observations from this series were surprising. First, a binding site (NPXY) for PTB domains in adaptor proteins is required for MuSK activity. This result was surprising because similar mutations of other kinases such as trkB or erbB3 impair but do not abolish activity. Therefore, it will be important to learn which PTB protein(s) bind(s) to MuSK, and what signaling components they recruit to the complex. Second, a carboxy-terminal binding site (VXV) for PDZ domains in scaffolding proteins is dispensable both for MuSK-activated AChR clustering and for association of MuSK with AChR–rapsyn clusters. This result was also unexpected in view of our evidence that MuSK can bind PDZ proteins through its carboxy-terminal VXV motif (Torres, R., E.D. Apel, H. Zhou, D.J. Glass, J.R. Sanes, and G.D. Yancopoulos, unpublished observations) and that PDZ proteins play multiple roles in assembly of the postsynaptic apparatus at neuron–neuron synapses . We favor the possibility that PDZ domain proteins do play roles in MuSK signaling, but that these roles are not readily detected in the assays we have used to date. Our new results, along with those presented previously , suggest a model in which MuSK plays critical roles in three distinct steps that together lead to the formation of the postsynaptic membrane . First, a signal from the nerve leads to formation of a primary synaptic scaffold of which MuSK is a component, and for which MuSK is required . The observation that MuSK clusters beneath nerve terminals in rapsyn-deficient mutant mice, whereas other components of the synaptic membrane do not, demonstrates that the primary scaffold is assembled by mechanisms distinct from those responsible for AChR clustering. The neural signal that induces formation of the primary scaffold is unknown; it may be agrin, but no published data bear directly on this point. Second, agrin released from the nerve terminal interacts with MuSK, presumably via MASC, to activate MuSK kinase. This interaction requires sequences in or near the first immunoglobulin-like domain of MuSK, and may also depend on the second immunoglobulin-like domain. Once phosphorylated, MuSK recruits PTB domain–containing proteins to initiate a signaling process that leads to formation of aggregates that contain AChRs, rapsyn, and other components of the postsynaptic membrane and cytoskeleton. Third, AChR–rapsyn clusters are recruited to the primary scaffold. This step is mediated by RATL, and requires juxtamembranous sequences of the MuSK ectodomain. It is agrin-independent in that it is mediated by constructs unable to interact with agrin. Although shown as a late step in the model, it probably occurs in parallel with formation of the primary scaffold in vivo . We believe this model is attractive because it accounts for a cardinal difference between synaptogenesis and other biological responses mediated by receptor tyrosine kinases. MuSK-dependent aggregation of postsynaptic specializations is not fundamentally different from other kinase-dependent developmental programs. What is different is that MuSK localizes the specialized domain with a submicron level of precision. By using separate accessory proteins to catalyze formation of the postsynaptic apparatus and to recruit that apparatus to subsynaptic sites, MuSK can independently control the composition and the location of the synapse, both of which are critical for synaptic function. | Study | biomedical | en | 0.999998 |
10477766 | The A431 squamous carcinoma cell line was obtained from the American Type Culture Collection and maintained in DMEM with 10% fetal calf serum, at 37°C in a humidified atmosphere containing 5% CO 2 . The following antibodies were used in this study: mouse mAb 2B7 (integrin α6–specific) was prepared in our laboratory 50 ; rat GoH3 mAb (integrin α6–specific) was purchased from Immunotech; rat mAb 439-9B (integrin β4–specific; reference 18) was provided by Dr. Rita Falcioni (Regina Elena Cancer Institute, Rome, Italy). A peptide-specific antiserum elicited against the last 20 amino acids of the carboxy terminus of the β4 subunit was prepared commercially. A rabbit polyclonal antibody specific for the EGF receptor was purchased from Santa Cruz Biotechnology. The phosphotyrosine-specific antibodies PY20 and 4G10 were purchased from Transduction Labs and Upstate Biotechnology Incorporated, respectively. Mouse monoclonal antibodies specific for BPAG-1 (R185), BPAG-2 (1D1), and HD1/plectin (121) 26 40 were a gift of Dr. Owaribe (Nagoya University, Japan). Anti-actin polyclonal antibody, pan-cytokeratin mAb, rat IgG, and mouse IgG were purchased from Sigma Chemical. Laminin-1, prepared from the EHS sarcoma was provided by Dr. Hynda Kleinman (NIDR, Bethesda, MD). Collagen type I was purchased from Collagen Corp. Human recombinant EGF was purchased from Sigma Chemical. The PKC inhibitor Gö6976 was obtained from Alexis Corp. PMA was obtained from Calbiochem-Novabiochem. Chemotaxis was analyzed using 6.5-mm Transwell™ chambers, 8-μm pore size (Costar). The separating membrane was coated with laminin-1 (20 ug/ml) on both sides for 2 h at room temperature and then blocked with 1% albumin in PBS for 30 min. A431 cells (3 × 10 4 ) were resuspended in DMEM containing 0.1% albumin. In some experiments, antibodies (10 μg/ml of 2B7 or mouse IgG control) were added to the resuspended cells. The cells were added to the top wells of the Transwell™ chambers and allowed to settle on the filters for 1 h at 37°C, before EGF (1 ng/ml) was added to the lower chamber. In some cases, Gö6976 (1 μM) or vehicle alone (DMSO) was added 30 min before EGF stimulation. After a 2-h incubation, cells that had not migrated from the upper surface of the membrane were removed using cotton swabs and the remaining cells on the lower side of the membrane were fixed in methanol, dried, and stained with a 0.2% solution of crystal violet in 2% ethanol. Migration was quantified by digital analysis as described below. A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence or absence of antibody (2B7 or IgG, 10 μg/ml). The antibodies were added 30 min before EGF stimulation. The lamellar area, defined as a characteristic flat and thin protrusion of the cell containing no vesicles, was measured using a Nikon Diaphot 300 inverted microscope with phase contrast optics. This microscope was connected to a CCD camera (Dage-MTI), a frame-grabber (Scion), and a 7600 Power Macintosh computer to capture the images. Images were collected and analyzed with IPlab Spectrum image analysis software. Lamellar area was determined by tracing the lamellae contour and quantifying the area digitally. For each individual experiment 50–80 cells were analyzed. Bacteriological dishes were coated with 20 μg of laminin-1 or collagen type I for 2 h at room temperature and the dishes were then blocked with PBS containing 1% bovine serum albumin (BSA) for 30 min. A431 cells were resuspended in serum-free RPMI 1640 medium containing 10 mM Hepes and 0.1% BSA. The cells were plated at low density (2 × 10 4 cells/cm 2 ) on the matrix-coated dishes and allowed to adhere for 1–2 h in a humidified atmosphere with 5% CO 2 at 37°C. When indicated, Gö6976 (0.5–1 μM) in DMSO or vehicle alone was added 30 min before stimulation. The cells were then stimulated with either EGF (0.5–100 ng/ml) for 15 min or PMA (25–50 ng/ml) for 30 min. Cells were fixed with a buffer containing 2% paraformaldehyde, 100 mM KCl, 200 mM sucrose, 1 mM EGTA, 1 mM MgCl 2 , 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 15 min. In some cases, the cells were extracted before fixation with a buffer containing 0.2% Triton X-100, 100 mM KCl, 200 mM sucrose, 10 mM EGTA, 2 mM MgCl 2 , 200 μM sodium vanadate, 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 1 min. After fixation, the cells were rinsed with PBS and incubated with a blocking solution that contained 1% albumin and 5% goat serum in PBS for 30 min. Cells that were to be stained for HD1/plectin, BPAG-1 or BPAG-2, were fixed with acetone/methanol 1:1 (vol/vol) instead of paraformaldehyde. Either primary antibodies or FITC phalloidin (20 μg/ml) in blocking solution were added to the fixed cells separately or in combination for 30 min. The cells were rinsed three times and either a fluorescein-conjugated donkey anti–mouse or a rhodamine-conjugated donkey anti–rat IgG (minimal cross-reaction inter-species; Jackson ImmunoResearch Laboratories) in blocking buffer (1:150) were used separately or in combination to stain the cells for 30 min. Cells were rinsed with PBS and mounted in a mixture (8:2) of glycerol and PBS (pH 8.5) containing 1% propylgallate. The dishes were cut into slides and examined by confocal microscopy. To obtain a fraction enriched in cytokeratins 9 19 35 , A431 cells (2 × 10 6 ) were incubated on laminin-1–coated dishes as described above for 1–2 h. In some cases, Gö6976 (1 μM) or vehicle alone was added 30 min before stimulation. EGF (0.5–2 ng ml) was added and the cells incubated at 37°C for an additional 15 min. The cells were initially extracted with a buffer containing 0.2% Triton X-100, 100 mM KCl, 200 mM sucrose, 10 mM EGTA, 2 mM MgCl 2 , 200 μM sodium vanadate, 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 1 min (membrane/soluble fraction). The cells were rinsed several times before adding a second buffer containing 1% Tween-40, 0.5% deoxycholate, 10 mM NaCl, 2 mM MgCl 2 , 20 mM Tris-HCl, pH 7.5, for 10 min (cytoskeletal fraction). The cells were rinsed and the residual fraction was extracted with a third buffer containing SDS 0.4%, 10 mM NaCl, 20 mM Tris-HCl, pH 7.5, sonicated and then boiled before adding Triton X-100 to a final concentration of 1% (vol/vol; cytokeratin fraction). The samples were immunoprecipitated with the β4-specific polyclonal antibody, resolved by SDS-PAGE (6 or 8%) and immunoblotted with the same polyclonal antibody. Immune complexes were detected using a secondary antibody conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham, Inc.). To examine tyrosine phosphorylation using phosphotyrosine-specific antibodies, A431 cells were plated on laminin-1–coated dishes for 1 h at 37°C as described above. Cells were then stimulated with EGF (1–100 ng/ml) for 15 min, extracted with RIPA buffer containing 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM sodium vanadate, 1 mM PMSF, 10 ug/ml each of leupeptin, pepstatin A, and aprotinin, and 50 mM Tris-HCl, pH 7.5. The samples were immunoprecipitated using the 439-9B antibody, resolved by SDS-PAGE and immunoblotted using a combination of both the PY20 and 4G10 phospho-specific antibodies. Immune complexes were detected using a secondary antibody conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham, Inc.). Subsequently, the membranes were stripped and immunoblotted with the β4-specific polyclonal antibody. For metabolic radiolabeling with 32 PO 4 , A431 cells (2 × 10 6 ) were plated on laminin-1–coated dishes for 30 min as described above. Subsequently, the medium was removed and replaced with a phosphate-deficient medium (GIBCO). After a 1-h incubation in this medium, 32 PO 4 [0.5–2.0 mCi/ml (NEN)] was added and the cells were incubated for an additional 2 h. When indicated, Gö6976 (0.5–1 μM) in DMSO or vehicle alone was added 30 min before stimulation. The cells were then stimulated with either EGF (0.5–100 ng/ml) for 15 min or PMA (25–50 ng/ml) for 30 min. The cells were extracted with RIPA buffer as described above and the extracts were immunoprecipitated with 439-9B antibody, resolved by PAGE (6% gels) and transferred to PVDF membranes (Immobilon-P; Micropore). The membranes were exposed to x-ray film, developed, and then immunoblotted with the β4-specific polyclonal antibody to control for equivalent amounts of protein in the samples. A quantitative analysis of the relative intensities of the radioactive bands was made using a 2D electronic counter (Instant Imager; Packard, Meriden, CT). For phosphoamino-acid analysis, the area of the membrane that contained the β4 subunit was excised with a razor, acid hydrolyzed, and the hydrolysate was separated using 2D-thin layer chromatography following standard techniques 49 and exposed to x-ray film. Wild-type and myristoylated PKC-α constructs were generated by PCR using a bovine PKC-α cDNA as a template. The wild-type PKC-α was subcloned into the pCMV5 mammalian expression vector using the EcoRI and XhoI sites. The FLAG epitope (DYKDDDDK) was added to the carboxy terminus of the PKC-α cDNA by PCR. The myristoylated PKC-α cDNA was constructed by adding the relevant PCR fragment of bovine PKC-α to the XbaI and EcoRI sites of pCMV6 and by adding the Src myristoylation site (MYPYDVPDYA) at the amino terminus. All sequences were confirmed by DNA sequencing. To assess the activity of the PKC-α cDNAs, human embryonic kidney 293T cells were transfected with 1 μg of either the vector alone (pCMV5), PKC-α-FLAG cDNA, or myristoylated PKC α-FLAG cDNA using calcium phosphate for 6 h. Subsequently, the cells were cultured in the absence of serum for 24 h and extracted in a 1% NP-40 buffer 13 . The PKC-α proteins were immunoprecipitated using a FLAG M2 mAb (Sigma Chemical Co.) and a mixture of protein A and G beads. The beads were washed stringently and immune complex kinase assays were performed on the washed beads using MBP as the substrate (see reference 13 for details). No lipid cofactors were added to the reaction mix. The kinase assay was resolved on a 12.5% SDS gel and phosphorylated MBP was detected by autoradiography. Relevant expression of PKC-α was detected by immunoblotting total cell extracts with a PKC-α specific polyclonal Ab (Santa Cruz). To analyze the effects of PKC-α expression on A431 cells, these cells were transfected with 5 μg of each construct using Superfect (Qiagen) according to manufacturer's guidelines. After 24 h, the cells were fixed as described above and double stained with anti-FLAG mAb and either GoH3 mAb or mAb 121. Indirect immunofluorescence microscopy revealed that the α6β4 integrin on the ventral surface of A431 cells plated on either laminin-1 or collagen (not shown) is localized primarily in discrete structures and plaques in areas that exclude stress fibers. This pattern of staining for α6β4 is characteristic of its localization in hemidesmosomes 11 46 . Moreover, a distinct colocalization of α6β4 with the hemidesmosomal components BPAG-1 , BPAG-2 , and HD1/plectin was evident in these structures. These data establish that α6β4 is localized in structures that are characteristic of hemidesmosomes on the ventral surface of adherent A431 cells. Our previous data established that α6β4 participates in carcinoma migration through its ability to interact with the actin cytoskeleton 44 . For this reason, we examined the involvement of this integrin in the chemotactic migration of A431 cells towards EGF because these cells express high levels of the EGF receptor and this growth factor is known to stimulate their migration 32 53 . As shown in Fig. 2 A, relatively low concentrations of EGF (1 ng/ml) stimulated a robust migration response in A431 cells. However, EGF did not stimulate significant migration when used at higher concentrations (>10 ng/ml) . The effect of EGF on cell migration was mostly chemotactic in nature because the chemokinetic element represented <20% of the total migration (measured as the migration occurring in the presence of EGF in both chambers to disrupt gradients at the optimal dose of 1 ng/ml; data not shown). The fact that A431 cells express α6β4 and no α6β1 integrin ( 1 , 17 , and data not shown) enabled us to use function-blocking, α6-subunit specific antibodies to examine the contribution of α6β4 to A431 chemotaxis. Treatment of A431 cells with the 2B7 mAb inhibited chemotaxis toward EGF on laminin-1 by 60% . This mAb did not inhibit the attachment of the cells to laminin-1 (data not shown), indicating a distinct function for α6β4 in the chemotactic migration of A431 cells. Lamellipodial protrusions are thought to be critical in cell migration and are the basis for generating lamellae, which are larger protrusions that are associated with the direction the cell migrates 36 52 . Indeed, A431 cells displayed a striking increase in lamellipodia and ruffle formation for sustained periods of time in response to treatment with EGF at a concentration that stimulate optimal chemotaxis (1 ng/ml) . High concentrations of EGF (>5 ng/ml), which were inefficient in stimulating chemotaxis, caused the cells to round up quickly after a short period of protrusive activity, leaving behind numerous retraction fibers (data not shown). For this reason, we used low concentrations of EGF to stimulate A431 cells in subsequent experiments (0.5–2 ng/ml). The importance of α6β4 in the formation of such protrusions is supported by the fact that the lamellar area of A431 cells was substantially reduced by pretreatment with the 2B7 mAb before plating on laminin-1 . The findings that α6β4 is localized in hemidesmosomes in adherent A431 cells and that EGF stimulated their α6β4-dependent migration raised the possibility that EGF also altered the localization and cytoskeletal interactions of this integrin. Under these conditions of EGF stimulation, a striking change in the localization of α6β4 was apparent. Specifically, this integrin was substantially reduced in hemidesmosomes on the ventral surface , but it was readily apparent in the lamellipodia and ruffles that are formed in response to EGF stimulation . We observed also that EGF stimulation results in a reduction of HD1/plectin staining in hemidesmosomes, indicating a disassembly of hemidesmosome structure . Our observation that α6β4 is redistributed from hemidesmosomes to lamellipodia and membrane ruffles in response to EGF stimulation prompted us to examine its association with cytokeratins and F-actin in more detail using an in situ extraction scheme that solubilizes proteins to an extent that correlates with their cytoskeletal associations 9 19 . Specifically, membrane, actin, and cytokeratin fractions were obtained from A431 cells that had been either left untreated or stimulated with EGF using sequentially a Triton X-100 buffer (fraction 1, membrane), a two-detergent buffer (1.0% Tween-40/0.5% deoxycholate, fraction 2, actin) that removes the bulk of the actin cytoskeleton but not cytokeratins and associated proteins, and a third buffer containing SDS that solubilizes cytokeratins and associated proteins (fraction 3, cytokeratin; references 9, 19). The relative amount of α6β4 present in each fraction was detected by immunoprecipitation and subsequent immunoblotting with β4-specific antibodies. The relative distribution of actin and cytokeratin among the three fractions was also determined to assess the efficiency of the fractionation . As expected, the cytokeratins were present largely in fraction 3 and actin was distributed between fractions 1 and 2, which represent the G-actin and F-actin pools, respectively. Importantly, EGF stimulation did not alter this relative distribution of cytoskeletal proteins among the three fractions. However, as shown in Fig. 3 , EGF stimulation resulted in a substantial reduction in the amount of α6β4 in fraction 3 (cytokeratin) and an increase in the amount of α6β4 in the actin fraction in comparison to unstimulated cells. Densitometric analysis of the β4-specific bands in this figure revealed an approximate 63% reduction of α6β4 in the cytokeratin fraction and a 48% increase in the actin fraction. These observations provide evidence that the mobilization of α6β4 from hemidesmosomes that we detected by indirect immunofluorescence microscopy is associated with a disruption in its association with cytokeratins and an increase in its association with F-actin. The localization of α6β4 in membrane ruffles and lamellipodia that form in response to EGF stimulation prompted us to explore the possibility of its association with F-actin in these structures because we had previously observed such an association in colon carcinoma cells 44 . We found that the colocalization of α6β4 with F-actin in cell protrusions detected by immunofluorescence was retained in a significant number of protrusions after extraction of EGF-stimulated cells with a Triton X-100 buffer that preserves the actin cytoskeleton . However, extraction of these EGF-stimulated cells with the Tween-40/DOC buffer described above eliminated both the F-actin and α6β4 staining (data not shown). Taken together, these findings indicate that EGF stimulates a dissociation of α6β4 from cytokeratin-associated hemidesmosomes, as well as the formation of lamellipodia and ruffles that contain α6β4 in association with F-actin. Our findings that EGF stimulation mobilizes α6β4 from hemidesmosomes and promotes α6β4-dependent chemotaxis suggested a possible association between α6β4 and the EGF receptor. To address this possibility, EGF-stimulated A431 cells were stained for both α6β4 and the EGF receptor. As shown in Fig. 5 A, a striking colocalization of these two receptors was evident in membrane ruffles and lamellipodia. The specificity of this colocalization is evidenced by the finding that another surface protein, the HLA antigen, was not present in these F-actin–rich structures (data not shown). We were unable, however, to detect a specific, physical association between α6β4 and the EGF receptor by coimmunoprecipitation (data not shown). Given the fact that the EGF receptor is a tyrosine kinase and the report that EGF stimulation of A431 cells results in a substantial increase in the tyrosine phosphorylation of the β4 integrin subunit 31 , we explored the distribution of phosphotyrosine in both EGF-stimulated, as well as unstimulated, A431 cells using indirect immunofluorescence. In unstimulated cells, most of the phosphotyrosine staining on the ventral surface is seen at the cell periphery in radial arrangements similar to focal adhesions . A consistent colocalization of either α6β4 or EGFR (data not shown) with phosphotyrosine in these structures was not evident. Moreover, significant phosphotyrosine staining was not evident in hemidesmosomes . EGF stimulation, however, resulted in the colocalization of phosphotyrosine and α6β4 in lamellipodia and ruffles . From these results, we can conclude that α6β4 is associated with more phosphotyrosine-containing proteins in cell protrusions than in hemidesmosomes. The colocalization of α6β4 with phosphotyrosine in the lamellipodia and ruffles of EGF-stimulated A431 cells prompted us to examine the phosphorylation of α6β4 induced by EGF. For this purpose, α6β4 was immunoprecipitated from stimulated A431 cells with the 439-9B mAb and the immunoprecipitates were blotted with two phosphotyrosine-specific Abs (PY20 and 4G10). In these experiments, the cells were extracted with RIPA buffer because we observed a nonspecific interaction between α6β4 and the EGFR using a Triton X-100 buffer (data not shown). Under these conditions, we detected no phosphotyrosine in the β4 subunit in response to the concentration of EGF (1 ng/ml) that induced α6β4 redistribution to lamellipodia and ruffles, and that stimulated optimal A431 chemotaxis . Moreover, even high concentrations of EGF (100 ng/ml) that induced a rapid rounding-up of adherent A431 cells and did not stimulate chemotaxis induced only a marginal increase, at best, in the phosphotyrosine content of the β4 subunit as detected by these antibodies . Similar results were obtained with other α6 and β4-specific mAbs including GoH3, 2B7, A9, as well as a β4-specific polyclonal antibody (data not shown). These findings are in contrast to the report that EGF stimulation induces a substantial increase in the tyrosine phosphorylation of the β4 subunit in A431 cells 31 . To exclude the possibility that the phosphotyrosine-specific Abs we used were unable to detect significant tyrosine phosphorylation of the β4 subunit after EGF stimulation, A431 cells were labeled metabolically with 32 P-orthophosphate and then stimulated with EGF. As shown in Fig. 6 B, EGF stimulation did increase the phosphorylation of the β4 subunit substantially with half-maximal phosphorylation observed at ∼1 ng/ml of EGF. The discrepancy between the phosphotyrosine-specific Ab results and the metabolic labeling results prompted us to do a phosphoamino acid analysis of the radiolabeled β4 subunit. Surprisingly, we found that the β4 subunit is phosphorylated almost exclusively on serine . Indeed, both the basal and EGF-induced increases in β4 phosphorylation that we detected in Fig. 6 B result from serine phosphorylation . This phosphoamino-acid analysis in conjunction with the phosphotyrosine antibody data provide convincing evidence that EGF stimulation induces significant phosphorylation of the β4 subunit on serine but not tyrosine residues. The above findings indicated that EGF activates a serine protein kinase that is involved in β4 phosphorylation and that could also be involved in the redistribution of α6β4 from hemidesmosomes to lamellipodia and membrane ruffles. We hypothesized that a likely candidate for this kinase is PKC because its activation by EGF is well documented 2 . As an initial test of this hypothesis, we examined the effect of PMA stimulation on α6β4 localization in A431 cells. PMA stimulation mobilized α6β4 from hemidesmosomes , and increases the formation of α6β4-containing lamellipodia and ruffles (data not shown) as assessed by indirect immunofluorescence microscopy. These data were substantiated biochemically by analyzing the amount of β4 that remains associated with the cytokeratin fraction after stimulation with PMA, using the detergent extraction procedure described above. PMA stimulation markedly reduced the amount of α6β4 in the cytokeratin fraction in comparison to unstimulated cells . Consistent with a role of PKC-dependent phosphorylation in the redistribution of α6β4, we found that PMA stimulation itself increased the phosphorylation of the β4 subunit significantly as assessed by 32 P-orthophosphate labeling . The fact that we detected no tyrosine phosphorylation of β4 in response to PMA stimulation using the phosphotyrosine-specific antibodies (data not shown) indicates that the increase in 32 P-orthophosphate labeling can be attributed to serine phosphorylation. If PKC activation is required for the mobilization of α6β4 from hemidesmosomes, inhibition of PKC activity should inhibit this process. To establish this causality, we used Gö6976, an inhibitor of the conventional isoforms of PKC (α, β, γ) 33 42 . The effects of this inhibitor on α6β4 localization were profound. As shown in Fig. 7G ö6976 blocked the release of α6β4 from hemidesmosomes on the ventral surface of A431 cells in response to either EGF or PMA stimulation. Consistent with a role for serine phosphorylation in the release of α6β4 from hemidesmosomes, we found that Gö6976 reduced the EGF-stimulated phosphorylation of the β4 subunit by ∼50% , a reduction that corresponds to the level of β4 phosphorylation observed in the absence of EGF stimulation . Moreover, Gö6976 inhibited the EGF-induced dissociation of α6β4 from the cytokeratin fraction as assessed by the detergent extraction approach described above . This inhibition was evident for both EGF and PMA-stimulated cells . The above data suggested the participation of a conventional PKC isoform in the disassembly of the hemidesmosome and mobilization of α6β4 integrin. To obtain additional evidence for PKC involvement, we examined the possibility that activation of PKC-α, a widely distributed conventional PKC isoform, was sufficient to disassemble hemidesmosomes in the absence of EGF stimulation. For this purpose, we constructed a constitutively active PKC-α cDNA that contained the Src myristoylation site at its amino terminus. This myristoylated PKC-α exhibited a high level of in vitro kinase activity relative to the wild-type enzyme . Subsequently, we expressed both the myristoylated and wild-type PKC-α cDNAs in A431 cells and analyzed the effect of these cDNAs on hemidesmosome structure. As shown in Fig. 10A 431 cells that expressed myristoylated PKC-α, as evidenced by expression of the epitope tag (FLAG), showed a striking reduction in hemidesmosomes. More specifically, expression of both HD-1 and α6β4 was markedly reduced on the basal surface of these cells. In contrast, the cells that expressed the wild-type PKC-α, showed little change in the formation of hemidesmosomes. These results suggest that activation of PKC-α is sufficient to cause redistribution of the α6β4 integrin and other components of the hemidesmosome. A key implication of the above findings is that PKC activity is required for EGF-stimulated chemotaxis of A431 cells because the PKC-dependent redistribution of α6β4 is a necessary event in the mechanism of chemotaxis. We tested this implication by analyzing the effect of Gö6976 on the chemotactic response of A431 cells to EGF. The results in Fig. 8 D reveal that Gö6976 inhibited EGF-stimulated chemotaxis by >80%. It is important to note that Gö6976 at the concentrations used did not inhibit the attachment or spreading of A431 cells . These results support the involvment of PKC in the chemotactic signal elicited by EGF, and they substantiate the importance of a regulated and dynamic redistribution of the α6β4 integrin in chemotactic migration. The data we present provide insight into the mechanism of cell migration, especially the migration of epithelial and carcinoma cells. Recent work by our group has established that α6β4 participates in the chemotactic migration of carcinoma cells by interacting with F-actin at their leading edges and regulating essential signaling pathways 39 44 51 . This function underlies the contribution of this integrin to carcinoma invasion (reviewed in 43). An issue that needed to be resolved, however, is the relationship between α6β4 function in the hemidesmosomes of epithelial-derived cells and its ability to promote the migration of these cells. Epithelial cells use hemidesmosomes to anchor to the basal lamina. These multi-protein structures connect the substratum with cytokeratins to form rigid adhesion complexes. The α6β4 integrin is an essential component of hemidesmosomes and it is necessary for mediating their adhesive function 4 16 23 . Given this ability of α6β4-containing hemidesmosomes to generate stable adhesive contacts, it is reasonable to assume that their function needs to be disrupted to facilitate cell migration similar to the disruption of focal contacts and reduction in the strength of cell-substratum adhesion that occurs during fibroblast migration 41 59 . In fact, there is evidence that epithelial cells lose their hemidesmosomes when they are induced to migrate in response to wounding 22 46 , and that metastatic carcinoma cells often lack hemidesmosomes 43 . Also, the results obtained in our study were foreshadowed in a previous study that found that EGF stimulation disrupted α6β4-associated hemidesmosomes in 804G bladder carcinoma cells 31 . Our findings demonstrate that a chemotactic stimulus will not only disassemble hemidesmosomes but also promote the formation of α6β4-containing lamellipodia and membrane ruffles, thus establishing a mechanism for the dichotomy of α6β4 function in stably adherent and migrating epithelial-derived cells. An important implication of this model is that chemotactic factors can drive the migration of invasive carcinoma cells by mobilizing α6β4 and disassembling hemidesmosomes. The redistribution of α6β4 from hemidesmosomes to lamellipodia and ruffles that we observed in response to EGF stimulation, implied the existence of EGF-mediated signaling events responsible for this redistribution. Indeed, an important finding in this study is that redistribution is dependent on the activity of PKC and that it is associated with serine phosphorylation of the β4 subunit. The serine phosphorylation of β4 by EGF stimulation provided an important clue in our identification of the kinase activity involved in the redistribution of α6β4. It is well established that EGF can activate PKC through the PLC-γ–mediated formation of diacylglycerol and inositol trisphosphate 25 38 . In fact, there is evidence that PLC-γ participates in EGF-stimulated cell migration 12 , as well as in the motility induced by other growth factors such as PDGF 28 and IGF-1 3 . Based on this knowledge, we found that PMA is able to mimic the effect of EGF by altering the localization of α6β4 from hemidesmosomes to lamellipodia and ruffles and stimulating the phosphorylation of the β4 subunit on serine residues. Most likely, the conventional PKC isoform, PKC-α, is involved in the redistribution of α6β4 and the disassembly of hemidesmosomes. Gö6976, a specific inhibitor of the conventional PKC isoforms (α, β, γ) 33 42 , was able to impede the mobilization of α6β4 from hemidesmosomes and inhibit the EGF-stimulated phosphorylation of the β4 subunit. Indeed, hemidesmosomes were well preserved in EGF-stimulated A431 cells that had been pretreated with Gö6976. Consistent with the notion that the preservation of hemidesmosomes impedes cell migration, Gö6976 also inhibited the chemotactic response of A431 cells to EGF. In addition to these data, we observed that activation of PKC-α by expression of a constitutively active, myristoylated form of the enzyme, was sufficient to induce the redistribution of α6β4 and disassembly of hemidesmosomes. An issue that remains to be addressed is the nature of the signaling pathway that links the EGF receptor to activation of PKC-α and the mobilization of α6β4 from hemidesmosomes. As mentioned above, PLC-γ is a likely intermediary 12 25 38 . However, we observed that the PLC-γ inhibitor U73122 (1 μM) had rather drastic effects on the morphology of A431 cells and could not be used to assess the involvement of this phospholipase in the dynamic behavior of α6β4. Similarly, other widely used inhibitors such as wortmannin induced morphological changes in A431 cells even in the absence of EGF stimulation. We did observe, however, a partial inhibition of the EGF-induced redistribution of α6β4 with the MEK inhibitor PD98059 (data not shown). Although this observation needs to be established more rigorously, it does suggest the possible involvement of the MAP kinase pathway in the EGF-induced mobilization of α6β4 from hemidesmosomes. Interestingly, PD98059 can abrogate EGF-induced focal adhesion disassembly and cell motility in fibroblasts 59 . Although we cannot exclude a role for tyrosine phosphorylation of the β4 subunit in the EGF-induced chemotaxis of A431 cells, the data we obtained argue strongly against such a role primarily because we did not detect tyrosine phosphorylation of β4 using EGF at concentrations that promote optimal chemotaxis. In fact, when we used much higher concentrations of EGF (100 ng/ml) we detected an additional increase in β4 serine phosphorylation, but only a marginal increase, at best, in β4 tyrosine phosphorylation. These results contrast with a previous study by Mainiero et al. 31 that reported a striking increase in the tyrosine phosphorylation of the β4 subunit in A431 cells stimulated with high concentrations of EGF (10–250 ng/ml). At this point, we are unable to explain the discrepancies between our findings and those of Mainiero et al. 31 . It is worth noting, however, that in our study, the use of such high concentrations of EGF to stimulate A431 cells failed to induce chemotaxis and caused the cells to round-up, making it difficult to correlate phosphorylation of β4 with cell migration under these conditions. The EGF-stimulated phosphorylation of the β4 subunit on serine residues that we identified is novel and it provides an impetus for investigating the nature of this phosphorylation and its role in regulating the cytoskeletal interactions of the α6β4 integrin in more detail. For example, the extremely large β4 cytoplasmic domain contains multiple consensus motifs for PKC phosphorylation based on our analysis of the human β4 cDNA sequence using Prosite (data not shown). The presence of these motifs supports the possibility that PKC may phosphorylate α6β4 directly. It is also worth considering the possibility that PKC may activate another downstream serine kinase that is involved in β4 phosphorylation because there are also consensus sites in the β4 sequence for other serine kinases such as casein kinase II. Another important issue to be studied is whether phosphorylation of the β4 subunit is essential for either its mobilization from hemidesmosomes or its recruitment into lamellipodia and ruffles. The data we provide in this study provide a strong correlation of serine phosphorylation with these events. Definitive proof of involvement will require identification of the specific serine residue(s) in the β4 subunit that are phosphorylated by EGF stimulation and the subsequent mutational analysis of these sites. The possibility that PKC-dependent serine phosphorylation also influences other components of hemidesmosomes should be considered. For example, there is evidence that PKC can mobilize BPAG-2 from hemidesmosomes 27 , and that it can phosphorylate HD1/plectin and weaken its interaction with intermediate filaments 20 . The EGF-induced release of α6β4 from hemidesmosomes and its association with F-actin in lamellipodia and membrane ruffles are probably independent events. This idea is derived from our finding that Gö6976, an inhibitor of the conventional PKC isoforms, did not block the EGF-induced formation of α6β4-containing membrane ruffles and lamellipodia (data not shown), even though it prevented α6β4 mobilization from hemidesmosomes . This observation suggests that the PKC-dependent mobilization of α6β4 from hemidesmosomes is independent of the recruitment of α6β4 into the lamellipodia and ruffles that are formed in response to EGF stimulation. As we have shown, however, Gö6976 did inhibit EGF-induced chemotactic migration. Collectively, these findings underscore the hypothesis that the increased formation of cell protrusions in the form of ruffles and lamellipodia is not enough to generate movement and that the destabilization of hemidesmosome function mediated by PKC is an essential component of the migration process. An interesting issue that arises is the role that EGF plays in the recruitment of α6β4 to the lamellipodia and ruffles. Most likely, EGF stimulates the formation of new cell protrusions that contain α6β4 rather than promoting the preferential incorporation of α6β4 into such structures. This assumption is based on our finding that the few lamellipodia that form in the absence of EGF do incorporate α6β4, and they have a similar intensity of α6β4 expression as those lamellipodia that are formed in response to EGF stimulation . Thus, we suggest that α6β4 is transported to and concentrated at the leading edges by mechanisms intrinsic to lamellipod formation, and that EGF increases the number of protrusions while providing a pool of α6β4 liberated from hemidesmosomes. One possible mechanism by which α6β4 could be recruited to the leading edge is exemplified by the transient association of small aggregates of β1 integrins with the actin cytoskeleton that occurs in neuronal growth cones. These aggregates of β1 integrins are transported on the dorsal surface in a directed way to the leading edge 48 . Although we do not have direct data to support a transient association of α6β4 with the actin cytoskeleton, the fact that only a fraction of the α6β4 in lamellipodia is resistant to extraction with a Triton X-100 buffer may be the reflection of a dynamic equilibrium attained by the constant association and dissociation of α6β4 with F-actin. Another model for the recruitment of membrane proteins into motile structures involves the concentration of recycling proteins in ruffles by directed exocytosis induced by EGF through a Rac-dependent pathway 7 . In this model, the ability of membrane proteins to be recycled is essential for their recruitment into ruffles. This model is of particular interest to our findings because there is evidence that α6β4 is recycled on the cell surface 6 , and we have implicated Rac in the α6β4-dependent migration and invasion of carcinoma cells 51 . Moreover, the recycling of the population of α6β4 that is released from hemidesmosomes by EGF could provide a pool for newly forming ruffles and lamellipodia. If the mobilization of α6β4 from hemidesmosomes constitutes one essential component of the EGF-stimulated migration of A431 cells, the second component of migration in which α6β4 participates is the actual process of migration itself. Indeed, a function for α6β4 in A431 migration is supported by its localization in lamellipodia and ruffles and, more directly, by our finding that an α6-specific mAb inhibited both chemotactic migration and lamellae formation on laminin-1. These results are consistent with our previous work on colon carcinoma cells that established a role for α6β4 in the formation and stabilization of lamellae and filopodia 44 . In this study, we observed that protruding filopodia, which anchor to the substrate using α6β4, are frequently followed by the extension of the lamella towards the anchoring point. This function of α6β4 may relate to the concept of a molecular clutch that anchors actin bundles to the substrate providing traction tracks for myosin II motors 36 . Because a retrograde flow of actin filaments from the periphery towards the nucleus occurs in most motile cells, the net effect of this clutch action on actin polymerization would also be to generate a forward protrusive force, as has been recently shown in a growth cone model 56 . In addition to these mechanical functions, we have defined at least two distinct signaling pathways regulated by α6β4 that are essential for lamellae formation, chemotactic migration and invasion of carcinoma cells. These pathways involve PI3-kinase/Rac 51 and a cAMP-specific phosphodiesterase 39 . It is also worth noting that we have observed apparent ligand-independent effects of α6β4 on lamellae formation and chemotactic migration. For example, expression of α6β4 in a breast carcinoma cell line stimulates lamellae formation and chemotaxis on collagen in response to a chemoattractant, processes that are not inhibited by α6-specific Abs 39 . The implication of these findings is that α6β4 can regulate key signaling pathways independently of its adhesive functions. The colocalization of the EGFR, α6β4, and phosphotyrosine in membrane ruffles and lamellipodia suggests the presence of an active signaling complex involved in cell migration. In fact, there is evidence that the activated form of the EGFR is localized in ruffles and lammelipodia, and that these structures are also sites of PLC-γ activation stimulated by EGF 10 15 . Several other EGFR substrates, such as ezrin, spectrin, and calpactin II are recruited to membrane ruffles and have been detected in their phosphorylated form in these structures 5 8 . The fact that we observed a striking colocalization of α6β4 with the EGF receptor in lamellipodia and ruffles suggests that these two receptors may interact to facilitate their ability to signal chemotactic migration. This possibility, which remains to be demonstrated for α6β4 and the EGFR, is supported by the recent finding that the α6β4 and α6β1 integrins associate with the erbB-2 receptor in several carcinoma cell lines 18 . Nonetheless, it is evident from our data that activation of the EGFR has a profound effect on the localization of α6β4, and the close proximity of these two receptors in F-actin–rich areas, along with the other signaling molecules mentioned above, suggests cooperation in their signaling of chemotactic migration. In summary, our findings describe a mechanism for the chemotactic migration of carcinoma cells that assemble hemidesmosomes. An important implication of our findings is that the mobilization of the α6β4 integrin from hemidesmosomes by a PKC-dependent mechanism, is an essential step in the migration process, presumably because it releases the strong and stable adhesion mediated by hemidesmosomes and allows for the dynamic adhesive interactions that are required for migration. Importantly, we also demonstrate that the α6β4 integrin can associate with F-actin in lamellipodia and membrane ruffles, and participate in the migration process itself. Collectively, our results explain how this integrin can mediate both stable adhesion and cell migration. They also suggest that growth and motility factors that are known to promote tumor progression may function, in part, by changing the cytoskeletal interactions and localization of this unique receptor. | Study | biomedical | en | 0.999997 |
10477767 | Long bones obtained from neonatal Wistar rats killed by decapitation were curetted into Hepes-buffered Medium 199 containing Hank's salts (GIBCO-BRL) and heat-inactivated fetal bovine serum (FBS, 5% vol/vol; Sigma Chemical Co.) (M199-H). The resulting suspension was dispersed onto devitalized cortical bone slices or 22-mm, 0-grade, glass coverslips (Libro/ICN). Osteoclasts attached to the respective substrate within 15 min (37°C) and contaminating cells were removed by gentle rinsing. Osteoclasts were identified readily by their large size, multinuclearity, complex morphology, densely ruffling edges, and response to calcitonin . Purified rabbit osteoclasts were prepared by the method of Kakudo et al. 1996 from unfractionated bone cells obtained according to the procedure described by Tezuka et al. 1992 . In brief, cell suspensions obtained from minced long bones of 10-d-old rabbits (Japan White; Saitama Experimental Animal Supply Co.) were agitated by vortexing and plated in 10-cm tissue culture dishes (Becton Dickinson) coated with 24% collagen gel (Nitta Gelatin Co.). After a 3-h incubation, adherent non-osteoclasts were removed from the collagen gel by sequential treatment with pronase E (0.001% wt/vol) and collagenase (0.01%, wt/vol; Wako Pure Chemical Industries). The remaining osteoclasts were then collected by 0.1% (wt/vol) collagenase solution treatment and replated. When these cell suspensions were seeded onto tissue culture dishes, osteoclasts attached and spread out. The purity of the osteoclast preparation was judged before membrane isolation by staining for an osteoclast-specific marker, tartrate-resistant acid phosphatase (TRAP) using a leukocyte acid phosphatase kit (Sigma). In line with previous experiments of Kameda et al. 1997 , we found that the purity of TRAP-positive multinucleated cells (>3 nuclei) was >99%. These cells have been shown to resorb bone and express specific osteoclast markers, cathepsin K, and calcitonin receptors . A rabbit osteoclast cDNA library containing 1 × 10 10 independent clones was used for PCR amplification . Two oligonucleotide primers were designed from the known rat CD38 cDNA sequence : forward primer, 5′-CCTGTTGCTGTGTTCTGGA-3′ (569–588 ), and reverse primer, 5′-GGTCGGTAGTTATCCTGG C-3′ (861–843) (GIBCO-BRL). The coding region of rabbit CD38 cDNA fragment was then amplified by PCR. In brief, the standard reaction mixture (50 μl) contained: 0.1 μl of rabbit osteoclast cDNA library, 1 μl of each oligonucleotide (50 μl), and 1 μl (5 U) of AmpliTaq (Promega Corp.). The GeneAmp PCR System 2400 (Perkin Elmer) was programmed as follows: a 5-min cycle at 95°C, then 40, 1-min cycles at 95, 55, and 72°C. The PCR products were separated by agarose gel electrophoresis. A ∼300-bp DNA fragment was isolated from excised gel slices using a QIAquick Gel Extraction Kit (QIAGEN Inc.) and ligated into EcoRV-cut pBluescript II SK (+/−) vector (Stratagene Ltd.) to produce the plasmid, pBS-CD38, which was then transformed into competent DH5α cells. 293 bp of pBS-CD38 insert was confirmed through the DNA Sequencing Facility at the University of Pennsylvania. To obtain the full-length CD38 cDNA, the 293-bp CD38 coding region DNA fragment was used as probe to screen our osteoclast cDNA library . For this, the probe was labeled with α-[ 32 P]CTP (3,000 Ci/mmol) (NEN™ Life Science Products Inc.) using the Red prime Random Prime Labeling Kit (Amersham Pharmacia Biotech Inc.). Duplicate filters, which covered 1 × 10 7 independent clones, were then hybridized overnight at 42°C with prehybridization solution (50% formamide, 6× SSC, 5× Denhardt's, 0.5% SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA) to which a labeled probe was added 3 h later. After a high-strigency wash at 68°C for 1 h, the filters were exposed to x-ray film with intensifying screens for 20 h at −70°C. Positive recombinant plaques were purified from phage plate lysates according to the Lambda ZAP II library's instruction manual (Stratagene). The DNA clones were confirmed by PstI-KpnI restriction analysis and direct nucleotide sequencing. We and others have recently applied in situ RT-PCR cytoimaging successfully to study IL-6 and IL-6 receptor expression in osteoblasts, osteoclasts, and bone marrow stromal cells . We now utilize this technology to examine CD38 expression in mature rat osteoclasts (primer sequences, as above). As a positive control, we also examined the expression of a housekeeping gene and an osteoclast-specific gene . Their primer sequences were: GAPDH, forward: 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ (51–76), reverse: 5′-CATGTAGGCCATGAGGTCCACCAC-3′ ; cathepsin K, forward: 5′-CCCAGACTCCATCGACTATCG-3′ (345–365), reverse: 5′-CTGTACCCTCTGCATTTAGCTGCC-3′ (674–651). Osteoclasts were incubated on glass coverslips (22 mm, 0 grade) in Medium 199 with Earle's salts (6.6 mM Na 2 CO 3 , M199-E) for 6 h (37°C, 5% humidified CO 2 , pH 7.4). In separate experiments, the cells were incubated with either vehicle or IL-6 (10 ng/liter or 10 μg/liter). The cultures were washed with M199-E, fixed with paraformaldehyde (4% vol/vol) in phosphate-buffered saline (PBS) (20 min, 4°C), and washed twice with cold PBS. The fixed cells were treated with 0.2 N-HCl (20 min, 20°C), washed with DEPC-water (Sigma Chemical Co.), and treated with proteinase-K (5 mg/liter in 10 mM Tris-HCl, pH 8, 15 min, 37°C) and cold paraformaldehyde (4% vol/vol, 30 min, 4°C). Before being air-dried, the cells were dehydrated by sequential 1-min immersions in graded aqueous ethanol solutions, 70, 80, 90, and 100% (vol/vol). They were then incubated overnight (37°C) with RNase-free DNase I (1,500 units/ml; Boehringer Mannheim) to remove genomic DNA. The DNase I was finally washed out with DEPC-water and inactivated by heating (90°C, 10 min). First-strand cDNA was synthesized by incubating cultures with RT mixture (50 μl) comprising 1 mM dNTP, 0.01 M DTT, 400 nM reverse primer (above), DEPC-water, and 14 U/ml Superscript RT II (GIBCO-BRL) (42°C, 60 min). An AmpliCover disc was used to cover each sample. The samples were then treated separately with PCR mixture (50 μl) comprising 0.2 mM dNTP, PCR buffer, 2.5 mM MgCl 2 , 0.1 U/μl Taq polymerase, 400 nM forward and reverse primers, 10 μM digoxigenin (DIG)-labeled-11-dUTP (Boehringer Mannheim), and DEPC-water. Each sample was then covered gently with an AmpliCover disc ensuring the absence of air bubbles. The GeneAmp In situ PCR System 1000 (Perkin Elmer) was programmed as follows. A 4-min soak at 94°C was followed by 40 cycles of: 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min. Incorporated DIG-11-dUTP in the PCR product was detected by an alkaline phosphatase (AP)-conjugated anti-DIG antiserum and AP substrates, 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indoyl-phosphate (BCIP) using a DIG Nucleic Acid Detection Kit (Boehringer Mannheim) per manufacturer's protocol. Negative controls, in which primers were omitted, were run in parallel. Messenger RNA expressing cells stained dark purplish brown, while negative controls did not stain. We then performed an analysis of the staining intensity using a blinded observer. Osteoclasts were scored on a scale from 0 to 4 . The results from three experiments were plotted as a frequency histogram. This allowed us to determine the proportion of cells that lay in a certain intensity range. A similar analysis has been used by us previously to demonstrate the effects of Ca 2+ on IL-6 and IL-6 receptor mRNA expression. Notably, GAPDH, a housekeeping gene, was used to control for the effects of IL-6 on CD38 expression. Also note, that, as previously, the few retracted osteoclasts were discarded from the analysis to prevent a biased intensity assessment. Dr. F. Malavasi (Torino, Italy) kindly provided the monoclonal anti-CD38 antibody, A10. A10 was raised by immunizing mice with Burkitt's lymphoma Daudi cells . The antibody recognizes a 46-kD CD38 molecule on T and B lymphocytes also enhancing their activation and proliferation (hence the term, agonist) . An antagonist antibody (Sigma Chemical Co.) was also used to establish specificity for CD38 detection in the ADP ribosyl cyclase assay. The control anti-ryanodine receptor antibody, Ab 34 , was raised against a cytosolic calmodulin-binding RyR epitope. Therefore, it does not stain nonpermeabilized osteoclasts . Osteoclasts were incubated with normal goat serum (in 10 mM PBS, 1:10, pH, 7.4, 15 min) in multiwell dishes and washed with HBSS (GIBCO-BRL). The cells were either incubated without antibody, or with nonimmune mouse IgG, Ab 34 (anti-RyR antibody) (all controls), or A10 (anti-CD38 antibody) (in M199-H, 1:100). In the same experiment, CD38-negative fibroblasts were also incubated with the same antibodies. The coverslips were rinsed gently with HBSS, drained, reincubated with goat anti–mouse FITC (Sigma Chemical Co.; 1:100, in HBSS, 60 min), washed gently, and finally, drained. The number of fluorescent osteoclasts was first determined in a laser confocal scanning microscope, at λ ex = 495 nm and λ em = 525 nm (Leica Inc.). To localize staining to the osteoclast membrane, 1-μm-thick optical sections were obtained in the cell's coronal plane in selected experiments. Finally, trypan blue (1 mM, 961 Da; Sigma Chemical Co.) was applied to exclude membrane damage that could allow antibody access into the cell. For isolation of plasma membranes, cells were first scraped and homogenized in TKM solution (50 mM Tris-HCl, pH 7.5, 25 mM KCl and 5 mM MgCl 2 ) supplemented with 0.25 mM sucrose. The homogenate was centrifuged (3,000 g , 10 min), the pellet resuspended in sucrose (70% wt/vol), and then rehomogenized (12 strokes) with a glass/Teflon homogenizer. The sucrose solution was then layered as follows: the homogenate was overlaid with 12 ml of 48% (wt/vol) sucrose, followed by 6 ml of 42% (wt/vol) sucrose. This was then centrifuged at 27,700 rpm for 70 min in a SW-28 swinging bucket rotor. The plasma membrane fraction banding at the interface of 70%/48% sucrose was collected and suspended in 70% (wt/vol) sucrose solution. The entire process was repeated twice to purify the plasma membranes. SDS-PAGE was performed using 12% separating and 4% stacking polyacrylamide gels using a minigel system (BioRad Laboratories). Plasma membranes prepared from osteoclasts and osteoblasts (30 μg protein) were heated for 5 min at 95°C in Laemelli's sample buffer (2% SDS, 2% β-mercaptoethanol, 10% vol/vol glycerol and 50 mg/liter bromophenol blue in 0.1 M Tris-HCl buffer, pH 6.8). Electrophoresis was performed at 20 mAmps per gel. The proteins thus resolved were stained with Coomassie Brilliant Blue (Sigma Chemical Co.) or transferred electrophoretically onto OPTITRAN-supported nitrocellulose membrane (Schleicher and Schuell) at 15°C for 1 h at 100 volts. The membranes were blocked with Tween 20 (0.3% vol/vol) in PBS at 20°C and incubated with the anti-CD38 antibody (1:3,000) (Sigma Chemical Co.). After rinsing, the blot was incubated for 1 h with HRP-conjugated anti–mouse antibody. The blot was developed using Pierce SuperSignal Ultra Chemiluminescence Kit, per manufacturer's instructions. ADP-ribosyl cyclase activity was measured in osteoclast plasma membranes isolated as above. We measured the cyclization of the NAD + surrogate, NGD + , to its fluorescent derivative, cGDPr. Plasma membranes (25 μg) were incubated, for 20 min at 37°C, in 20 mM Tris-HCl (pH 7.4) with 100 μM NGD + . The reaction was stopped with 5 μl of 100% (vol/vol) trichloroacetic acid. Fluorescence in the supernatant was measured using a high-sensitivity spectrofluorometer (λ ex = 300 nm; λ em = 410 nm). The amount of cGDPr formed was plotted as mean ± SD in nmol/ml/mg protein. To establish specificity of the assay, we incubated membranes in with anti-CD38 antibody (1:1,000; Sigma Chemical Co.) and NAD + (400 μM). Mouse IgG5 was used as control. Glass coverslips containing freshly isolated osteoclasts were incubated in serum-free medium (30 min, 37°C) with 10 μM fura 2/AM (Molecular Probes), then washed in M199-H and transferred to a Perspex bath positioned on the microspectrofluorometer stage. The latter was previously constructed from an inverted microscope (Diaphot; Nikon) . Prewarmed test solutions of the anti-CD38 antibodies (A10, agonist and antagonist; Sigma Chemical Co.) (1:500), NAD + (0.5, 1, or 10 mM), ryanodine (5 μM), caffeine (250 μM and 1 mM), or thapsigargin (4 μM) were applied in various protocols, as described in Results. The cells were exposed alternatively to excitation λ s of 340 or 380 nm. The emitted fluorescence was deflected through a 400-nm dichroic mirror and subsequently filtered at 510 nm. The signal was converted to 25 ns, 5V transistor-transistor-logic (TTL) pulses in a photomultiplier tube (PM28B; Thorn EMI). The resulting pulses were counted by a dual photon counter (Newcastle Photometrics) and recorded every second to give a ratio of emitted intensities at excitation λ s of 340 and 380 nm, F 340 /F 380 . The cytosolic Ca 2+ measuring system was calibrated using an established protocol for intracellular calibration . In brief, fura 2–loaded osteoclasts were bathed in Ca 2+ -free, EGTA-containing solution containing 130 mM NaCl, 5 mM KCl, 5 mM glucose, 0.8 mM MgCl 2 , 10 mM Hepes, and 0.1 mM EGTA. 5 μM ionomycin was first applied to obtain the minimum ratio due to lowest cytosolic Ca 2+ (R min ) and the maximum fluorescence intensity at 380 nm (F max ). 1 mM CaCl 2 was then applied with 5 μM ionomycin to obtain values of the maximum ratio due to an elevated cytosolic Ca 2+ (R max ) and the minimum fluorescence intensity at 380 nm (F min ). The dissociation constant K d for Ca 2+ and fura 2 is 224 nM (20°C, 0.1 M, pH 6.85). The values were substituted into the equation: [Ca 2+ ] = K d × [(R − R min )/(R max − R)] × [(F max /F min )]. Mean changes (Δ) in the cytosolic Ca 2+ concentration ([Ca 2+ ]) were then calculated by subtracting peak from basal cytosolic [Ca 2+ ]. Statistical comparisons of cytosolic Δ [Ca 2+ ] were made by Analysis of Variance (ANOVA) with Bonferroni's Correction for Inequality. Bone resorption was measured using the pit assay . In brief, the bones from 24- to 48-h-old rats were sliced in 3.5 ml M199-H, and the resulting cell suspension was settled onto devitalized cortical bone slices (4 mm × 4 mm) for 30 min. After the removal of nonadherent cells by gentle rinsing, the slices were transferred to a multiwell dish containing M199-E (with 10% FBS vol/vol). Either vehicle or anti-CD38 antibody (1:500 or 1:5,000) were applied together with 1 mM NAD + . The slices were incubated for 24 h in humidified CO 2 (5%) (pH 6.9), after which they were fixed with glutaraldehyde (10% vol/vol) and stained for the presence of tartrate-resistant acid phosphatase (TRAP) using a kit (Kit 386A; Sigma Chemical Co.). The number of osteoclasts with two or more nuclei was determined on each slice using a light microscope (Olympus). The cells were removed by treating the slices with NaOCl (5 min), and the slices rinsed with distilled water followed by acetone, and then air-dried. The slices were stained subsequently with toluidine blue (1% vol/vol, in 1% wt/vol borate, 5 min). The number of resorption pits was determined on each slice by light microscopy. Notably, each experiment was performed with osteoclasts obtained from three animals with five or six bone slices per treatment. The number of pits or osteoclasts per bone slice was expressed as a mean ± SEM. Student's unpaired t test was used to analyze the effect of treatment, which was considered significant at P < 0.05. Osteoclasts on coverslips were bathed in a multiwell dish containing 500 μl M199-E (with 10% FBS vol/vol) for 6 h in the presence of either vehicle or anti-CD38 antibody (1:500) and NAD + (1 mM). The culture medium was removed and its IL-6 level was measured with an ELISA kit . In brief, 96-well plates coated with a polyclonal anti–mouse IL-6 antiserum were used to accommodate 50 μl of assay diluent (buffered protein) and 50 μl of standard, control, or sample. After incubation (20°C, 2 h), the wells were aspirated, washed repeatedly, and loaded with 100 μl horseradish peroxidase–conjugated anti–IL-6 antibody. After a further incubation (20°C, 2 h), 100 μl of substrate solution containing H 2 O 2 and tetramethylbenzidine, was added to each well. Finally, a further incubation (30 min) was followed by the addition of 100 μl of dilute HCl to stop the reaction. The optical density of each sample was measured at 450 nm on a microplate reader (BioRad). IL-6 was estimated from the standard curve in triplicate experiments and represented as mean ± SEM. Differences between control and treatment were assessed by the Student's unpaired t test. To obtain full-length CD38 cDNA clones, a rabbit osteoclast cDNA library was screened. A 293-bp CD38 cDNA coding region DNA fragment was initially cloned and used as probe. A single positive cDNA clone was identified after screening 1 × 10 7 independent phage recombinants; this contained a 2.8-kb EcoRI-XhoI insert in the plasmid pBluescript-SK (termed SL385). The sequence of the full-length SL385 CD38 insert was obtained. Sequence analysis confirmed the presence of the CD38 coding sequence and extended into 3′-untranslated region . The osteoclast CD38 cDNA sequence was 71, 69, and 66% similar to corresponding full-length CD38 cDNA sequences of mouse, rat, and human CD38 (obtained from the GenBank database) . No significant homology was found, however, between the sequence of the insert and any other sequence in the GenBank database. Fig. 2 shows the predicted amino acid sequence of the full-length rabbit osteoclastic CD38. There was a 59, 59, and 50% similarity between this sequence and that of mouse, rat, and human CD38 (GenBank), respectively. The relative sequence divergence suggests that the amplified DNA product codes for a yet uncharacterized member of the CD38 family of cyclases. CD38 mRNA expression in isolated single osteoclasts was investigated by in situ RT-PCR cytoimaging using the same primers as used for PCR cloning (above). Fig. 3 shows light micrographs of histostained osteoclasts after RT-PCR. Panel i shows an unstained osteoclast (negative controls) in an experiment in which primers were omitted from the reaction mixture. Panels ii and iii show osteoclasts in which the intense bluish-brown staining represents, respectively, mRNA expression for cathepsin K (cell-specific positive control) or GAPDH (housekeeping gene). Panels iv to vi show intense CD38 mRNA histostaining in osteoclasts that were either incubated with vehicle (iv), 10 ng/liter IL-6 (v), or 10 μg/liter IL-6 (vi). Fig. 4 shows a semi-quantitative analysis of staining intensity using a method modified from that reported by Adebanjo et al. 1998 . Osteoclasts staining for CD38 mRNA were thus assessed by a blinded observer who assigned an intensity level to the staining as a number between 0 and 4 (no staining to intense staining). Three experiments were pooled to derive frequency histograms relating the number of cells to their assigned intensity score. Osteoclasts that underwent in situ RT-PCR without added primers (control) showed a skewed distribution to the left (i, n = 26 cells). Cells incubated with primers, but without treatment for 6 h with IL-6 (ii, n = 49 cells) or those treated with 10 ng/liter IL-6 (iii, n = 53 cells), showed a normal (Gaussian) distribution of their assigned scores. The data became significantly skewed ( P < 0.05) to the right when osteoclasts were treated with 10 μg/liter IL-6 (iv, n = 29 cells). In contrast, the expression of mRNA for GAPDH, the housekeeping gene, followed a similar distribution in all three experimental sets, namely no treatment, 10 ng/liter IL-6, and 10 μg/liter IL-6 (not shown). Taken together, the results showed that a much larger proportion of cells stained intensely with 10 μg/liter IL-6 compared with 10 ng/liter IL-6, suggesting that the former concentration of IL-6 might enhance CD38 mRNA levels. As in our earlier study , we must emphasize that the results are semi-quantitative at best, due not only to the inherent pitfalls of the in situ RT-PCR technique, but also because the cells may undergo slight margin retraction resulting in scoring artifacts. Again, as before, we have excluded obviously retracted cells, as in these cells, staining is likely to appear more intense due to cytoplasmic condensation. We next examined whether our highly specific anti-CD38 antibody, A10, bound to the surface of intact live osteoclasts. This agonist antibody has previously been shown to bind to, and activate, the CD38 antigen in several systems . Fig. 5 (B–D) shows confocal microscopic images taken at 1-μm intervals in the coronal plane of CD38-positive osteoclasts. Intense, strictly peripheral, immunostaining was visualized distinctly reminiscent of plasma membrane staining. Notably, CD38-negative fibroblasts were found not to stain with the antibody (not shown). Also of note is that every one of the ∼20 osteoclasts examined in each different experiment showed positive staining. Furthermore, all cells remained negative for trypan blue, excluding membrane damage that would otherwise permit antibody access into the cytosol. Control experiments were performed by (a) not including the anti-CD38 antibody (not shown); (b) using preimmune mouse IgG instead of the antibody (not shown); (c) using an irrelevant anti-ryanodine receptor antibody, Ab 34 . That osteoclasts did not stain with any such treatment provided clear evidence for specificity. Note that Ab 34 was raised against a cytosolic calmodulin-binding sequence of the RyR, and hence, is known not to stain the surface of nonpermeabilized osteoclasts . A negative result with the latter antibody further attests to an intact plasma membrane. We further confirmed that the CD38 protein was present in isolated osteoclast plasma membranes by Western blotting using a different antagonist anti-CD38 antibody (Sigma Chemical Co.). A ∼46 kD band was observed when plasma membranes purified by sucrose gradient centrifugation were electrophoresed and immunoblotted . A further, significantly weaker, band of a smaller molecular weight (∼39 kD) was also seen; this may represent a degradation product, but we are unclear of its identity. The latter band was not obvious when post-nuclear membranes from osteoblastic MC3T3-E1 cells were similarly immunoblotted. Note that the purity of the osteoclastic preparations was >99% based on TRAP staining (see Materials and Methods). CD38 is not only an ADP-ribosyl cyclase that converts NAD + to cADPr, but is also an ADP hydrolase converting active cADPr to inactive ADP-ribose. It is difficult to separate the two reactions that proceed simultaneously. We have therefore used an assay that monitors cyclization of NGD + to cGDPr, a nonhydrolyzable cADPr surrogate. Thus, the rate of cGDPr formation, in the absence of its breakdown, will more accurately reflect the ADP-ribosyl cyclase activity of CD38. Furthermore, cGDPr is a fluorescent compound that can be quantitated by fluorimetry. We found that osteoclast plasma membranes synthesized cGDPr at a rate of 4.3 nmoles/min/mg protein . The anti-CD38 antagonist antibody (Sigma Chemical Co.) inhibited cGDPr formation significantly, thus attributing the observed ADP ribosyl cyclase activity to CD38. Enzyme activity was also inhibited significantly by addition of NAD + (400 μM), indicating a possible competition between the two nucleotides. Similar results were obtained from postnuclear membranes prepared MC3T3-E1 osteoblasts (not shown). Having established the presence of CD38 in the osteoclast plasma membrane, we next investigated the effects of its activation by the agonist anti-CD38 antibody. Thus, we measured changes in cytosolic [Ca 2+ ] in response to application of NAD + (substrate) in the presence of the agonist antibody. Expectedly, only in the presence of the antibody (1:500), did 1 mM NAD + trigger a cytosolic [Ca 2+ ] elevation . This result was consistent with an activated CD38/ADP-ribosyl cyclase that catalyses cADPr generation from NAD + . In separate experiments, the anti-CD38 antibody itself, in the absence of NAD + , did not elevate cytosolic [Ca 2+ ], indicating that the substrate, NAD + , was necessary for CD38-induced Ca 2+ signaling (not shown). Finally, 1 mM NAD + failed to trigger a cytosolic Ca 2+ signal in the presence of the control anti-RyR antibody, Ab 34 , further confirming response specificity. At higher, 10 mM, NAD + concentrations, a marked elevation in cytosolic [Ca 2+ ] was noted even in the absence of the antibody . This response was significantly different ( P = 0.013) to the control response (1 mM NAD + alone), but did not differ significantly ( P = 0.22) from the response triggered by 1 mM NAD + with antibody . We further demonstrated CD38-specificity of the NAD + -induced cytosolic Ca 2+ response by preincubating osteoclasts with the anti-CD38 antagonist antibody (Sigma Chemical Co.) before application of 10 mM NAD + . The antagonist antibody attenuated the magnitude of the cytosolic Ca 2+ response significantly . To determine whether NAD + triggered the release of Ca 2+ from intracellular stores, we carried out experiments with 10 mM NAD + in the presence or absence of 2 mM EGTA (to chelate extracellular Ca 2+ to near-nanomolar levels) or thapsigargin (a microsomal membrane Ca 2+ -ATPase inhibitor that is known to deplete intracellular Ca 2+ stores). The response to 10 mM NAD + in Ca 2+ -free, EGTA-containing medium remained unchanged compared with that to 10 mM NAD + in 1.25 mM Ca 2+ ( P = 0.335). Furthermore, Fig. 8 B shows that when cells were treated with 4 μM thapsigargin, there was a significant attenuation of the cytosolic Ca 2+ response to 10 mM NAD + . However, it is notable that thapsigargin did not completely abolish the cytosolic Ca 2+ response to NAD + suggesting that the Ca 2+ signal was not completely dependent upon the fullness of intracellular Ca 2+ stores. Taken together, the results suggested that NAD + primarily triggered the release of Ca 2+ from intracellular Ca 2+ stores, although Ca 2+ influx may also play a role. We next attempted to test the hypothesis that NAD + -induced cADPr generation resulted in the activation of intracellular ryanodine receptors. For this, we examined whether the known cell permeant ryanodine receptor modulators, ryanodine and caffeine, inhibited the response to applied NAD + . Both ryanodine (5 μM) and caffeine (250 μM and 1 mM) significantly inhibited the cytosolic Ca 2+ response to NAD + . Taken together, the results suggest that RyR-gated Ca 2+ stores were being emptied in response to NAD + , implicating, though not proving, a direct role of cADPr as a second messenger. This is consistent with our direct demonstration of cADPr-forming, ADP-ribosyl cyclase activity in the osteoclast plasma membrane as assessed by the NGD→cGDPr assay . It should be emphasized that thapsigargin, ryanodine, and caffeine have all been used as tools to understand the mechanism of NAD + -induced Ca 2+ signaling, and in view of their other cellular actions would not be expected to reverse the effect of NAD + on bone resorption and IL-6 release. We have shown that while a cytosolic Ca 2+ change triggers resorption inhibition, it elevates IL-6 synthesis and release . Our goal, therefore, was to examine the effect of CD38 activation by NAD + (in the presence of its agonist antibody, A10) on bone resorption and IL-6 release. In the presence of A10, at either dilutions (1:5,000 or 1:500), 1 mM NAD + inhibited osteoclastic bone resorption significantly ( P = 0.034 and P = 0.025, respectively, compared with vehicle-treated cells) . Expectedly, osteoclast number per slice did not change significantly ( P > 0.05 for either antibody dilution) , excluding an effect of the antibody on osteoclast formation or demise. In separate experiments, the 1 mM NAD + (in the presence of A10, 1:500), caused a dramatic and highly significant threefold elevation ( P < 0.001) of IL-6 release . Taken together, the results appear consistent with the paradoxical effects of Ca 2+ on bone resorption and IL-6 release . The multifunctional ectoenzyme, CD38, is known to modulate lymphocyte functions as critical as adhesion, proliferation and cytokine production . It also functions as a counter-receptor for CD31, presumably facilitating cell-to-cell communication . It is also an ADP-ribosyl cyclase that catalyzes the formation of cADPr from NAD + . Several reports have suggested that the latter is a cellular second messenger, somewhat akin to IP 3 . We show that CD38 regulates osteoclastic bone resorption via the production of cADPr. Specifically, we show that a novel CD38 homologue is located in the rabbit osteoclast plasma membrane; that it possesses ADP-ribosyl cyclase activity; that its activation results in cytosolic Ca 2+ elevation through ryanodine receptor activation; and that the cytosolic Ca 2+ change is accompanied, quite expectedly, by an elevation in IL-6 release and resorption inhibition. CD38 catalyzes the cyclization of NAD + not only to cADPr , but also to the more recently described, dimeric ADPr . While the classical action of cADPr is to release Ca 2+ from RyR-bearing Ca 2+ stores, dimeric ADPr potentiates this effect . In the osteoclast, we have shown that cADPr triggers both Ca 2+ release and Ca 2+ influx through its action, respectively, on microsomal membrane RyRs and a uniquely positioned surface RyR-2 . Apart from being activated by cADPr, the uniquely positioned osteoclast surface RyR-2 appears also to sense changes in the cell's ambient Ca 2+ concentration during resorption . Any rise in cytosolic Ca 2+ in the osteoclast triggers rapid cell retraction, diminished enzyme release, and reduced acid secretion, culminating finally, in the inhibition of bone resorption . However, an increased cytosolic Ca 2+ also enhances IL-6 secretion, possibly to release an osteoclast from the resorption inhibition induced by a high Ca 2+ . The observed effect of CD38 activation in inhibiting bone resorption and elevating IL-6 release thus mirrors that of Ca 2+ . Notably, both agents act by elevating cytosolic Ca 2+ . Interestingly, however, cADPr-induced Ca 2+ release also mediates the effect of CD38 in inducing other cytokines, including IL-6, interferon-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), and IL-10 . Cyclic ADPr also promotes secretion of hormones, such as insulin from pancreatic β cells, and cytokines from T cells . Nevertheless, it is unclear as to how these released cytokines, in turn, affect CD38 expression and cADPr formation. We provide new evidence that IL-6 enhances the expression of CD38 mRNA. This appears consistent with a NF-IL-6 site in the CD38 gene promoter . Our in situ RT-PCR results, however, must be treated with caution in view of the known technological pitfalls and possible artifacts, which we have tried to avoid. The Ca 2+ -like effects of CD38 might also be relevant physiologically in the metabolic control of bone resorption via NAD + . It is noteworthy that the energy requirement of a resorbing osteoclast is high due to its active secretion of acid and enzymes and its intense motile activity. It is therefore possible that large amounts of NAD + are being generated intracellularly during resorption. Significant amounts of this NAD + may indeed extrude from the osteoclast. Indeed, Zocchi et al. 1999 have demonstrated the existence of a saturable and bidirectional NAD + transport system in a variety of eukaryotic cells; the same could be true for osteoclasts. Alternatively, neighboring cells undergoing apoptosis may release much NAD + . The extracellularly located catalytic domain of the CD38/ADP-ribosyl cyclase could then sense the NAD + , and by catalyzing its conversion to cADPr, limit further osteoclastic resorption. Franco et al. 1998 have demonstrated a role of CD38 in NAD + sensing in HeLa cells and human erythrocytes. Our evidence for the production of cADPr through NAD + catalysis by CD38 is twofold. First, we have directly demonstrated that osteoclast plasma membranes that are positive for CD38 immunoreactivity contain ADP-ribosyl cyclase activity. This has been assessed using an assay that allows for the catalytic conversion of the NAD + surrogate, NGD + , to its nonhydrolyzable and fluorescent derivative, cGDPr. We showed that the observed ADP-ribosyl cyclase activity could be inhibited noncompetitively by an antagonist antibody to CD38, confirming directly, a role for CD38 in cGDPr formation. That NAD + also significantly inhibited NGD + catalysis confirmed further that the two molecules most likely shared the same substrate-binding site. cGDPr formation in osteoclast plasma membranes thus appears truly reflective of the ADP-ribosyl cyclase activity of CD38. Second, and in line with the above, we have shown that NAD + application to osteoclasts triggers cytosolic Ca 2+ release mostly from intracellular stores that are sensitive to inhibition by RyR modulators, ryanodine and caffeine. This, albeit indirect, demonstration for a role of RyRs in NAD + -induced Ca 2+ release further suggests a second messenger role for the generated cADPr. Despite our molecular and biochemical demonstration of functionally active CD38/ADP ribosyl cyclase in the osteoclast plasma membrane, it remains unclear how any cADPr synthesized extracellularly could act on intracellular RyRs. Two explanations have been offered in other models . First, the CD38 catalytic compartment may become internalized after its recognition of substrate, thus generating cADPr intracellularly . In fact, Zocchi et al. 1999 have shown, using endocytic vesicles, that NAD + first internalizes through a saturable transport system independent of CD38, and once, within the vesicle, is catalyzed to cADPr. The latter is then pumped out into the cytosol to affect Ca 2+ release from RyR-gated Ca 2+ stores. It has been suggested that agonist antibodies, such as A10, may aid such internalization . Indeed, A10 is known to enhance the activation and proliferation of B and T lymphocytes through enhanced cADPr production (hence the term, agonist) . Such a mechanism provides one likely explanation for the synergistic effects of the A10 and NAD + on cytosolic Ca 2+ . An alternative possibility, however, also exists. This is that cADPr is first generated extracellularly, and then traverses the cell membrane to interact with intracellular RyRs. Effects of extracellularly applied cADPr on cellular function have been described in rat cerebellar cells , murine B lymphocytes and rat osteoclasts . Franco et al. 1998 have shown, however, using human erythrocyte membranes and CD38-reconstituted proteoliposomes that CD38 is a selective transporter of catalytically generated, but not exogenously added cADPr. Indeed, CD38 internalization is currently the favored hypothesis and could explain our results fully. However, the major goal of this study has not been to probe this mechanism; instead, it has been to identify a plausible role of CD38/ADP ribosyl cyclase in the control of osteoclastic bone resorption. We have provided evidence that the NAD + -induced Ca 2+ signal is made up of two components, Ca 2+ release from RyR-gated intracellular stores, and Ca 2+ influx possibly through the uniquely positioned plasma membrane RyR-2. The role of ryanodine receptors has been generally confirmed through experiments demonstrating that the cytosolic Ca 2+ response to NAD + is inhibited strongly by both ryanodine and caffeine . However, these experiments have not allowed us to determine whether the respective modulators block the intracellular RyRs, or the surface RyR-2, or both. Nonetheless, we show here that the NAD + -induced cytosolic Ca 2+ response is maintained in Ca 2+ -free, EGTA-containing medium, suggesting its dependence on intracellular Ca 2+ release. Our experiments with thapsigargin, a microsomal membrane Ca 2+ -ATPase inhibitor known to deplete intracellular Ca 2+ stores, appear more conclusive. These results show that thapsigargin attenuates, but does not abolish the cytosolic Ca 2+ signal, suggesting that there is a component of extracellular Ca 2+ influx. This, however, remains to be established. In conclusion, we have documented a new function for osteoclastic CD38. We believe that its activation at the osteoclast plasma membrane results in cytosolic Ca 2+ release from RyR gated intracellular Ca 2+ stores via cADPr generation from NAD + . The released Ca 2+ then signals a reduction in bone resorption and a paradoxical elevation of IL-6 release. It is therefore possible that the CD38/Ca 2+ /IL-6 pathway may have a critical role in coupling an osteoclast's metabolic activity with its resorptive function. Our current studies with CD38−/− mice should shed more light on the function of CD38 in osteoclast control . | Study | biomedical | en | 0.999996 |
10477768 | L1-deficient mice are described in Cohen et al. 1998 . In brief, a targeting construct with 5.5 kb of homology with the L1 locus, a thymidine kinase gene and a neomycin resistance gene was used to disrupt the endogenous L1 gene at exon 13 in SV-129 mice. Mice were genotyped by Southern blot analysis of tail DNA. 0.6 kb of L1 cDNA was used as a probe and detected an 11-kb DNA fragment in wild-type and an 8-kb DNA fragment in L1-deficient mice when digested with EcoR1 restriction enzyme. MAG-deficient mice are described in Li et al. 1994 . In brief, a targeting construct with 7.5 kb of homology with the MAG locus, a neomycin resistance gene, and the thymidine kinase gene was used to disrupt the endogenous MAG gene at exon 5 in the 129 × CD1 mouse strain. Southern blot analysis was used to identify the MAG genotype. A 1.6-kb region of the MAG cDNA was used as a probe and detected a 7.5- and a 5.5-kb DNA fragment from wild-type mice and a 5.5- and a 4.5-kb DNA fragment from MAG-deficient mice when digested with BamH1-HindIII restriction enzymes. Genomic DNA was isolated from 1 cm of mouse tail by phenol/chloroform extraction. 20 μg of genomic DNA was digested with restriction enzymes (Boehringer-Mannheim) and run on an 0.8% agarose gel. The DNA was transferred to a Zeta Probe-GT membrane (Bio-Rad Laboratories) and probed with the respective 32 P-labeled cDNAs using High Prime (Boehringer-Mannheim). Hybridization was performed at 65°C for 18 h in the presence of 0.5 M NaPO 4 , 7% SDS, 1% BSA, 1 mM EDTA, 200 μg/ml denatured salmon sperm DNA, and 32 P-radiolabeled probe. Blots were washed at 65°C for 1 h with 40 mM NaPO 4 , 5% SDS, 0.5% BSA, and 1 mM EDTA and exposed to Kodak X-omatAR film with intensifying screens (48 h). P60 control, L1-deficient, MAG-deficient, and L1/MAG-deficient mice were killed by cervical dislocation. Brain and sciatic nerves were harvested immediately, placed in 4% SDS, and dissociated with a Polytron homogenizer. 20 μg of CNS and PNS protein were run on 4–20% SDS–polyacrylamide gradient gels (Novex) and transferred to PVDF membrane (Amersham). Membranes were pretreated with 10% nonfat dry milk and 0.1% Tween-20 in PBS for 30 min at 4°C and then transferred to a solution containing primary antibody, 5% nonfat dry milk, 0.1% Tween-20 in PBS, and incubated for 18 h at 4°C. Blots were washed and incubated in appropriate horseradish peroxidase–conjugated secondary antibodies (Amersham) at a dilution of 1:1,000. Labeled protein was detected with an enhanced chemiluminescence kit (ECL; Amersham) according to the manufacturer's directions on Kodak BiomaxMR film. The MAG and L1 antibodies used to assay protein expression are well characterized. P7 and P60 control, L1-deficient, MAG-deficient, and L1/MAG-deficient mice were perfused intracardially with 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.08 M Sorenson's phosphate buffer. Lumbar dorsal roots 4 and 5 (L4, L5), sciatic nerve, sural nerve, superior cervical ganglia (SCG), and cervical sympathetic trunk (CST) were dissected and postfixed for 1 h. The tissue was then processed for electron microscopy by standard procedures and embedded in Epon (EMS). Ultrathin sections (80 nm thick) were placed on slot grids (EMS), stained with lead citrate and uranyl acetate, and examined in a Philips 100CX electron microscope. Montages of the entire L4 and L5 dorsal roots were constructed from electron micrographs printed at a final magnification of 4,200×. Total unmyelinated axons were quantified in L4 and in L5 dorsal roots from five control, four L1-deficient, and four L1/MAG-deficient mice. The data was analyzed by the Student's t test. The von Frey test uses Semmes Weinstein Monofilaments (Stoelting Co.) to measure skin sensitivity to an applied pressure. This test is used in clinical neurology to assess light touch and deep pressure cutaneous sensation. Unmyelinated C fibers contribute to these sensations as well as deep burning pain, extreme cold and heat, and crude touch . The monofilament will exert an increasing pressure until it begins to bend. Once bending occurs, a constant force is applied to the region, which allows for a reproducible force level for each filament tested. The filaments give a linear scale of perceived intensity and correlate to a log scale of actual grams of force. To administer the test a mouse is scruffed and turned upside down to allow accessibility to the hind paws. A filament is then used to touch the glabrous region of the paw 10 times in 10 s. A response to any of the 10 monofilament applications (toe curling, paw withdrawal) is scored as a positive sensory response. The data was analyzed by the Student's t test. P60 wild-type and L1-deficient mice were perfused intracardially with 4% paraformaldehyde. The dorsal roots, dorsal root ganglia (DRG), sciatic nerves and SCG's were removed and cryoprotected in 2.3 M sucrose and 30% polyvinylpyrrolidone. 1-μm cryosections were cut on a Reichart UltracutS (Leica), placed on slides, and incubated in the following solutions: primary antibody overnight at 4°C, biotinylated secondary antibodies (1:500), Avidin/Biotin Complex (1:1,000) (both from Vector Laboratories), 3,3′-diaminobenzidine tetrahydrochloride (Sigma) and 0.4% osmium tetroxide (EMS). Tissue used for teased fiber preparations was postfixed for one hour in 4% paraformaldehyde, separated in 1% Triton X-100 with teasing needles, treated with Triton X-100 overnight at 4°C, incubated in primary antibody for 48 h at 4°C, and stained as described above. Tissue used for free floating sections was postfixed for 1 h after perfusion, cryoprotected in 20% glycerol overnight, and sectioned at a thickness of 20 μm on a Zeiss freezing sliding microtome. Tissue was incubated in primary antibody for 48 h at 4°C and then stained as described above or by immunofluorescent procedures. Sections processed for double-labeling were incubated in both fluorescein-conjugated donkey anti–mouse and Texas red donkey anti–rabbit (Vector Laboratories) secondary antibodies at 1:500 and mounted in Vectashield mounting media (Vector Laboratories). The polyclonal L1 anti-sera was used at a concentration of 1:2,000 for Western blots and 1:6,000 for immunocytochemistry. MAG polyclonal antisera was used at a concentration of 1:10,000 for both Western blots and immunocytochemistry. The monoclonal CGRP was purchased from Research Biochemicals International and used at a concentration of 1:1,000. The nonphosphorylated neurofilament (SMI-32) was purchased from Sternberger Monoclonals and used at a concentration of 1:15,000. Sciatic nerves segments (4 mm long) were removed from control or L1-deficient mice and sutured into the sciatic nerve of wild-type, L1-deficient, or nude mice as described previously . Surgery was performed under sterile conditions and the mice were housed in a sterile environment until they were killed at 60 d after transplantation. No immunosuppression was necessary. For wild-type or L1-deficient recipient mice, cyclosporin A (Sandoz) was injected daily at a dose of 17 mg/kg. This dosage was shown previously to immunosuppress without affecting nerve regeneration . Four different transplant paradigms were performed. Control or L1-deficient donor nerves were transplanted into nude mice. Control nerves were transplanted into control or L1-deficient mice (both of the SV-129 strain). At 60 d after surgery, the sciatic nerves were harvested and immersed in 3% glutaraldehyde for 30 min. The transplanted portion of the nerve was identified by the sutures used to secure the end-to-end anastomosis. In reference to the transplanted portions, the regions of the sciatic nerve are designated proximal, transplant, and distal. Each portion of the nerve was cut into an ∼2-mm portion, postfixed for 3 h and embedded in Epon for electron microscopic analysis as described above. 1-μm and ultrathin sections were obtained from the middle of each nerve segment . Unmyelinated axons were analyzed by transmission electron microscopy in all three regions of each transplanted nerve ( n > 3) and were scored as either greater than one-half ensheathed or less than one-half ensheathed by Schwann cells. In each segment from each transplant paradigm 300 to 1,100 axons were examined and the data was analyzed by the Student's t test. Mice deficient for both MAG and L1 were generated by initially breeding SV-129 L1 heterozygote females (− x /+ x ) with 129 × CD1 MAG-deficient (−/−) males. From these breedings, female L1 heterozygotes (− x /+ x )/MAG heterozygotes (+/−) were identified and bred to MAG-deficient (−/−) males. Female L1 heterozygotes (− x /+ x )/MAG-deficient (−/−) mice were identified and bred with MAG-deficient males to produce L1/MAG-deficient males (− x / y for the L1 gene and −/− for the MAG gene). MAG-deficient, L1-deficient, and L1/MAG-deficient mice were identified in Southern blots probed with MAG and L1 cDNAs . When probed with the L1 cDNA, wild-type and MAG-deficient DNA (digested with EcoR1) contained an 11-kb band while DNA from L1-deficient and L1/MAG-deficient males contained a band at 8 kb. L1 heterozygote females had bands at both 11 and 8 kb. Wild-type and L1-deficient DNA (digested with BamHI and HindIII) contained two MAG bands (7.5 and 4.5 kb). The MAG-deficient and L1/MAG-deficient mice showed a diagnostic 5.5-kb band as well as the 4.5-kb band. DNA from L1 heterozygote/MAG-deficient females also contained bands at 5.5 and 4.5 kb. The absence of L1 and MAG protein in the respective null mice was confirmed by Western blot analysis of CNS and PNS protein extracts . MAG antibodies detected an appropriate band at 100 kD only in wild-type and L1-deficient mice. L1 antibodies detected a band of ∼200 kD only in wild-type and MAG-deficient mice. A large percentage of the L1-deficient and L1/MAG-deficient males were runted from birth and remained smaller than the male littermates until approximately one month of age. The ultrastructural appearance of unmyelinated fibers was compared in the sciatic nerve and dorsal roots of P60 L1-deficient and wild-type mice. As previously described , nonmyelinating Schwann cells in wild-type sciatic nerve ensheathed individual axons in separate cytoplasmic troughs (data not shown). In P60 dorsal roots from wild-type mice, many unmyelinated axons still remained in polyaxonal Schwann cell pockets . Unmyelinated fibers in MAG-deficient dorsal roots had a similar ultrastructure as the unmyelinated fibers in wild-type dorsal root (data not shown). In L1-deficient dorsal roots examined at P60, many unmyelinated axons were not surrounded or were partially surrounded by Schwann cell processes. Unensheathed axons often contained fragments of basal lamina on their surface indicating former Schwann cell ensheathment. Evidence of ongoing axonal degeneration including axonal swelling, dissociation of microtubules and neurofilaments, and clumping of axonal contents was apparent in the L1-deficient peripheral nerves . Small diameter axons without Schwann cells often abutted the basal lamina of myelinated fibers, and nonmyelinating Schwann cells extended processes passing near but not surrounding many naked axons . The basal lamina was often discontinuous on the surface of these L1-deficient nonmyelinating Schwann cell processes. Lateral adhesion between adjacent Schwann cells of unmyelinated fibers was also disrupted (data not shown). Similar phenotypes were also present in sciatic and sural nerves of L1 and L1/MAG-deficient mice. Myelinated fibers in the L1-deficient mouse appeared similar to those in wild-type mice. Myelin membranes were tightly compacted and the periaxonal space was appropriately maintained at 12–14 nm. Myelination proceeded normally in the L1/MAG-deficient mice and fibers analyzed at P60 showed MAG-deficient phenotypes in the myelinated fibers and the L1-deficient phenotypes in the unmyelinated fibers. Unmyelinated fibers in the cervical sympathetic trunk (CST), a predominantly unmyelinated autonomic nerve were also analyzed. Wild-type nonmyelinating Schwann cells of the CST engulf single axons in cytoplasmic troughs. At P60, the ultrastructure of wild-type and L1-deficient CST unmyelinated fibers was similar. Because L1 may function in neural migration and axon guidance, the phenotype associated with the L1-deficient unmyelinated fibers could be related to a developmental abnormality. This possibility was investigated by analysis of young L1-deficient mice. In electron micrographs of sural nerves from P7 mice, the ultrastructural relationship between Schwann cells and axons in wild-type and L1/MAG-deficient mice was identical and there was no apparent loss of unmyelinated axons in the L1/MAG-deficient mice. At P14, some small diameter axons were not surrounded by Schwann cell processes and by P28 clusters of small diameter naked axons (data not shown) were present in L1- and L1/MAG-deficient mice. Ultrastructural changes in L1-deficient unmyelinated axons were consistent with ongoing axonal degeneration. To extend this observation, the number of nonmyelinated axons was quantified in electron micrograph montages of the entire fourth and fifth lumbar dorsal roots from five wild-type, four L1-deficient, and four MAG/L1-deficient mice . Lumbar dorsal roots 4 and 5 (L4 and L5) from L1-deficient and L1/MAG-deficient mice showed a 30 and 35% decrease of unmyelinated axons compared with wild-type L4 and L5 dorsal roots. This difference was statistically significant ( P < 0.01) using the Student's t test. Myelinated fiber number was similar in control, L1-deficient, and L1/MAG-deficient dorsal roots (data not shown), indicating that the loss of unmyelinated axons is not a result of an increase in myelinated fibers. In addition, the number of nonmyelinating Schwann cell nuclei per unit area was not significantly different between control and L1-deficient mice (data not shown). Loss of sensory axons could result from axonal degeneration, neuronal degeneration, or both. Loss of sensory neuronal perikarya is preceded by cytoplasmic vacuolation, followed by cell necrosis, neuronophagia, and proliferation of satellite cells resulting in residual nodules of Nageotte . In L1-deficient P60 L4 and L5 dorsal root ganglion, pathological evidence of small sensory neuron degeneration was not detected indicating a primary axonal degeneration. Loss of axons without neuronal degeneration is consistent with early stages of a dying-back axonopathy. Possible sensory deficiencies due to the loss of unmyelinated axons was investigated by the von Frey Pressure Test which measures the perception of a predetermined applied force to the skin. The glabrous skin of the hind paws of P60 wild-type ( n = 9) and L1-deficient mice ( n = 7) was tested three times each to assess the response to different diameter monofilaments. All wild-type mice responded to 1.02 g of force by toe curling and paw withdrawal. The L1-deficient mice responded to monofilament forces between 2.041 and 11.749 grams of pressure. This difference was statistically significant ( P < 0.015) using the Student's t test. To investigate if the different phenotype in sensory and sympathetic unmyelinated fibers was due to differential expression of L1, the immunocytochemical localization of L1 was compared in dorsal roots and cervical sympathetic trunks from P60 wild-type mice. Unmyelinated fibers in teased fiber preparations from dorsal roots were positive for L1 . L1 immunoprecipitate was clearly delineated in the nonmyelinating Schwann cells. In transverse one micron thick cryosections from P60 wild-type mouse sciatic nerve L1 antibodies also stained the Schwann cells of unmyelinated fibers . However, the optical resolution of immunostaining in these preparations was insufficient to determine whether L1 was also present in the small diameter axons. Myelinated fibers were not labeled with the L1 antibody. Exclusive detection of L1 in unmyelinated fibers in the PNS was consistent with previous reports . To determine the distribution of L1 on sensory and sympathetic neurons and unmyelinated axons, free floating sections (20 μm thick) of P60 mouse DRG and SCG were double-labeled with L1 and neurofilament antibodies and analyzed by confocal microscopy. Neurofilament antibodies stained large diameter myelinated axons and neuronal perikarya . L1 antibodies labeled the surface of small diameter neurons , the unmyelinated portion of axons as they exit and encircle small dorsal root neurons , and unmyelinated fibers . Small diameter axons of the unmyelinated fibers were also stained for neurofilament, but at this magnification they were not resolvable against the intense L1-staining. Consistent with previous results , L1 was not detected on the satellite cells surrounding the DRG neuronal cell bodies. L1-positive DRG neuronal perikarya were of small diameter (between 20–25 μm), a characteristic feature of neurons that give rise to unmyelinated axons. To extend this observation, we immunostained DRG sections for L1 and calcitonin gene-related product (CGRP), a neuropeptide in neurons that give rise to unmyelinated axons . L1 was detected on the surface and axons of CGRP-positive small diameter neurons , but not large diameter CGRP-positive neurons. These data locate L1 on both Schwann cells and axons of unmyelinated sensory fibers. The nonmyelinating Schwann cells that surround SCG axons were also L1-positive . However, L1 was not detected on the surface of SCG neurons or axons leaving their cell bodies. Therefore, L1 is located on Schwann cells but not axons in unmyelinated sympathetic fibers. The inability of Schwann cells to maintain axonal ensheathment in L1-deficient mice could result from a lack of a homophilic binding between Schwann cell-L1 and axonal-L1, heterophilic binding of Schwann cell-L1, or heterophilic binding of axonal-L1. These possibilities were tested in sciatic nerve transplant paradigms that isolated L1 to either the Schwann cell or the sensory axon. To maintain wild-type nerve transplants into L1-deficient nerves, mice were immunosuppressed with cyclosporin A. To test if cyclosporin A treatment affected axonal regeneration, sciatic nerves from a wild-type SV-129 mice were also transplanted into the sciatic nerves of T cell–deficient nude mice. At 60 d after surgery, axons had regenerated through the graft and donor Schwann cells ensheathed unmyelinated axons and maintained adhesion in both sets of animals. Four percent or less of the unmyelinated axons in all three regions of the transplanted nerves were less than one-half ensheathed by Schwann cells. As described previously in sciatic nerve transplants , these data indicate that axonal regeneration, Schwann cell ensheathment of unmyelinated axons, and myelination (data not shown) occurs in sciatic nerve transplants and is not affected by cyclosporin A treatment. In transplants where L1-positive axons regenerated through a region of L1-deficient Schwann cells , unmyelinated axons were normally ensheathed and could not be distinguished from the proximal or distal region of this control nerve. Two percent of all unmyelinated axons were less than half ensheathed by Schwann cells in all three regions (proximal, transplant, and distal) in this transplant paradigm. In an environment where L1-deficient axons regenerated through a region of L1-positive Schwann cells , 28% of the unmyelinated axons were less than half ensheathed by Schwann cells, indicating that L1-positive Schwann cells are unable to normally maintain ensheathment of L1-deficient axons. In the regions proximal and distal to the transplant where L1 is absent from both the axon and the Schwann cell, 36 and 25% of the unmyelinated axons were less than half ensheathed. The differences in Schwann cell ensheathment of control and L1-deficient axons were statistically significant using the Student's t test. These data indicate that loss of axonal-L1 is responsible for the lack of adhesion between Schwann cell and unmyelinated sensory axons. The objective of the present study was to elucidate the function of L1 in unmyelinated fibers of the PNS by analysis of L1-deficient mice. Our data establishes that L1 maintains Schwann cell ensheathment of sensory but not sympathetic unmyelinated axons. This null mutation phenotype was reproduced when normal Schwann cells were transplanted into L1-deficient sciatic nerve, but not when L1-deficient Schwann cells were transplanted into wild-type sciatic nerve. These data demonstrate that heterophilic adhesion of axonal-L1 is essential for maintaining Schwann cell ensheathment of sensory unmyelinated axons . The L1 null mutation also results in a late onset degeneration of unmyelinated axons and decreased sensory function, consistent with axonal L1 functioning as an adhesion and signaling molecule. Normal ensheathment of unmyelinated sympathetic fibers in L1-deficient mice indicates that the mechanism of adhesion between Schwann cells and unmyelinated axons can be mediated by mechanisms unrelated to L1. The present study established that axonal-L1 is essential for maintaining adhesion, ensheathment, and axonal viability of unmyelinated sensory fibers. L1-deficient mice on a C57Black6 background also have altered Schwann cell ensheathment of sensory unmyelinated axons . L1-mediated adhesion can occur by both homophilic and heterophilic binding and a disruption of either may result in the phenotype seen in the L1-deficient mice. These hypotheses were directly tested by sciatic nerve transplant paradigms. Normal ensheathment of sensory axons by transplanted L1-deficient Schwann cells established that adhesion between Schwann cells and sensory unmyelinated axons does not occur by L1 homophilic nor Schwann cell-L1 heterophilic binding. In contrast, when L1 was absent from the axon and present on the transplanted Schwann cells, a phenotype similar to the L1-deficient mouse developed in the grafted nerve. This data isolates axonal-L1 as the molecule responsible for maintenance of sensory axon-Schwann cell ensheathment and indicates a heterophilic mechanism of adhesion between axonal-L1 and a Schwann cell molecule. While the number of axons in the corticospinal tract is reduced in L1-deficient mice , most CNS and PNS axons reach appropriate targets and form appropriate fiber tracts in developing L1-deficient mice . Analysis of P7 mice in the present study indicates that unmyelinated fibers develop normally in the absence of L1. However, by P60, L1-deficient mice have a significant decrease in sensory unmyelinated axons and decreased sensory function. Whereas loss of axonal-L1 is responsible for abnormal axonal ensheathment, several mechanisms could contribute to loss of axons. Schwann cells can provide extrinsic trophic factors or cell surface ligands that regulate the maturation, normal function, and survival of axons. Adhesion between Schwann cells and sensory axons may be crucial for delivery of Schwann cell trophic factors and the loss of this contact may contribute to axonal degeneration in L1-deficient mice. The majority of mature small diameter DRG neurons express the nerve growth factor (NGF) receptors, p75 and trkA . A small percentage of these neurons also express the brain-derived neurotropic factor (BDNF)/neurotrophin 4 (NT4) receptor, trkB . Schwann cells of unmyelinated fibers secrete NGF and before myelination Schwann cells secrete BDNF and CNTF . NT4 mRNA is present at low levels in sciatic nerve , but it is not known what cell types make and secrete NT4. Lack of ensheathment might disrupt expression or delivery of trophic factors, such as NGF or NT4 and ultimately lead to axonal degeneration. The Schwann cell periaxonal membrane may contain a ligand that helps stabilize the axonal cytoskeletal architecture and thus assures normal axonal function and survival. In support of this mechanism, myelinated axons in MAG-deficient mice have marked alterations in neurofilament phosphorylation, neurofilament spacing, and axonal diameter that precede a late-onset axonal degeneration . While our transplantation studies indicate that Schwann cell L1 is not essential for axonal ensheathment, it is possible that Schwann cell-L1 or another molecule functions as a ligand that helps stabilize axonal cytoskeleton and thus assures axonal viability. Chronic disruption of this trophic effect could contribute to axonal degeneration. The loss of axons in L1-deficient mice may also result from an axonal deficit in processing Schwann cell trophic signals. The correlation between loss of axonal-L1, disruption of Schwann cell ensheathment, and axonal degeneration supports the possibility that axonal-L1 functions as an adhesion and signaling molecule. In the appropriate environment, the cytoplasmic domain of L1 can initiate Ca 2+ influx during neurite outgrowth and Ca 2+ influx is an early event in Wallerian degeneration . It is also possible that the L1 cytoplasmic domain is essential for maintaining the normal architecture of the axonal cytoskeleton by binding to ankyrin . Ankyrin is enriched beneath the axolemma of unmyelinated sensory axons , and L1 and ankyrin colocalize at regions of cell–cell contact . In the optic nerves of ankyrin-B null mice, L1 was initially expressed by optic nerve axons, but was not detected at P7 when the optic nerves began to degenerate . Based upon these correlations, axonal-L1 may assure axonal viability by coordinating signaling events through its cytoplasmic domain or by stabilizing microfilament attachment to the axolemma. The chronic nature and partial penetrance of the axonal degeneration phenotype suggest that the mechanisms responsible for axonal loss are complex and possibly regulated by several interaxonal pathways. Schwann cells can be considered as polarized epithelial cells with their abaxonal surface attached to the basal lamina, their adaxonal surface attached to axons, and their lateral surface attached to adjoining Schwann cells . When basal lamina formation is inhibited by vitamin C deprivation, Schwann cells do not myelinate or appropriately ensheathe axons . While initial polarization of unmyelinated Schwann cells occurs in L1-deficient mice, functional polarization is not maintained in adult sensory fibers. The basal lamina is discontinuous, axonal adhesion and lateral adhesion of Schwann cells to neighboring Schwann cells is lost, and Schwann cell processes extend into the endoneurial space. L1-deficient nonmyelinating Schwann cells, therefore, are not able to maintain a mature, nonmigratory, polarized status. However, this effect was not observed when L1-deficient Schwann cells were transplanted into L1-positive axons. Therefore, axonal-L1 appears to stabilize the polarization of Schwann cell surface membranes via heterophilic binding to a Schwann cell molecule. Schwann cells express L1 as they colonize peripheral nerves and sort axons into to polyaxonal pockets or one-to-one relationships . Myelinating Schwann cell membranes that form the initial one and a half spiral wraps around an axon are also L1-positive . As myelination proceeds, L1 is no longer detected in Schwann cell membranes. L1 antibodies inhibited myelination of DRG axons maintained in vitro , suggesting that L1 was essential for axonal ensheathment and initial spiral wrapping of myelin membranes. Normal myelination of two lines of L1 null mice (C57Black6 and SV-129) indicate that L1 is not essential for initial axon ensheathment nor myelination . Furthermore, it was hypothesized that L1 compensated for loss of MAG during myelination in MAG-deficient mice . The present study demonstrated normal myelination in the absence of both L1 and MAG. The molecule responsible for spiral wrapping of membranes during myelination is not known. The data presented in this report and normal ensheathment and myelination in MAG/NCAM-deficient mice demonstrate that MAG, L1, and N-CAM do not play an irreplaceable role during spiral wrapping of myelin membranes or initial ensheathment of unmyelinated and myelinated axons. | Study | biomedical | en | 0.999998 |
10477769 | All protocols involving mice were approved by the Johns Hopkins University Animal Care and Use Committee (Baltimore, MD). Transgenic mice expressing either the K16 cDNA or the K16-C14 cDNA under the control of the human K14 promoter have been previously described 39 . In short, the cDNAs were subcloned into a modified version of the K14 cassette expression vector which contains 2 kb of human K14 promoter sequence, the rabbit β-globin intron, and 0.6 kb of human K14 poly A sequence 52 . The purified constructs were micro-injected into fertilized C57B6/BalbC3 F 2 mouse embryos. K14 null mice were generated in the 129/SV background as previously described 25 . Two K16-C14 and four K16 transgenic lines were generated with each having a single insertion site. The approximate transgene copy number for the K16 lines ranged from ∼10 (no. 13 line), to ∼15 (no. 6 and no. 10 line), to ∼45 (no. 21 line). To generate the replacement mice, mice heterozygous at the insertion site for the transgene were mated with mice that were heterozygous for the K14-targeted (null) allele 25 . Offspring were screened for the presence of the transgene and the K14-targeting event by Southern blotting as previously described 25 39 . The desired mice were then bred until the replacement offspring were obtained. To determine the levels of transgene mRNA in the various mice, total RNA was isolated from the skin of 7-d-old mice. Skin obtained from killed animals (∼250 mg) was frozen in liquid nitrogen and crushed using a mortal and pestle kept cold with dry ice. The pulverized pieces were homogenized in 2 ml of Trizol reagent (GIBCO BRL) and the total RNA was isolated according to the manufacturer's protocol. Equivalent amounts of isolated RNA (30–40 μg) were electrophoresed using a 1% agarose gel containing formaldehyde and transferred to a nylon membrane (NEN Life Science Products). Membranes were prehybridized for 30 min at 65°C in hybridization buffer . Hybridization was carried out overnight at 65°C. Blots were washed for 20 min at 65°C four times with 0.1× SSC, 0.5% SDS. A 500-bp HindIII-BamHI fragment from the K14 cassette 51 was used as a probe to specifically detect the human transgene mRNAs (K16 or K16-C14 mRNA). An ∼140-bp FspI-HindIII fragment from a plasmid containing the mouse K14 cDNA 18 was used to detect mouse K14. Mouse actin was detected using a probe derived from DNA from Ambion. Urea soluble proteins were isolated from dorsal skin of killed animals as previously described 39 . Equivalent amounts of isolated proteins (∼20 μg, as determined by spectrophotometry and confirmed by Coomassie blue staining) were resolved via 8.5% SDS-PAGE and transferred to nitrocellulose. Western blotting was performed using the alkaline phosphatase method (Bio-Rad Laboratories). Human K16 was detected using the previously described rabbit polyclonal no. 1275 58 . Mouse K16 was specifically detected using RPmK16, a rabbit polyclonal antibody 43 . Mouse K14 and human K16-C14 were detected with the mouse monoclonal antibody LL001 44 that recognizes an identical sequence shared between the human and mouse K14 tail domains. Mouse K5 was detected using the rabbit polyclonal antibody no. 5054 24 while K6 was detected using the rabbit polyclonal K6 general antibody 29 . K17 was detected with the rabbit polyclonal α-K17 antibody 29 and K15 was detected with the rabbit polyclonal antibody UC54 25 . For the solubility experiments, skin from killed animals was crushed as previously described and homogenized in a buffer containing 1% Triton X-100 in PBS and 5 mM EDTA. After centrifugation, the supernatant was isolated (soluble fraction) and the pellet was homogenized in HSE buffer 26 . After another centrifugation, the insoluble pellet was homogenized in buffer A 39 . This urea soluble fraction is referred to as the insoluble fraction. Primary cultures of skin keratinocytes were established as previously described 39 48 . 1–4 d after plating, the primary keratinocytes were processed for immunofluorescence. The coverslips were washed three times in PBS and fixed for 15 min with 100% methanol at −20°C. After three washes with PBS, coverslips were blocked with 5% normal goat serum in PBS. Primary antibodies were diluted in blocking buffer and incubated for 45 min at room temperature. FITC-conjugated goat anti–mouse and rabbit, rhodamine-conjugated goat anti–mouse and rabbit, and FITC-conjugated goat anti–guinea pig secondary antibodies were used to detect bound primary antibodies. Coverslips were mounted onto slides and analyzed via fluorescence microscopy. Primary antibodies used were mouse monoclonals LL001, LL025 (anti-K16; reference 23 ), K8.60 (anti-K10; Sigma Chemical Co.), K8.12 (anti-K16; Sigma Chemical Co.), and rabbit polyclonals no. 1275, K6 general, α-K17, and no. 5054. Mouse tissues were fixed in Bouin's overnight at 4°C. The fixed tissues were embedded in paraffin and 5-μm sections were stained with hematoxylin and eosin or immunostained using the horseradish peroxidase procedure (HRP) by following the manufacturer's protocol (Kirkegaard and Perry Labs.). Primary antibodies used were mouse monoclonals LL001 and K8.60, and rabbit polyclonals no. 1275, K6 general, and α-K17. For electron microscopy, dorsal and ventral skin tissues were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, post-fixed in aqueous 1% osmium tetroxide, and embedded in LX112 epoxy-resin (Ladd Research Industries Inc.). Ultrathin sections (50–70 nm) were placed on copper grids, counterstained with uranyl acetate and lead citrate, and visualized using a Zeiss EM10 transmission electron microscope operated at 60 kV. Mice were anesthesized with avertin and their backs were depilated with Nair (Carter-Wallace). PMA treatment (phorbol-12-myristate-13-acetate; Sigma Chemical Co.) was performed by topically applying 200 μl (50 μm stock in acetone) on days 1, 3, 5, and 7 56 . The tissue was harvested for analysis on day 7. Two methods were employed to test the mechanical integrity of back skin. Scotch Guard tape (3M) was repeatedly applied (∼20 times) to back skin and removed with a sudden motion. The stratum corneum could be seen attached to the tape. Alternatively, depilated back skin was vigorously rubbed using a thumb and forefinger for 30 s. This was repeated every other day (days 1, 3, 5, and 7) over the course of a week. It was also performed right before the tissue was harvested for analysis (day 7). The four previously described K16 ectopic transgenic lines, no. 6, 10, 13, and 21 and the two K16-C14 ectopic transgenic lines, no. B1 and C1 in the C57B6/BalbC3 background 39 were bred with the 129/SV K14 null mice 25 to generate the K16 and K16-C14 replacement mice. Mice heterozygous for the K16 or K16-C14 transgene (Tg+/−) were mated with mice that were hemizygous null at the K14 locus (K14+/−). The desired F1 offspring (Tg+/−, K14+/−) were mated with each other to generate the replacement mice. While a total of nine different F2 genotypes are possible from this mating, the three of interest were: (a) K14 null mice (Tg−/−, K14−/−); (b) heterozygous replacement mice (Tg+/−, K14−/−); and (c) homozygous replacement mice (Tg+/+, K14−/−). In addition, wild-type mice (Tg−/−, K14+/−, or K14+/+) were included as controls. The K14 null mice develop gross blistering over their body surface and die ∼2 d after birth 25 . Thus, this genetic background provides a clear readout for epidermal keratin function in the skin. All four of the K16 replacement lines and the two K16-C14 replacement lines were able to rescue the young mice from death. In addition, either replacement genotype (Tg+/− or Tg+/+, K14−/−) rescued the K14 null phenotype. No replacement mice displayed skin blistering at any body site at an early age. The hyperproliferative phenotype of the K16 ectopic mice is most severe ∼7 d after birth 39 . To determine if there were any comparable morphological or molecular aberrations at the same age in the replacement mice, trunk skin from 7-d-old K16 and K16-C14 replacement mice was examined by light microscopy . Skin tissue sections were analyzed by hematoxylin and eosin staining or by immunohistochemistry. The skin of both types of replacement mice appeared equivalent and normal compared with wild-type skin . In both cases, there were abundant hair follicles correctly oriented in the hypodermis and the epidermides were of normal thickness. No aberrations could be observed in any layers of the epidermis in either type of replacement mouse. In addition, there was no evidence of blistering in the basal layer. The localization of the transgenic proteins was determined by performing immunohistochemical analysis with tail domain specific antibodies. Both transgenes were detected in the outer root sheath of hair follicles and in the basal layer of the epidermis . There was no signal corresponding to K14 in the K16 replacement sample and there was no signal corresponding to K16 in the K16-C14 replacement sample as expected. Based on these data, the transgenes were correctly expressed in a K14-like fashion 39 62 . K10 was used as a marker to determine if early terminal differentiation was normal in the replacement mice . Both samples featured suprabasal staining of the epidermis using the K8.60 antibody, which was identical to a wild-type sample (data not shown). Filaggrin staining was also performed to determine if there were any differences in late terminal differentiation in the replacement mice. No differences were noted when compared with the wild-type sample (data not shown). Keratins K6 and K17, which are commonly associated with hyperproliferation or altered differentiation in skin 55 57 66 were also analyzed. The expression of both was restricted to the outer root sheath of the hair follicles in both cases (data not shown). In addition, BrdU labeling indicated no differences in the number of mitotic nuclei between the two replacement samples and a wild-type sample (data not shown). Based on these multiple criteria, the skin appears to develop and self-renew normally in both types of replacement mice at an early age. These results are in stark contrast to those observed in the K16 ectopic mice that featured multiple hyperproliferative abnormalities and aberrant keratin expression in the skin at ∼7 d 39 , suggesting that the keratin composition of a keratinocyte is crucial in determining the effects of K16 in the skin. To finely assess whether young replacement epidermis was truly similar to wild-type epidermis, ventral skin from 7-d-old wild-type and K16 replacement mice was examined by transmission electron microscopy. There were no discernible morphological differences between replacement and wild-type basal keratinocytes. K16 replacement basal keratinocytes had a low-columnar, cuboidal shape and were tightly packed together. There were no obvious alterations in cell-cell or cell-matrix adhesion. Keratin filaments were loosely bundles and distributed throughout the cytoplasm. These same morphological characteristics were observed in both wild-type and K16-C14 replacement basal keratinocytes (data not shown). There was also no evidence of cell lysis or epidermal blistering. Spinous keratinocytes in the replacement epidermis (K16 or K16-C14) were also normal. Filament bundling occurred with the concomitant increase in the number of desmosomes (data not shown). Cells in the granular layer and the stratum corneum of replacement epidermis were also similar to wild-type (data not shown). By all morphological criteria, the replacement epidermides are equivalent to wild-type epidermis. This is in stark contrast to what was observed in the basal keratinocytes from ectopic K16 phenotypic epidermis in which the hypertrophic basal keratinocytes had large aggregations of keratin filaments and major decreases in cell-cell adhesion 39 . The electron microscopy results prompted the examination of the replacement keratinocytes in culture in order to analyze the global organization of the keratin filaments within the context of an intact keratinocyte. Primary cultures of newborn keratinocytes from K16 and K16-C14 replacement mice were established and analyzed by immunofluorescence to further determine if there were any possible abnormalities in the keratin networks of the K16 replacement mice. K16 replacement keratinocytes appeared normal after one day in culture (data not shown). All cells were positive for K5, K6, human K16, and K17, and negative for K14 (data not shown). As the cells remained in culture, however, they began to display time-dependent changes in their keratin filament networks. In a subset of cells (<50%), the keratin filament networks began to appear fragmented and even absent in some areas of the cytoplasm . There were also large bundles of filaments that were distributed throughout the cell rather than preferentially located adjacent to the nucleus as previously noted for the K16 ectopic keratinocytes 39 . There was no evidence of keratin reorganization, fragmentation, or loss in any of the K16-C14 replacement keratinocytes regardless of the time spent in culture. These keratinocytes featured filament networks that were indistinguishable from wild-type keratinocytes . These data suggest that in the absence of K14, K16 is not able to support a keratin filament network and that over time this fragile network is susceptible to fragmentation and loss. Furthermore, they provide further evidence that the carboxy-terminal ∼105 amino acids of K16 are responsible for these differences in filament organization. We have previously shown that the expression of K16 protein at levels comparable to endogenous mouse K14 was able to cause the hyperproliferative skin phenotype of the K16 ectopic mice 39 . The lack of a phenotype in the replacement mice at an early age raised the possibility that transgene expression was reduced in these mice. To test this idea, urea extractable proteins were isolated from the back skin of 7-d-old heterozygous and homozygous replacement mice from the no. 10 line and compared with wild-type, heterozygous, and homozygous ectopic mice from the same line (no. 10) by Western blot analysis using the no. 1275 antibody which specifically reacts with the tail of human K16 and exhibits partial cross-reactivity with mouse K16 8 30 . As previously documented, the amount of K16 transgene expressed in the homozygous ectopic mouse sample was approximately twice the amount expressed in the heterozygous ectopic sample 39 . Interestingly, there was no difference in transgene expression between the heterozygous and homozygous replacement mice. Furthermore, the level of transgene expression was lower than that observed in the homozygous ectopic sample but higher than the heterozygous ectopic sample. This possible difference between the replacement and the homozygous ectopic samples may be accounted for by cross-reactivity of the 1275 antibody with mouse K16, which is induced in the homozygous ectopic sample . The same type of Western blot analysis was performed with mice from the K16-C14 no. B1 ectopic and replacement lines using the LL001 antibody which detects the K14 moiety of the chimera . As was the case with the K16 transgenics, the amount of K16-C14 approximately doubled from the heterozygous ectopic sample to the homozygous ectopic sample (the upper band in these and the control sample is endogenous mouse K14). The level of transgene expression was similar between the heterozygous and homozygous samples also. However, in contrast to the results observed in the no. 10 line, the amount of transgene expressed in the replacement background was greater than in the homozygous ectopic sample. Further Western blot analysis of urea extractable proteins from 7-d-old trunk skin from the four K16 replacement lines was performed to determine if there were differences in transgene expression between the various replacement lines or between the two types of replacement genotypes (Tg+/− and Tg+/+). Equivalent amounts of protein from both heterozygous and homozygous replacement mice from each of the four K16 lines were analyzed using rabbit polyclonal antibody no. 1275. As was the case with the no. 10 line , the amount of transgene expression was the same within a given line regardless of the genotype . However, the level of expression varied slightly between the lines. Compared with the ectopic mice, the maximal K16 transgene levels are slightly reduced in the replacement background with the exception of the no. 13 line in which the levels are increased (data not shown). Thus, in the K14 null background there appears to be a tightly regulated range of transgene protein levels allowed in a keratinocyte which may be modulated by the levels of K5 and possibly K6 59 . Collectively these data strongly suggest that K14 has a dramatic impact on the regulation of K16 (or K16-C14) in a keratinocyte. The fact that K14 has an impact on K16 levels prompted the mating of the two types of replacement mice to generate double replacement mice that express both transgenes. Urea extractable proteins from 7-d-old double replacement trunk skin was analyzed by Western blot analysis to determine if the two transgenes could influence the expression levels of each other. Proteins from K16, K16/K16-C14, and K16-C14 replacement mice were probed with the 1275 or the LL001 antibody to detect the transgenes. The levels of both K16 and K16-C14 in the double replacement sample were equivalent to the amount produced in the single replacement samples . Although the chimeric transgene contains the last ∼105 amino acids of K14, it was not able to affect the levels of K16 transgene expression. In addition to transgene expression, the levels of other epidermal keratins were examined by Western blot analysis to determine if there were any differences in keratin expression that may account for the phenotypic differences observed between the ectopic and replacement mice at 7 days of age. Equivalent amounts of urea proteins from trunk skin of various ectopic and replacement samples were probed with specific antibodies against K5, K6, K15, mouse K16, and K17 . K5 levels were equivalent in all samples except in the homozygous ectopic and double replacement where the amount of K5 was slightly increased. K6 and mouse K16 levels were equal in all mice except for the homozygous ectopic sample where both were greatly increased. This is consistent with the fact that the epidermis of these mice is hyperproliferative and that K6 staining was observed in the suprabasal layers of these mice 39 . While K17 levels were equivalent among all samples, K15 was dramatically reduced in the homozygous ectopic sample. K15 is expressed in a pattern analogous to K14 in the epidermis but at much lower levels 25 . It is interesting to note that a type I keratin that is such a minor component of basal cells is decreased to such levels while K14 levels remain unchanged 39 . It has recently been reported that K15 mRNA levels in hyperproliferative human skin are decreased 64 . These results suggest that the keratin profiles of the replacement samples are very similar to control and nonphenotypic, heterozygous ectopic samples for those keratins examined. We performed Northern blot analysis on total RNA isolated from 7-d-old ectopic and replacement mouse skin to determine if the protein levels were an accurate reflection of the steady state levels of transgene mRNA. Equivalent amounts of total RNA were analyzed using a transgene specific probe that detects both the K16 and K16-C14 messages but does not cross react with nontransgenic control RNA . The heterozygous ectopic no. 10 sample featured one band that reacted strongly with the transgene probe. The intensity of the K16 transgene in the no. 10 homozygous ectopic sample was increased at least fourfold compared with the heterozygous sample which is not consistent with the protein results. However, when the same samples were analyzed using a probe specific for mouse K14 the amount of this message was also increased in the homozygous sample . These findings were reproducible as other comparable sets of samples from the no. 10 and no. 21 lines yielded similar results. This is consistent with a previous report that showed that the activity of the K14 promoter increases during hyperproliferation 42 . In addition, the mRNA levels of K6 and mouse K16 are elevated only in the ectopic homozygous samples (data not shown). Thus, the human K14 promoter that drives transgene expression is stimulated in the homozygous ectopic sample because of the hyperproliferative conditions prevalent at 7 days of age. The steady state level of K16 transgene mRNA in the homozygous replacement no. 10 sample was lower than that observed in the two no. 10 ectopic samples despite the fact that the amount of K16 protein in the no. 10 replacement line is intermediate between the two heterozygous ectopic samples . The level of transgene mRNA from a homozygous replacement no. 21 sample was much greater compared with the no. 10 replacement sample. However, the amount of transgene protein expressed in skin is essentially equivalent for the two lines suggesting that despite the wide range in steady state transgene mRNA levels between the lines there is a limit to the amount of K16 protein that can be expressed in a replacement basal keratinocyte. In the no. B1 chimera line, there was more transgene mRNA in the homozygous ectopic sample than in the heterozygous replacement sample despite the protein levels being higher in the heterozygous replacement sample . These results provide further evidence that the presence of K14 protein in the skin is acutely critical in determining the levels of transgene protein expression and that the mechanisms responsible may occur at both the transcriptional and post-transcriptional levels. We have previously shown that a single proline residue in the 1B rod domain of human K16 (residue 188) completely accounts for the reduced stability of K16-containing heterotetramers under denaturing buffer conditions in vitro 65 . The reduced stability of these tetramers correlated with the diminished ability of K16 to form 10-nm filaments efficiently in vitro. This led to the possibility that the solubility and partitioning of K16 between the soluble and insoluble keratin pools in the epidermis may be an important factor in determining its effect on keratinocytes 65 . To determine if the partitioning of K16 was different among the various types of mice, skin from 7-d-old mice was lysed and the soluble and insoluble (cytoskeletal) fractions were isolated and subjected to Western blot analysis using various keratin antibodies . There was very little detectable human K16 protein in the soluble fractions from any of the mice. The small amount detectable was proportional to the amount observed in the corresponding insoluble fractions. Thus, there were no obvious differences in K16 solubility among the samples. The same results were observed for mouse K16, which does not contain a destabilizing proline residue in the 1B rod domain (McGowan, K., K. Hess, and P.A. Coulombe, unpublished data, also see reference 43). There were also no differences observed in the partitioning of K5, K6, and K17 (data not shown). The only difference observed was that more K15 appeared to be soluble in the two replacement samples compared with the ectopic samples. These results suggest that there are no major differences in keratin solubility among the types of mice analyzed that could account for the phenotypic differences observed and that the mechanism by which K16 functions probably does not involve a change in its in vivo solubility. Beginning as early as 4–5 wk after birth, the K16 replacement mice begin to lose hair . This alopecia generally initiates at the crown of the head and proceeds in a head-to-tail fashion. The loss occurs primarily on the dorsal surface but also occurs to a lesser extent on the ventral surface. Once lost, the hair does not regrow. While ∼80% of the K16 replacement mice exhibit alopecia, about one in three develop severe skin lesions characterized by epidermal ulceration and scar contraction. These lesions are more prevalent in areas of hair loss and frequent physical contact (limbs, paws, eyes, nose, and mouth areas). Interestingly, the amount of K16 transgene expressed in skin does not change as a function of time. In fact, K16 transgene levels from hairless or lesional skin from the 8-mo-old mice were equivalent to the levels observed in 7-d-old skin (data not shown). These phenotypes occur in mice of either the heterozygous or homozygous replacement genotype, which is expected given that transgene expression levels are equivalent. In addition, the phenotypes observed occur in all four of the K16 replacement lines. However, this phenotype is not fully penetrant as ∼20% of the K16 replacement mice do not exhibit hair loss or skin lesions. This incomplete penetrance may partially be due to the mixed genetic background of the mice (see Materials and Methods). On the other hand, only ∼33% of the K16-C14 replacement mice exhibit minor alopecia and none have developed lesions. As mentioned, a subset of the K16 replacement mice exhibit hair loss and the development of skin ulcerations. To understand these further, tissue samples from 8-mo-old K16-C14 control and phenotypic K16 replacement mice were taken from skin and a variety of other stratified epithelia and examined by light microscopy. Nonphenotypic, hairy skin from a K16 replacement mouse was similar to wild-type and K16-C14 control skin . The epidermis was thin and there were many hair follicles. However, the hair follicles were in the telogen stage as opposed to wild-type skin in which the follicles were in anagen (data not shown). K16 transgene expression was still restricted to the basal layer (data not shown). There was no evidence of hyperproliferation as the epidermis did not stain for K6 and K17 (data not shown). In contrast, K16 replacement skin that exhibited hair loss in the absence of any visible lesions was abnormal in many respects . The epidermis was significantly thickened due to acanthosis and an increase in the number of cell layers. While there were many anagen staged hair follicles that extended deep into the hypodermis, some of the follicles were misoriented and some were without hair shafts . In addition, there appeared to be more sebaceous glands than in the wild-type. K6 expression was observed in the suprabasal layers indicative of hyperproliferation (data not shown). Lesional K16 replacement skin also displayed many abnormalities . The epidermis and the outer root sheaths of the hair follicles were hyperplastic and acanthotic. There was also strong K6 and K17 expression in the suprabasal layers of the epidermis (data not shown). Embedded in the dermis were many large cysts derived from pilosebaceous units. Migration in the K16 replacement epidermis was not inhibited as epithelial sheets appeared to be migrating into an epidermal ulcer . The dermis also featured increased cellularity suggestive of an ongoing inflammatory response. The hair follicles in the lesional skin also extended very deeply into the hypodermis. While nonphenotypic hairy skin from a K16-C14 mouse was normal in most morphological aspects , skin from a hairless region displayed many of the same abnormalities observed in the K16 replacement hairless skin . The epidermis was thickened and the hair follicles were anagen staged. There appeared to be more sebaceous glands and there were also hair follicles that lacked hair shafts (large asterisk). There was, however, no evidence of cyst formation. In addition to skin, other stratified epithelia that feature expression of the K16-C14 control and K16 transgenes in the basal layer, including forestomach and cornea, were examined. Forestomach from a K16 replacement mouse exhibited extensive blistering and basal layer cytolysis along the length of the tissue. On the other hand, forestomach from a K16-C14 replacement mouse appeared completely normal when compared with wild-type (data not shown). Some of the older K16 replacement mice had obvious corneal opacities (data not shown). When corneal tissue from a K16 replacement mouse with opacities was observed under the light microscope, the normal morphology was completely absent . The underlying connective tissue had a dramatic increase in the cellularity, probably due to an inflammatory response. It was also very difficult to discern basal, suprabasal, and differentiating keratinocytes in the cornea. These data clearly demonstrate that K16 expression in the basal layer of various stratified epithelia in the absence of K14 does not result in the normal differentiation and maintenance of these tissues. Hair follicles from K16 replacement and wild-type mice were examined using transmission electron microscopy to determine if there were ultrastructural changes that might account for the hair loss observed. Outer root sheath keratinocytes from the isthmus (permanent) portion of a telogen staged wild-type hair follicle had a normal morphology and the hair fiber stained darkly with OsO4, uranyl acetate, and lead citrate, as expected for wild-type hair 46 . In contrast, anagen staged hair follicles from hairless K16 replacement skin exhibited severe vacuolization within the hair fiber which accounts for the improper formation and absence of hair. In addition, the outer root sheath of the follicle had been invaded by inflammatory cells . There was also vacuolization observed in the K16 replacement keratinocytes of the outer root sheath. Hair follicles from hairless skin that did not exhibit as severe vacuolization in the hair fiber still featured extensive vacuolization in the keratinocytes of the outer root sheath. The vacuolization observed in the keratinocytes of the outer root sheath may be an event that precedes the improper formation of hair observed in the hairless skin from K16 replacement mice. The electron microscopy data from hairless K16 replacement skin suggested that there were major morphological alterations in the outer root sheath of the hair follicle, the hair shaft, and in the correct cycling of the follicles. Hairless and hairy regions of K16 replacement skin were treated with a depilatory agent to remove existing hairs and to stimulate the anagen phase of the hair cycle . Immediately after treatment the regions of skin that were previously hairless had a much darker color compared with the areas that were hairy . As early as 2 d after treatment there were signs of hair regrowth in the region that was previously devoid of hair (data not shown). Five days after treatment with the depilatory agent hair regrowth was obvious in the previously hairless region while hair regrowth had not occurred in the previously hairy regions. After 2 wk the regrown hairs were quite long and hair growth in some of the previously hairy regions had also occurred. Hair regrowth in these regions was slower and more sporadic as some of these areas remained hairless for many weeks after the treatment (data not shown). These results clearly indicate that hair cycle progression and hair growth is aberrant in the K16 replacement mice. Replacement skin was also subjected to mechanical trauma to determine if the epidermis was weakening with age. Skin from the three and 5-mo-old wild-type, K16, and K16-C14 replacement mice was subjected to acute mechanical trauma by tape stripping of the epidermis or to extensive mechanical trauma by vigorously rubbing the skin between a thumb and forefinger over the course of a week. Regardless of the treatment, there was no evidence of blistering in any sample as analyzed by light microscopy (data not shown). Despite the fact that alopecia and epidermal ulcers occur, the K16 replacement skin is still resistant to vigorous acute and prolonged mechanical trauma. Replacement skin was treated with PMA, a phorbol ester which stimulates hyperproliferation (56, and references therein), to determine if there were changes in the proliferative capacity of replacement basal keratinocytes as a function of age. 3-mo-old wild-type, K16, and K16-C14 mice were treated with 50 μm PMA over the course of one week 56 . The epidermides of all three samples were dramatically thickened to a comparable extent after PMA treatment and some spinous keratinocytes exhibited vacuolization (data not shown). However, there were no differences observed among the three samples. These data suggest that the ability of replacement skin to respond to the PMA treatment regimen applied is not compromised. The absence of K14 protein in the basal layer of mouse epidermis results in a lethal phenotype that resembles epidermolysis bullosa simplex 25 . Neonatal mice exhibit severe blistering over their body surface and generally die early after birth. In the absence of K14, the residual keratin filament network (K5/K15) is not able to provide sufficient mechanical support to basal cells, which renders them susceptible to trauma induced rupturing 3 25 49 . Thus, the integrity of basal cells in the epidermis is dependent on the formation of a keratin filament network containing K5 and K14. These results raise the question as to whether basal cells specifically require K14 to achieve a functional keratin filament network. Among the type I epidermal keratins, K14, K16, and K17 share the highest amino acid identity. The identity between human K14 (472 amino acids) and K16 (473 amino acids) is 91%-86%-38% along their tripartite head-rod-tail domain structure 30 60 . Despite this high identity, they are quite distinct in both their assembly properties 40 and in their expression patterns 35 66 . Based on these contrasting facts, we wanted to determine if K16 could provide an equivalent function in the basal layer of mouse epidermis and substitute for K14 by rescuing the lethal blistering phenotype. Expression of K16 in the basal layer of K14 null mice resulted in the rescue of the lethal skin blistering phenotype. The skin developed normally and there was no evidence of blistering at an early age. As the mice aged they exhibited extensive alopecia, developed chronic skin ulcerations in areas of repeated physical contact, and displayed morphological alterations in other stratified epithelia. Comparatively, a small percentage of replacement mice that expressed the K16-C14 chimeric protein exhibited only minor alopecia. While no obvious abnormalities were observed in the filament networks of replacement basal keratinocytes in situ, transmission electron microscopy of ultra-thin sections does not provide the optimal context in which to evaluate their three dimensional organization. In fact, when newborn K16 replacement keratinocytes were placed in primary culture, multiple abnormalities in the organization of their keratin filament networks were observed. Despite the fact that the K16 replacement skin is susceptible to hair loss and epidermal ulcers in areas of frequent trauma, and that an internal stratified epithelia such as the forestomach featured blistering and cytolysis of basal cells, we were unable to induce blistering in the epidermis using a variety of methods. To provide mechanical support to the tissue, keratinocytes from a particular stratified epithelium may have different mechanisms of organizing keratin filaments. These results indicate that K16 can functionally substitute for K14 to a significant, yet incomplete fashion, and that the carboxy-terminal ∼105 amino acids are responsible for a significant fraction of the functional differences between these two keratins. These features are likely to be maintained in mouse K16 as the sequence identity between the orthologs is high (McGowan, K., K. Hess, and P.A. Coulombe, unpublished data, also see reference 43). The multiple anomalies observed in the hair of the K16 replacement mice may indicate a role for keratins in hair growth and follicle cycling. Hair follicles from K16 replacement mouse skin were consistently out of phase compared with those from control mice (data not shown). K16 replacement hairless skin featured large cysts derived from pilosebaceous units and anomalies in outer root sheath keratinocytes that likely preceded the loss of hair. The treatment of hairless K16 replacement skin with a depilating agent stimulated the regrowth of hair. These observations suggest that progression through the hair cycle is partially impaired in the K16, and to a lesser extent, in the K16-C14 replacement mice. A possible explanation for these changes may reside in the outer root sheath where the transgene is expressed. The transition from telogen to anagen is initiated when a subpopulation of K14-expressing keratinocytes in the bulge region of the outer root sheath is induced to migrate down into the dermis when stimulated by a specialized group of fibroblasts, the dermal papillae 5 . It may be that in this context K16 affects the ability of these cells to promote a timely progression through the stages of the hair cycle. Recently, a similar experiment was performed in which the human simple epithelial type I keratin K18 was introduced into the epidermis of K14 null mice 16 . While K5 and K18 were able to form keratin filaments both in vitro and in vivo, the resulting network was not strong enough to withstand mechanical trauma and only partially complemented the lethal phenotype 16 . Paw skin, a body site of constant mechanical friction, exhibited extensive skin blistering in the K18 replacement mice. The cytoplasm of the basal cells were devoid of keratin filaments and featured the presence of densely packed keratin aggregates, which is reminiscent of dominant negative keratin mutants in mice and in Dowling-Meara EBS patients 1 63 . In contrast, no blistering was observed in back skin where the majority of basal cells featured seemingly normal keratin filament networks that were well distributed throughout the cytoplasm and attached at desmosomes and hemidesmosomes. However, when back skin was subjected to acute mechanical trauma, the clumping and aggregation of keratin filaments along with basal cell lysis and blistering occurred 16 . These data suggest that a K5/K18 filament network can form in basal cells but that it is unable to withstand normal mechanical stress and is susceptible to blistering. These results provided evidence that epidermal and simple epithelial type I keratins are not equivalent in vivo. While this may have been predicted considering the comparatively low sequence identity between K14 and K18 (48%) 16 , it contrasts sharply with the K16 replacement mice results. These observations along with the K16 and K18 replacement mice will provide a context for examining the relationship between keratin filament organization and the susceptibility of keratinocytes to mechanical trauma. It has been proposed that the multiplicity of keratin sequences arose from successive gene duplication events 10 . Given their remarkably high sequence identity and their proximity within the type I keratin gene cluster 11 34 52 , it is highly probable that the genes for human K14, K16, and K17 arose from a common ancestor. Our evidence strongly argues that in addition to possessing distinct mechanisms of transcriptional regulation, the genes for K14 and K16 have also evolved to provide a combination of shared and distinct functions at the protein level. These conclusions may apply to the entire family of keratin genes. Another large group of evolutionarily conserved genes, the Hox family of transcription factors, are also organized in a tandem fashion with adjacent genes sharing common functions 15 20 , suggesting that this may be a general feature of large multigene families. To date we have produced three different types of transgenic mice that express human K16 in skin. The tissue specific overexpression of human K16 (under the control of its own promoter) led to widespread follicular keratosis in the skin beginning one week after birth 8 58 . Aberrant keratinization in the outer root sheath of hair follicles resulted in the hyperproliferation and aberrant differentiation of the adjacent inter-follicular epidermis. The appearance of keratin filament aggregates near the nucleus and cytoplasmic areas devoid of filaments in suprabasal keratinocytes preceded the phenotypic changes observed in the skin 40 . In addition, cells were hypertrophic and there were decreases in the number of desmosomes at the cell surface that correlated with blister formation. The ectopic expression of K16 directed to the basal layer of the epidermis (using the K14 promoter) lead to a phenotype consisting of scaly, wrinkled skin that lacked fur 39 . The epidermis was severely thickened and hair follicle morphogenesis and hair production was delayed. Basal cells were hypertrophic, hyperproliferative, and featured keratin filament aggregation. Cell-cell adhesion was also drastically altered in the basal and suprabasal layers of the epidermis. The phenotype improved beginning ∼5 wk after birth. These effects were very reminiscent of what occurs when the EGF receptor signaling pathway is activated in skin. In contrast to the other two mouse studies, K16 expression in basal keratinocytes lacking K14 (this study) did not result in phenotypic epidermis at an early age. There was no evidence of hyperproliferation or developmental and morphological anomalies. It was not until after ∼5 weeks of age that the K16 replacement mice began to exhibit alopecia and lesion formation. What becomes apparent by comparing and contrasting these three different transgenic mouse phenotypes is that the effect that K16 can have on a keratinocyte depends on many factors. These include the location within the epidermis, the differentiation status, and the keratin composition of the keratinocyte. The level to which it is expressed is also critical. The properties of K16 can be classified into context dependent and independent effects. Independent of the expression context, K16 has the ability to reorganize keratin filaments and is able to perturb the normal development and cycling of hair follicles when it is expressed in the outer root sheath. Depending on the expression context, K16 can affect cell size and cell-cell adhesion. It can also affect proliferation, development, and differentiation within the epidermis. An exquisite example of the dramatic effects that the expression context can have on K16 function is proliferation. A recent report has stated that K16 has the ability to promote proliferation when transfected into a variety of cultured epithelial cell lines 41 . It further stated that K16 can antagonize the inhibitory effect that K10 has on proliferation. Our results have provided a direct in vivo test of the relevance of these claims. The presence of K16 in progenitor basal cells of the epidermis (including keratinocytes of the outer root sheath) can either have a positive, negative, or neutral effect on keratinocyte proliferation (39, this study). From these data, it appears that the relationship between K16 expression and the control of keratinocyte proliferation is complex and likely indirect. This interpretation is consistent with the regulation of K16 expression at the edges of skin wounds where the induction of K16 protein does not correlate spatially or temporally with enhanced proliferation in the epidermis 30 40 . We generated four different transgenic mouse lines covering a wide range of copy numbers (see Materials and Methods) that expressed the human K16 cDNA in the basal layer of the epidermis 39 . K16 protein levels were different in the four lines with the highest expressing lines having three times as much K16 as the lowest expressing line. The amount of K16 expressed in the highest expressing lines was approximately equivalent to the amount of endogenous mouse K14. Within each line, mice homozygous for the transgene expressed approximately twice as much transgene as mice heterozygous for the transgene. These data suggested that basal keratinocytes have the ability to accept a wide range of K16 protein. However, there appeared to be an upper limit as two of the lines (with different transgene copy numbers) expressed to approximately equivalent levels (no. 6 and no. 21). It is likely that the levels of the type II basal keratin K5, and possibly K6 59 , determine the limit of transgene expression at the protein level. The amount of K16 expressed in the four lines varied when bred into the K14 null background. However, there was no variation within a replacement line as either the heterozygous or homozygous genotype expressed equivalent amounts of K16 protein. In fact, the amount of K16 transgene expressed in a heterozygous replacement mouse was greater than the amount expressed in a heterozygous ectopic mouse (within the same line). These facts clearly indicate that the presence of K14 in basal cells strongly influences the amount of K16 protein that is present. Similar results were also observed with the K16-C14 replacement mice. One simple possibility that may explain these results is that K16 does not effectively compete with endogenous K14 and may turn over faster in basal cells of the epidermis and the outer root sheath. In fact, in the double replacement there was no evidence of competition or of one keratin influencing the levels of the other. Considering the structure of the two transgenes and the protein data, the region responsible for this behavior lies somewhere between the head domain and the end of the rod domain of K16. One potential biochemical determinant of this behavior may be the proline residue at position 188 65 . While this proline does not appear to influence the solubility of human K16 in vivo, it may still affect its stability. Despite the huge variation in K16 protein levels in the ectopic mice, the amount of K14 protein remained constant regardless of the transgene line and the genotype 39 . Even when K14 mRNA levels are increased due to hyperproliferation (this study, reference 42) or decreased twofold due to the targeted inactivation of the K14 allele (K14 hemizygous null epidermis, reference 25), K14 protein levels remained unchanged. These data imply that despite various genetic changes and biological contexts, the level of K14 protein is tightly regulated in mouse epidermis and is not affected by the presence of K16. In addition, in the absence of K14 protein, there is no mechanism that increases the amount of K15 or other type I keratin proteins in the epidermis to compensate for the absence of K14 25 . This renders the K14 knockout distinct from the targeted disruption of the genes for K4, K10, and K18 in which there were major changes in other keratins at the mRNA or protein level 27 37 42 . While K14 levels are tightly regulated, the amount of K15 protein was dramatically reduced in the phenotypic ectopic mice. This is consistent with a recent report stating that the levels of human K15 mRNA are greatly reduced in hyperproliferative epidermis 64 . Surprisingly, in the soluble pool of the replacement epidermis there was an increase in the levels of K15 suggesting that there may be competitive inhibition of its partitioning into the insoluble pool by K16. Therefore, there may be multiple mechanisms that limit K15 protein levels in the epidermis. Interestingly, K5 protein levels in the K14 null mice were not as dramatically reduced as might have been expected based on the decreased amounts of type I proteins and the fact that in cell culture studies, K5 protein is degraded in the absence of K14 3 22 24 . In fact, in the phenotypic ectopic and the double replacement mice, the amount of K5 increased correlating with an increased amount of type I keratins. These observations serve the underscore the complexity of keratin regulation in the basal layer of the epidermis. In addition to the regulation of K16 at the protein level, there also appear to be mechanisms that regulate it at the level of the mRNA. While the amount of transgene protein doubled from the heterozygous ectopic to the homozygous ectopic mouse, the amount of mRNA did not. In fact, the steady state mRNA levels in the homozygote were much higher (greater than fourfold) than expected compared with the heterozygote based on the protein data. This may be partially accounted for by the increase in the activity of the K14 promoter. Steady state levels of K16 transgene mRNA levels also varied widely among the different replacement lines despite the fact that the protein levels were similar. Cumulatively, these data suggest that there is not a straightforward relationship between the regulation of the K16 mRNA and the expression of the protein. The various transgenic mice that we have generated may provide significant insight into the mechanisms that regulate keratin expression at the level of both the mRNA and the protein. | Study | biomedical | en | 0.999996 |
10491385 | Ran is an abundant GTP-binding protein that is required for the trafficking of proteins and RNA in and out of the nucleus . In general, it forms a complex with nuclear transport signal (NLS) receptors and their cargoes and directs their movement through nuclear pores. Like other small GTP-binding proteins (e.g., Ras), Ran's GTP hydrolysis activity and guanine nucleotide affinity are modulated by accessory factors. GTP hydrolysis is stimulated by a GTPase-activating protein known as Ran-GAP1 and its accessory factor RanBP1 , while replacement of GDP with GTP is accomplished by the guanine nucleotide exchange factor (GEF) RCC1 . Together, these proteins comprise an enzymatic cycle by which Ran binds GTP, hydrolyzes it to GDP (due to the activity of Ran-GAP1), releases the GDP (due to RCC1 activity), and rebinds GTP (due to the presence of a relatively high GTP concentration in the cell). Ran's function as a nuclear transport factor depends upon the nature of its bound guanine nucleotide. Specificity of binding (i.e., to import or export receptors) and direction of movement is determined by whether Ran is bound to GTP or GDP. Ran-GDP typically associates with nuclear import receptors (e.g., importin α/β) and directs their movement from the cytoplasm into the nucleus. Conversely, Ran-GTP generally associates with nuclear export receptors (e.g., CRM1) and directs movement of their cargoes from the cytoplasm into the nucleus . According to this scheme, the Ran protein should be predominantly bound to GTP while in the nucleus and to GDP while in the cytoplasm. This is achieved by compartmentalization of Ran's GAP and GEF. Whereas Ran is localized throughout the cell, RCC1 is bound to chromatin in the nucleus , while Ran-GAP1 and RanBP1 are found exclusively in the cytoplasm . Consequently, Ran-GDP is prevalent in the cytoplasm due to stimulation of GTPase activity by Ran-GAP1 and RanBP1, whereas Ran-GTP is prevalent in the nucleus due to the nucleotide exchange activity of RCC1 . Thus, the localization of the GAP and GEF is paramount in regulating the proper function of Ran in nuclear transport. Whereas the nuclear transport activity of Ran has been extensively characterized, evidence for its role in microtubule regulation has, until recently, been largely circumstantial. An initial hint for Ran involvement in microtubule regulation was derived from the observation that, in the budding yeast Saccharomyces cerevisiae , overexpression of the RCC1 homologue Prp20 can suppress the toxic effects of certain hyperstable α-tubulin mutants . Moreover, yeast strains harboring temperature-sensitive mutant alleles of the yeast Ran-binding protein Yrb1 exhibit spindle misorientation defects due to a lack of astral microtubules . Thus, at a phenotypic level, some mutant forms of Ran-associated proteins can have an effect on microtubule structure in vivo. Well beyond this initial suggestion, a series of recent papers have converged upon the discovery that Ran and its cohorts are key components of aster formation and spindle assembly, especially for spindles assembled in the absence of centrosomes. An initial insight into this process came from the identification of a novel mammalian Ran-binding protein, RanBPM, that could elicit microtubule polymerization. Isolated on the basis of its interaction with Ran-GTP in a two-hybrid assay, RanBPM has been shown to associate with centrosomes, the microtubule-nucleating centers of mammalian cells. Interestingly, it has been observed that overexpression of RanBPM can induce ectopic aster formation in transfected cells. Such asters are structurally similar to normal centrosomal asters in that they contain centrosomal proteins such as γ-tubulin at their foci. Moreover, inhibition of RanBPM or Ran activity can prevent in vitro aster formation from a mixture of purified centrosomes and tubulin. Taken together, these results suggest that Ran and RanBPM somehow act together to effect microtubule nucleation. It has long been known that asters will readily grow from sperm centrioles in Xenopus egg extracts arrested in meiotic metaphase II. Surprisingly, this centriole-mediated process has now been found to be Ran-GTP–dependent. Reduction in the relative levels of Ran-GTP by immunodepletion of RCC1 or by addition of a mutant form of Ran that favors GDP binding , severely inhibits aster formation from centrioles. Therefore, the GTP-bound form of Ran is necessary for centriole-dependent aster formation in these extracts. Conversely, addition of high levels of purified Ran-GTP, GTP-locked forms of Ran (e.g. Ran-GTPγS; Ran G19V, Ran Q69L; RanL45E; see Table ), or high levels of RCC1 strongly stimulates centriole-associated aster growth in sperm-treated extracts as well as de novo aster formation in extracts lacking added chromatin or centrioles . Like the asters formed by overexpression of RanBPM , the asters formed by artificially raising the levels of Ran-GTP include typical centrosome-associated proteins (e.g., γ-tubulin, NuMA, XGRIP109, and XMAP215) at their foci . Strikingly, centrosome-free asters formed by high levels of Ran L45E are capable of forming into bipolar spindle-like structures . In contrast, asters formed without Ran (i.e., by chemically stimulating tubulin polymerization with agents such as dimethyl sulfoxide) do not require γ-tubulin or XMAP215 and do not assemble into higher-order structures . In sum, the findings that loss of Ran function blocks aster formation while overexpression of Ran-GTP induces ectopic formation of microtubule structures clearly implicate the Ran-GTPase cycle in microtubule assembly in M phase. Although it can induce aster assembly in Xenopus extracts, Ran-GTP does not stimulate microtubule polymerization from purified tubulin subunits . Thus, there must exist additional factors that more directly affect polymerization properties. The most plausible candidate now known for such an effector is RanBPM, which both associates with centrosomes and Ran-GTP in mammalian cells. In the absence of centrosomes, the simplest view is that a soluble (i.e., non-centrosomal) portion of RanBPM (or an as yet undiscovered relative) may associate with Ran-GTP generated by chromosomal RCC1, thereby promoting microtubule polymerization adjacent to chromosomes. Is there a role for GTP hydrolysis by Ran in microtubule assembly? It has been suggested that during nuclear transport, the nucleotide-bound state of Ran acts simply as a switch to delineate the direction of movement and that the energy of GTP hydrolysis is not strictly required . The observation that forms of Ran that do not hydrolyze GTP (e.g. Ran L45E, Ran-GTPγS) can induce aster formation in Xenopus extracts , suggests that a similar situation exists for microtubule assembly. However, while GTPγS-bound Ran can induce aster formation, such asters are considerably smaller than those formed by Ran-GTP . Thus, GTP hydrolysis by Ran may have some secondary role in the elongation of previously nucleated microtubules. The recognition that Ran-GTP may be a key component of microtubule nucleation allows resolution of an old cell biological problem: how chromatin can drive spindle assembly, especially in the absence of centrosomes . What now seems likely is that in the absence of a nucleus, chromatin-bound RCC1 and cytoplasmic RanGAP1 produce a natural gradient of Ran-GTP that is most concentrated at the chromosome surface . Consequently, a Ran-GTP–dependent factor such as RanBPM, would stimulate microtubule assembly adjacent to the chromosomes. Then, other cellular factors could act to organize these microtubules into spindles. For instance, it has been shown that the chromosome-associated, kinesin-like protein XKLP1 is required for the stable attachment of microtubules to chromatin . Such chromatin-bound, plus end–directed kinesins could, thus, attach to the newly formed microtubules and draw the rapidly growing plus ends to the surface of the chromosomes . Finally, a complex of cytoplasmic dynein, dynactin and NuMA, previously shown to be required for maintenance of focused microtubule arrays both in the presence or absence of centrosomes , could organize the chromosome-bound microtubules into spindles . This would arise through the microtubule cross-linking activity of NuMA and the retrograde motility of dynein acting together to draw the microtubules into poles at their minus ends. Such a model could explain the inside-out assembly of spindles during vertebrate oogenesis and plant mitosis, both of which are accomplished without centrosomes. Indeed, the works described herein offer some evidence in support of such a model. First there is evidence for the influence of chromatin-dependent components. While it had previously been shown that random segments of DNA attached to a solid-phase support (e.g., polystyrene beads) could initiate centrosome-free spindle assembly in CSF-arrested Xenopus extracts , Carazo-Salas et al. 1999 have now shown that this process requires generation of Ran-GTP by chromatin-associated RCC1. Second, concerning a role in centrosome-free pole formation, it has been demonstrated that microtubules formed by addition of purified Ran-GTP require the activity of cytoplasmic dynein for organization into aster-like structures . The linkage of Ran-GTP to M phase microtubule nucleation reinforces a principle long understood from nuclear transport. Compartmentalization of the Ran GAP and GEF modulate the function of Ran itself. Because of its association with chromatin, RCC1 can establish a high concentration of Ran-GTP exclusively in the vicinity of chromosomes during M phase. This, in turn, can serve as the initial positional cue for spindle formation in the absence of centrosomes. Thus, the observation that the GTP-bound form of Ran can stimulate microtubule polymerization offers a significant insight into the process by which chromosomes drive spindle assembly, especially in the absence of microtubule-organizing centers. | Study | biomedical | en | 0.999995 |
10491386 | Cells were grown at 37°C in an atmosphere of 5% CO 2 in RPMI supplemented with 10% FCS (HeLa S6 cells [provided by W.W. Franke, DKFZ, Heidelberg]), female human diploid fibroblasts (Hv provided by Professor J. Murken, LMU, Munich), SH-EP-N14 human neuroblastoma cells and CHO cells (provided by S. Müller, LMU, Munich). Primary human lymphocytes and C2C12 mouse myoblasts (provided by M.C. Cardoso, MDC, Berlin) were cultured in the presence of 20% FCS. The media were supplemented with antibiotics (100 μg/ml penicillin and 100 μg/ml streptomycin). 4 μg/ml phytohemagglutinin were added to cultures of primary lymphocytes. For synchronization in early S phase, 200 μM mimosin was added to the culture medium for 14–16 h. The block was released by adding fresh medium after washing the cells with PBS. For microinjection and microscopy of fixed cells, cells were grown on coverslips and fixed after replication labeling/nascent RNA labeling in PBS/3.7% formaldehyde for 10 min. Fixed cells were stored in PBS at 4°C and always kept wet during all of the following immunostaining/in situ hybridization procedures. For following the clonal inheritance of replication labeling patterns in living cells, the cells were cultured, microinjected, and imaged in cell culture dishes with a gridded coverslip (cellocate, Eppendorf-Netheler-Hinz GmbH) inserted into the bottom. Replication labeling was performed with exponentially growing cultures or cultures synchronized in early S phase. Replication labeling involving a single BrdU (bromodeoxyuridine) or Cy3-dUTP pulse (Cy3-dUTP was microinjected) or iododeoxyuridine (IdU)/chlorodeoxyuridine (CldU) double pulse labeling was performed as described in according to the time schedules described in the results. FITC-dUTP (Boehringer) was microinjected at a concentration of 100 μM diluted in CMF-PBS (PBS without Ca ++ and Mg ++ ). If synchronized cultures were used, the block was released either after microinjection or before BrdU or IdU was supplemented to the medium to obtain early S phase patterns. To obtain later S phase patterns, the cells were replication-labeled 2–9 h after release. Cells were fixed at the time points indicated in the results either during or after S phase. If necessary, detection of incorporated nucleotides was performed as described in (in the present study BrdU was only detected with a mouse anti–BrdU antibody and an anti-mouse TRITC-conjugated secondary antibody, see BrUTP detection). No detection procedures were necessary for visualizing incorporated fluorescent nucleotides (Cy3-dUTP, FITC-dUTP). Synchronized or unsynchronized cells from cultures of HeLa S6 or CHO cells were replication-labeled by microinjection of FITC-dUTP and grown after replication labeling for 14–19 h. Subsequently, replication-labeled cells were microinjected with 5-bromouridine-5′-triphosphate (BrUTP, Sigma Chemical Co., 50 mg/ml in CMF-PBS). After 10 min of microinjection, cells were fixed. For immunodetection of BrUTP, preparations were blocked for 30 min in PBS, 0.3% Triton X-100, 0.2% Tween 20, and 3% BSA, and subsequently incubated with a monoclonal anti–BrdU antibody (Becton and Dickinson, recognizes also BrUTP) diluted in blocking solution for 1 h. After washing the preparations three times for 5 min with PBS, 0.2% Triton cells were incubated with the secondary antibody (TRITC conjugated goat anti–mouse; Dianova) diluted in the blocking solution. After washing the cells three times for 5 min in PBS, preparations were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml in PBS) and mounted (Vectashield) for microscopy. Cells were replication-labeled either with Cy3-dUTP or BrdU and fixed. Fixed preparations were permeabilized and blocked in blocking solution (PBS, 0.2% Triton X-100, 0.2% Tween 20, 5% BSA) at room temperature for 1 h. Subsequently, cells were incubated with rabbit antiserum R 232/8 diluted 1:500 in blocking solution for 1 h at room temperature. R 232/8 is a high titer rabbit antiserum that is specific for histone H4 acetylated at lysine 8. It recognizes more highly acetylated H4 isoforms (mainly di- and triacetylated isoforms). Cells were washed three times for 10 min with PBS and 0.2% Triton X-100. Afterwards, cells were incubated with an FITC-conjugated goat anti–rabbit antibody (Dianova) diluted in blocking solution. Cells were washed three times for 10 min with PBS and mounted for microscopy if they were replication-labeled with Cy3-dUTP. In the case when cells were replication-labeled with BrdU, they were postfixed with PBS, 3.7% formaldehyde for 10 min. After fixation, DNA was denatured for BrdU detection by incubating the preparations for 10 min in 2 M HCl (for BrdU detection procedure see above). DNA from an H3 isochore fraction of human DNA was amplified and labeled by DOP-PCR with biotin-16-dUTP (Boehringer). Fragment length was checked by gel electrophoresis after DNase I digestion (3 μg/ml for ∼30 min at 15°C). 200 (for metaphase spreads) or 400 ng (for interphase nuclei) of labeled DNA was precipitated with 30 μl CotI-DNA (1 mg/ml; GIBCO BRL) and 5 μl of salmon testis DNA (11 mg/ml; GIBCO BRL). The dry pellet was dissolved in 10-μl hybridization solution (50% formamide, 10% dextran sulfate, 1× SSC). Before hybridization, the probe DNA was denatured in hybridization solution for 6 min at 75°C and preannealed for 20 min at 37°C. Metaphase spreads of SH-EP-N14 cells and primary lymphocytes were prepared according to standard protocols . After denaturation (2 min at 72°C in 70% formamide, 0.6× SSC, pH 7), the preparations were dehydrated in an ethanol series and air dried. Denatured probe in hybridization solution was applied and hybridized overnight at 37°C under a sealed coverslip. Fixed interphase nuclei were pretreated for hybridization and hybridized as described in Zink et al. 1998 . Detection of hybridized probe DNA followed the same procedure for metaphase spreads and interphase nuclei. Preparations were washed for 5 min in 2× SSC at 37°C and three times for 5 min in 0.1× SSC at 60°C and blocked for 1 h at 37°C in 4× SSC, 0.2% Tween 20 (SSCT) with 3% BSA. Cells were incubated with fluorescein avidin DCS (1:200 in SSCT, 1% BSA; Vector Labs, Inc.) for 45 min at 37°C. Cells were washed three times for 10 min with SSCT and incubated for 45 min with a biotinylated anti–avidin antibody (1:200 in SSCT, 1% BSA; Vector Labs, Inc.). After washing the preparations three times for 10 min in SSCT they were incubated again with fluorescein avidin DCS as described above. Cells were finally washed three times for 5 min in SSCT, counterstained with DAPI, and mounted for microscopy (see above). Cells prepared for in situ hybridization were only replication-labeled with Cy3-dUTP. Confocal imaging of nuclei was performed as described in Eils et al. 1996 . Epifluorescence microscopy was performed with an Axiovert microscope 135 TV (Zeiss) equipped with an Attoarc device (Zeiss) to regulate light intensity during living cell imaging and a CCD camera (MicroMAX; Princeton Instruments). For imaging, the Metamorph software (version 3.0; Universal Imaging Corp.) was used. Images were arranged using Adobe Photoshop (version 4.0). Previous studies suggested a nuclear higher order compartmentalization of mammalian genomes according to the replication timing of DNA sequences . The nuclear compartments appear to be established at late telophase/early G1 . However, the stability of nuclear compartments established after mitosis during subsequent interphase stages and the stability of their inheritance was not clear. We were interested in the question of whether compartments comprising DNA sequences with a similar replication timing are indeed reproducibly established after mitosis and stably maintained during all subsequent interphase stages. In this case, a specific S phase pattern produced by DNA pulse labeling during replication should be observed at all other interphase stages and in all daughter cell nuclei. To prove this prediction, we pulse-labeled DNA of different cell lines and primary cells derived from man, mouse, and hamster during the S phase. Studies were performed with the following cell types: primary human diploid fibroblasts (HDFs), HeLa S6 cells, human neuroblastoma cells (SH-EP N14), CHO cells, and C2C12 mouse myoblasts. Pulse labeling was performed with BrdU or Cy3-dUTP. Cells were fixed 30 min after supplementing with the modified nucleotides to obtain typical S phase patterns . Regarding S phase patterns, we mainly followed the classification of O'Keefe et al. 1992 and defined five different types of pattern reflecting an ordered time sequence during S phase progression (type I beginning and type V end of S phase): type I displays hundreds of small foci (∼300 nm in diameter) distributed throughout the DNA within the nuclear interior. DNA sequences located at the nuclear and nucleolar peripheries and minor accumulations of late replicating chromatin within the nuclear interior (see type IV and V patterns) are not labeled. Nucleoli are excluded in all types of patterns. The nuclear space occupied by the type I pattern will be regarded as the interior compartment in the following text. The type II pattern shows some labeling of DNA located at nuclear and nucleolar peripheries. The interior compartment is still partially labeled, but fewer foci are observed here compared with the type I pattern and unlabeled regions appear within the interior compartment. The type III pattern displays heavy labeling of nuclear and nucleolar peripheries (excluded from the type I pattern), whereas the interior compartment is almost devoid of label. The compartments labeled by the type III pattern will be regarded as the peripheral compartments. Type IV and V patterns are characterized by a few large (∼800 nm in diameter) foci that are located within the nuclear interior as well as in the peripheral compartments. Type V displays less labeling of the peripheral compartments than type IV. The totality of late replicating chromatin accumulations within a nucleus labeled by type IV and V patterns will be regarded as the late replicating compartments in the following text. All cell lines examined displayed type I–V patterns. These results confirmed for all cell types used the finding that DNA sequences with a defined replication timing occupy during S phase specific nuclear areas . This way, higher order nuclear compartments are established, comprising DNA with a similar replication timing (e.g., the interior compartment comprises the early replicating DNA sequences). To investigate the stability of these compartments during cell growth and distinct cell cycle stages, we pulse-labeled cells during the S phase as described above. In contrast to the previous experiment, cells were not fixed immediately but after a growth period of 1–5 d. The labeling patterns after this growth period were compared with typical S phase patterns . The patterns we observed 1–5 d after labeling were similar to typical S phase patterns . The distribution of numbers and sizes of foci was the same as that seen in cells fixed during S phase. 200 cells were examined for each cell type and no labeling patterns were observed that could not be classified according to the five types of patterns outlined above. Therefore, we extended the classification of the five types of patterns also to non-S phase cells. The only difference to typical S phase patterns observed in cells grown for 1–5 d was the appearance of unlabeled nuclear areas corresponding to unlabeled chromosome territories . The appearance of unlabeled chromosome territories is due to the random segregation of labeled and unlabeled chromatids beginning at the second mitosis after replication labeling . This phenomenon confirmed that the cells went through at least two mitoses after labeling, and excluded the possibility that cells maintained the patterns because they stopped cycling. As the patterns were often similar in neighboring cells the data suggested that nuclear compartments comprising DNA with a similar replication timing were clonally inherited. To exclude the possibility that cells displayed labeling patterns reminiscent of S phase patterns because of the fact that cells entered S phase again, exponentially growing CHO cells were pulse-labeled with Cy3-dUTP to obtain typical replication labeling patterns. Cy3-labeled cells were grown for 19 h, pulse-labeled with BrdU for 30 min, and fixed immediately . BrdU labeling revealed whether cells displaying typical type I–V Cy3-patterns had entered S phase again. As the cultures grew exponentially, some daughter cells of Cy3-dUTP replication-labeled cells were in S phase at the time point of fixation , whereas others were not. Nevertheless, all cells displayed the typical type I–V Cy3 labeling patterns (60 cells examined for each cell type), indicating that the corresponding nuclear compartmentalization is present during all interphase stages. Similar results were obtained with HeLa S6 cells. To confirm clonal inheritance of nuclear genome compartmentalization, single cells from HeLa S6 cultures exponentially growing on gridded coverslips were replication-labeled with Cy3-dUTP. Cells were not fixed but imaged each day to follow the inheritance of the initial labeling patterns in developing clones. Unfortunately, the motility of the cells usually did not allow the unequivocal identification of cells belonging to one clone (although usually neighboring cells and fields of cells were observed displaying similar patterns). However, this was possible in the case depicted in Fig. 3 where, indeed, all daughter and granddaughter cells displayed the initial type II labeling pattern. To further confirm the inheritance of patterns HeLa S6 cells were synchronized with mimosin in early S phase. Immediately after release of the mimosin, block cells were pulse-labeled for 0.5 h with IdU, chased for 9.5 h, and pulse-labeled for 0.5 h with CldU . We previously established that 93% of cells ( n = 1,000) displayed a type I pattern if they were labeled within the first hour after release. 50% of cells ( n = 1,000) displayed a type III pattern and 18% a type IV or V pattern if they were labeled 10 h later after release. Therefore, most cells after IdU/CldU double labeling according to the scheme outlined above, display a type I pattern (labeled with IdU) as well as type III–V pattern (labeled with CldU). Cells were fixed 14 or 69 h after the CldU pulse, respectively . Many cells fixed 14 h after CldU labeling went through mitosis. After cell division, daughter cells (39 cells examined) displayed the same nuclear compartmentalization as their mother cells after S phase labeling as revealed by the double labeling patterns. For example, cells with the typical morphology of early G1 cells displayed a type I IdU pattern in combination with a CldU type III pattern . These data show that nuclear compartments comprising DNA sequences with a similar replication timing are immediately established after mitosis according to the pattern present in the previous interphase. Similar compartmentalization patterns (IdU type I patterns with CldU type III–V patterns) were also present after at least two mitoses (indicated by the presence of unlabeled chromosome territories) 69 h after initial IdU/CldU labeling. Fig. 4d–f , shows a typical example of the 46 nuclei examined. As single replication-labeled chromosome territories can be observed the data also indicate how single chromosome territories, composed of DNA replicating at distinct time points during S phase, contribute to higher order nuclear compartments. Single territories display a polar organization with DNA replicating early during S phase (IdU-labeled) clustered at subterritorial positions located within the nuclear interior and DNA replicating at later S phase stages (CldU-labeled) clustered at subterritorial positions located at nuclear or nucleolar peripheries. Alignment of polar territories gives rise to higher order nuclear compartments. R-bands of mitotic chromosomes comprise DNA replicating early during S phase and G- or C-bands harbor DNA replicating at later S phase stages . Therefore, one would expect that DNA located within these distinct bands of mitotic chromosomes corresponds to the observed early and late replicating DNA at different subterritorial positions and contributes distinctly to the different higher order compartments. It has been previously shown that clustering of R- and G-band sequences at distinct subchromosomal positions leads to the formation of polar interphase chromosome territories . To confirm the contribution of DNA belonging to specific chromosomal bands to distinct higher order nuclear compartments, we performed in situ hybridization in combination with Cy3-dUTP replication labeling. As a probe for in situ hybridization we used the H3 isochore fraction of human DNA . The H3 fraction usually hybridizes with a subset of R-bands on human mitotic chromosomes . However, when we tested the specificity of hybridization on metaphase spreads of human chromosomes prepared from cultured primary lymphocytes as well as SH-EP N14 human neuroblastoma cells ( n = 20; data not shown) we obtained almost a complete R-banding pattern although different R-bands were stained with variable intensity . Even though the probe hybridized specifically to the whole set of R-bands, which was contrary to previous results (see Discussion), the result was highly reproducible under the conditions we used. As the probe under the conditions we used was an excellent and specific marker for R-band DNA, we hybridized it to replication-labeled nuclei of HeLa S6 and SH-EP N14 cells (in this case it was not possible to include hamster or mouse cells as the probe is specific for human DNA). The hybridization signal was spread all over the interior compartment , but was excluded from the peripheral compartments and the late replicating compartment . The data confirm that R-band DNA builds up the interior compartment, and reveal a clear correlation between the organization of mammalian genomes during interphase and the banded organization of mitotic chromosomes . As R-bands of mitotic chromosomes comprise most of the genes expressed during interphase, one would expect the transcriptionally competent chromatin within the interior compartment comprising the R-band sequences. Hyperacetylated isoforms of histone H4 are a characteristic feature of transcriptionally competent chromatin . To investigate whether transcriptionally competent chromatin is indeed confined to specific nuclear compartments, we stained replication-labeled nuclei with antiserum R 232/8 specifically recognizing hyperacetylated isoforms of histone H4 (see Materials and Methods). Fig. 7 summarizes the results obtained for female HDFs (100 nuclei examined) and mouse myoblasts (70 nuclei examined). Unsynchronized, exponentially growing cultures were fixed 27 h after BrdU pulse labeling and immunostained. Comparison of BrdU labeling patterns and the nuclear distribution of hyperacetylated histone H4 isoforms revealed a clear correlation. Hyperacetylated histone H4 isoforms were detected throughout the whole interior compartment labeled by the type I pattern , but were excluded from the peripheral and late replicating compartments . Also, in female HDFs, a heterochromatic structure that presumably is the Barr body was not stained by antiserum R232/8 . This is in agreement with previous data demonstrating a lack of histone H4 acetylation for mammalian inactive X chromosomes during mitosis . As the transcriptional competence of genome compartments is an important feature with regard to their functional characteristics, we extended the analysis to CHO and HeLa S6 cells. Unsynchronized, exponentially growing cultures of HeLa or CHO cells were fixed 1 or 23 h after replication labeling with Cy3-dUTP to confirm similarity of results between S phase and non-S phase cells. Cy3-dUTP labeling excludes an influence of artificial changes of chromatin structure induced by DNA denaturation (Zink, D., unpublished results) that is necessary for BrdU detection on the results. Cy3-dUTP–labeled CHO nuclei immunostained with R232/8 antiserum were examined and examples are shown in Fig. 8 , a–i. For both groups (fixation after 1 h [ n = 28] or 23 h [ n = 30]) we obtained similar results. There was the same correlation between nuclear compartments revealed by replication labeling and the nuclear distribution of hyperacetylated histone H4 isoforms as observed for HDFs or C2C12 cells . Hyperacetylated isoforms of histone H4 are confined to the interior compartment labeled by the type I pattern . Type III–V patterns do not overlap with regions immunostained by antiserum R 232/8 . Similar results were also obtained with HeLa S6 cells, fixed after 1 h ( n = 20) or 23 h ( n = 20) and double-labeled with Cy3-dUTP and antiserum R 232/8 . The results suggested that not only transcriptional competence, but also that the process of transcription itself also might be subject of higher order nuclear compartmentalization. Nuclear higher order compartments were visualized within HeLa S6 and CHO nuclei by replication labeling with FITC-dUTP (synchronized and unsynchronized cultures used). On the next day, FITC-labeled cells were microinjected with BrUTP and fixed after 10 min of microinjection. Immunostaining of incorporated BrUTP revealed only the interior compartment as the transcriptionally active compartment. . No considerable BrUTP labeling was visible within the peripheral compartments . There was also no overlap between type IV and V FITC patterns and BrUTP incorporation. Similar results were obtained for HeLa and CHO cells. Therefore, the interior compartment comprising the early replicating R-band sequences harbors the transcriptionally competent as well as the actively transcribed chromatin. The data are summarized in Fig. 10 . In this study, we characterized the functional compartmentalization of mammalian genomes during interphase. The data demonstrated that a specific pattern of spatial genome compartmentalization was present during all interphase stages and was clonally inherited. Distinct compartments comprised DNA sequences belonging to distinct chromosomal bands during mitosis. R-band sequences built up a coherent compartment within the nuclear interior, whereas G/C-band sequences localize to compartments at the nuclear and nucleolar peripheries as well as to minor internal compartments. The early replicating compartment comprising the R-band sequences harbored the transcriptionally competent chromatin. Detectable nascent RNA synthesis was confined to the interior compartment. Fig. 10 summarizes the correlations between functional higher order nuclear genome architecture and chromosome organization during mitosis and interphase. The functionally different higher order compartments were built up through the alignment of polar chromosome territories with clusters of early replicating DNA directed towards the nuclear interior and clusters of later replicating DNA located at the nuclear or nucleolar peripheries. We demonstrated recently that early replicating R- or later replicating G/C-bands of mitotic chromosomes are retained as distinct chromosomal domains termed subchromosomal foci (SF) within interphase chromosome territories . Within chromosome territories of cycling (G1) cells, we observed a clustering of R-SF or G/C-SF at distinct sites of the territories . Therefore, the emerging picture is as follows: distinct bands (in the megabase pair size range) alternating on mitotic chromosomes are retained as distinct domains during interphase (SF), but are reorganized within the territories. The distinct SF cluster within a territory and, therefore, give rise to a polar organization of this structure in the size range of several tens to hundreds megabase pairs . Alignment of these polar territories creates higher order functional genome compartments (size range: gigabase pairs) within mammalian cell nuclei . The results and conclusions of the present study are in agreement with the present understanding of chromosome organization on the one hand, and a growing body of evidence indicating functional higher order nuclear compartmentalization on the other. The fact that R-band sequences localize within the compartment harboring the transcriptionally competent and active parts of the genome is in agreement with the fact that R-bands contain the majority of genes and in particular those genes that are actively transcribed during interphase . Sequence identity was confirmed by in situ hybridization with DNA from the H3 isochore fraction . Under the conditions we used, this probe hybridized specifically to sequences present within most of the R-bands of the human karyotype. Although previous studies described hybridization only to sequences present within a subset of R-bands , the result may strongly depend on the actual hybridization conditions. Hybridization to metaphase spreads of human lymphocytes and neuroblastoma cells and the reproducible R-banding observed on chromosomes, demonstrated that the H3 isochore DNA fraction is a reliable probe for R-band DNA under the hybridization conditions we used. Thus, the data clearly demonstrated that R-band sequences localize to the interior compartment. However, the in situ hybridization data do not rule out that these sequences also contribute to other compartments. We presently cannot definitely exclude that a lack of signal in unlabeled compartments is due to accessibility problems. However, we think this is unlikely as we characterized R-band chromatin not only by sequence identity, but also by its replication timing and its content of hyperacetylated histone H4. Early replicating sequences do not obviously contribute to peripheral and late replicating compartments that are also not enriched in hyperacetylated histone H4. As both features are characteristic for R-band chromatin , together the data indicate that R-band chromatin does not make a major contribution to peripheral and late replicating compartments. Although we did not use specific DNA probes to localize G- and C-band DNA within the nucleus, a variety of data indicate that the corresponding sequences localize in the peripheral and late replicating compartments. First, many studies describe the detection of C-band sequences at the corresponding nuclear positions , whereas the localization of G-band sequences at these nuclear positions was suggested by replication labeling studies . G/C-band sequences are known to replicate during the second half of S phase . As our results show that DNA within the interior compartment replicates early in S phase, G- or C-band sequences cannot make a major contribution to this compartment and, therefore, have to be confined to the nuclear compartments comprising the transcriptionally inactive parts of the genome. This is in agreement with the fact that G-band DNA displays a low density of genes that are mostly not expressed and the fact that C-bands comprise highly repetitive sequences . The data are in agreement with many studies indicating that expressed sequences are generally located within the nuclear interior, whereas repressed sequences locate towards the nuclear periphery often in close association with constitutive heterochromatin (C-bands of mammalian chromosomes comprise the constitutive heterochromatin) . However, these studies were performed with a variety of eukaryotic taxa as different as yeast, flies, and mammals. Although the principle of compartmentalization with repressed sequences at perinuclear positions might be general, experimental data indicate that the type of DNA sequences and chromosomal structures located within the different nuclear compartments, as well as the mechanisms mediating genome compartmentalization, might be different in distinct taxa . With regard to the specific localization of chromatin observed in mammals, previous analyses indicated that the process of transcription might not be responsible for nuclear genome compartmentalization in mammals. The fact that the nuclear compartmentalization of mammalian genomes is strongly related to the banding patterns of mitotic chromosomes, and that the nuclear compartmentalization is established immediately after mitosis imply that chromatin belonging to the different bands is targeted to distinct nuclear positions when the nucleus reconstitutes. This process might take place independently for each chromosome . In this case, DNA or chromatin belonging to distinct bands of a chromosome has to be specifically recognized. Recognition could take place at the level of DNA sequence as R- and G/C-bands display a distinct isochore composition . However, we think that unlikely as the active (Xa) and inactive (Xi) X chromosomes of female mammals display a similar DNA sequence, but are affected by the nuclear genome compartmentalization regarding transcriptional competence and activity . As there are specific modifications of R- or G/C-band chromatin that affect also Xa and Xi (Xi is exceptional in the sense that chromatin modifications affect almost the whole chromosome rather than its distinct bands), specific recognition at this level seems possible. Although some chromatin domains might display dynamic relocalizations according to changes in their functional states, the stable maintenance of replication labeling patterns indicates that positional changes of chromosomes or chromosomal regions might be exceptional events within the nucleus. This is in full agreement with recent studies of DNA dynamics within nuclei of living mammalian cells . Nevertheless, rare large-scale movements of chromosomes or parts of them were observed in living cell studies . Such movements are compatible with the observed, stable compartmentalization of functionally defined chromatin, as long as they occur within or between corresponding compartments. Whereas the intranuclear location of the compartments is fixed, at least within similar cell types, there may be a degree of flexibility in the positioning of individual DNA sequences within the compartments. For example, if a centromeric C-band domain moves during interphase from the nucleolar to the nuclear periphery, the overall compartmentalization is not disturbed. In this regard, the observed stable compartmentalization is also compatible with the dynamic repositioning of nuclear domains observed in fixed cell studies . | Study | biomedical | en | 0.999997 |
10491387 | S . cerevisiae strains used in this work are listed in Table . Plasmids used in this work are listed in Table . Rich medium (YPD) was prepared as described . Synthetic complete medium (SC) lacking the appropriate nutrients for plasmid maintenance was as described with the following modifications. Tryptophan was added to 40 μg/ml, leucine to 100 μg/ml, and glutamic acid, aspartic acid, and serine were omitted. Ammonium and proline media, or media without ammonium or proline as a nitrogen source, were prepared using yeast nitrogen base without ammonium sulfate and amino acids supplemented with 2% glucose, 40 μg/ml tryptophan, 20 μg/ml histidine, 100 μg/ml leucine, and either 10 mM ammonium sulfate or 1 mg/ml proline, or no ammonium or proline. Rapamycin (gift of Sandoz Pharmaceutical) was dissolved at 1 mg/ml in drug vehicle (90% ethanol and 10% Tween-20) and added to liquid media to a final concentration of 200 ng/ml. Yeast transformation was performed by the lithium acetate procedure . Escherichia coli strain DH5α was used for propagation and isolation of plasmids as described . Restriction enzyme digests and ligations were done by standard methods. Enzymes and buffers were obtained commercially (Boehringer Mannheim). DNA was sequenced by the dideoxy-chain termination method with the T7 sequencing system (Pharmacia). HA-TAT2 (in pAS55, pAS64, or pTB287) encodes an NH 2 -terminally HA-tagged, fully functional TAT2 protein under control of its own promoter. HA-TAT2 was constructed by ligating a 0.65-kb PstI-XbaI PCR product, containing the TAT2 promoter, 5′ untranslated region, initiation codon, and double HA tag, to a 2.3-kb XbaI-EcoRI PCR product containing the TAT2 open reading frame and 3′ noncoding region. The PstI and EcoRI sites were natural sites flanking the TAT2 gene. The PCR primers used to generate the 0.65-kb fragment were 5′-CG TCTAGA TGCATA GTCCGGGACGTCATAGGGATAGCCCGCATAGTCAGGAACA - TCGTATGGGTA CAT ATGAGAGTGTGTTGCGTAATTTG-3′ (XbaI site in italics, antisense 2×HA open reading frame underlined and antisense initiation codon in bold) and the 1233 primer (New England Biolabs). The primers used to generate the 2.3-kb fragment were 5′-CG TCTAGA ACCGAAGACTTTATTTCTTCTGTC-3′ (XbaI site in italics) and the −20 primer (New England Biolabs). To create deletion variants, the following primers were used in combination with the −20 primer (New England Biolabs) to generate alternative 2.3-kb fragments: 5′-GG TCTAGA ATGCGTTCAAATGAGGAGCTG-3′ ( TAT2 Δ 10 ), 5′-GG TCTAGA ATGGAGCGAAAATCTAACTTTGG-3′ ( TAT2 Δ 17 ), 5′-CG TCTAGA ATGTCTAACTTTGGATTTGTAG-3′ ( TAT2 Δ 20 ), 5′-CG TCTAGA TCCAAGCAATTAACATCATC-3′ ( TAT2 Δ 29 ), 5′-CG TCTAGA TCCAGGCAATTAACATCATC-3′ ( TAT2 Δ 29 K31R ) and 5′-GG TCTAGA CAATTAATATCATCCTCATC-3′ ( TAT2 Δ 31 ). An internal deletion ( TAT2 Δ 17-31 ) was constructed in a two-step PCR with the complementary primers 5′-TCAAATGAGGAGCTGCAATTAACATCATCC-3′ and 5′-GGATGATGTTAATTGCAGCTCCTCATTTGA-3′, each in combination with a flanking primer and pTB287 as the template. TAT2 5K > R was reconstituted from two PCR fragments generated with primers 5′-GC GAGCTC CTCATTTGAACGC C TGACAGAAGA-3′ (antisense) and 5′-GAG GAGCTC A G GGAGCGAA G ATCTAACTTTGGATTTGTAGAATACA G ATCCA G GCAATTAACATCAT-CCTCATC-3′ (sense), each in combination with a flanking primer and pTB287 as the template. A silent nucleotide substitution that created a SacI site (italics) is indicated in bold, and point mutations that changed lysine codons to arginine codons are underlined. Vent DNA Polymerase (New England Biolabs) or Taq polymerase (Boehringer Mannheim) was used in PCR. Mutations, deletions, and introduction of the HA tag-encoding sequence in TAT2 were verified by sequencing. The import rates of radiolabeled amino acids ( l -[5- 3 H]-tryptophan (33 Ci/mmol), l -[4,5- 3 H]-leucine (58 Ci/mmol), and l -[2,5- 3 H]-histidine (42 Ci/mmol; Amersham) were measured as described , with the following modifications. Wild-type (JK9-3d) cells were grown to early logarithmic phase in YPD medium at 30°C, and the culture was split into six equal aliquots. Individual aliquots were incubated at 30°C in the presence of rapamycin for either 0, 15, 30, 45, 60, or 90 min. Uptake of labeled amino acids (% import/OD over time) was determined for cells of each aliquot. For the graph in Fig. 1 A the 10-min timepoint of each curve was plotted over the rapamycin preincubation time of the given aliquot. To prepare whole cell extracts for SDS-PAGE and Western analysis, cells were grown in SC medium to early logarithmic phase, resuspended in ice-cold extraction buffer (120 mM NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM PMSF, and 1% NP-40) and lysed with glass beads in a Mini-Beadbeater (Biospec Products). Unbroken cells and debris were removed by a 500- g spin, and protein concentrations were determined using the BioRad microassay. Samples were denatured at 37°C for 10 min. A total of 50 μg protein was loaded per lane for standard SDS-PAGE (10% acrylamide) and Western analysis . For detection of tagged proteins, a rat anti-HA antibody (clone 3F10; Boehringer Mannheim) and mouse anti-myc antibody (9E10; kindly provided by H.-P. Hauri, Biozentrum, University of Basel, Basel, Switzerland) were used. For signal detection, the Amersham ECL kit was used; stripping of blots and reprobing were performed as recommended by the manufacturer. Untreated logarithmically growing cells or cells treated with rapamycin for 60 min were fixed for 2 h in the growth medium supplemented with formaldehyde (3.7% final) and potassium phosphate buffer (100 mM final, pH 6.5). Cells were washed and resuspended in sorbitol buffer (1.2 M sorbitol and 100 mM potassium phosphate, pH 6.5). Cell walls were digested for 30 min at 37°C in sorbitol buffer supplemented with β-mercaptoethanol (20 mM final) and recombinant lyticase (5 mg/ml) or zymolyase 20T (12.5 mg/ml; Seigagaku Corporation), yielding identical results. Spheroblasts were fixed on poly- l -lysine–coated glass slides and permeabilized with PBT (53 mM Na 2 HPO 4 , 13 mM NaH 2 PO 4 , 75 mM NaCl, 1% BSA, and 0.1% Triton X-100). Immunofluorescence directed against the HA-epitope was performed by application of a high affinity monoclonal anti-HA antibody (clone 16B12; Babco) at a dilution of 1:1,000 in PBT for 2 h, and subsequently of a Cy3-conjugated rabbit anti–mouse IgG (Molecular Probes), diluted 1:1,000 in PBT, for 60 min. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) at a concentration of 1 μg/ml. Cells were visualized with a Zeiss Axiophot microscope (100× objective) and a video imaging system (MWG Biotech). Logarithmically growing wild-type cells (JK9-3d) expressing HA-TAT2 were diluted in 400 ml SC medium (starting OD 600 = 0.1) containing 2 mCi of L-[ 35 S]methionine (Easytag protein labeling system; NEN). After 4 h (OD 600 = 0.3), a 1,000-fold excess of cold methionine was added. The culture was split, and rapamycin or empty drug vehicle was added. Temperature-sensitive sec4 cells grown at 24°C to early logarithmic phase and expressing HA-TAT2 or HA-TAT25 K>R were chilled on ice, harvested by centrifugation, and immediately resuspended at an OD 600 = 5 in 12 ml of SC medium containing 2 mCi of l -[ 35 S]methionine instead of cold methionine. Cells were incubated for 3 min at 24°C, then shifted to 37°C for an additional 5 min. The cultures were chilled on ice, harvested, and resuspended at an OD 600 = 0.5 in prewarmed (37°C) SC medium containing cold methionine and rapamycin (200 ng/ml). Culture aliquots equal to 15 OD 600 of cells were removed after 0, 30, 60, and 90 min, and kept on ice for 10 min in the presence of 10 mM NaN 3 and 10 mM NaF. Cells were harvested and cell extracts prepared as described above. Extracts were precleared for 30 min with Sepharose CL-4B, and incubated for 2 h at 4°C with protein G immobilized on Sepharose CL-4B (Sigma) and monoclonal anti-HA antibodies (12CA5 or 16B12; Babco), in a volume of 1 ml. The beads were collected and washed extensively with extraction buffer. Bound protein was solubilized by incubating in SDS sample buffer for 10 min at 37°C. Gels were scanned and signals quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software. Ubiquitinated TAT2 was visualized in Western blots by use of monoclonal anti-myc antibody (clone 9E10). For this purpose, myc-tagged ubiquitin was expressed as described , in wild-type cells (JK9-3d) also expressing HA-TAT2, HA-TAT25 K>R , or untagged TAT2. The permease was immunoprecipitated from extracts of these cells (from a total of 1 mg of protein) as described above. We observed that amino acid prototrophic yeast strains are less sensitive to rapamycin than auxotrophic strains (Beck, T., A. Schmidt, and M.N. Hall, unpublished observation). Because auxotrophic strains rely on the uptake of externally added amino acids for growth, this suggested that rapamycin inhibits amino acid import. We measured the import of radiolabeled tryptophan in cells treated with rapamycin for various times, and found a significant and early decrease in tryptophan uptake . The time-dependent decrease in tryptophan import was followed, with a short time lag, by a downregulation in the level of TAT2 protein . Tryptophan import and TAT2 protein dropped to almost undetectable levels after 60 min of rapamycin treatment. As rapamycin causes starvation in yeast cells, the rapamycin-induced downregulation of TAT2 appeared to be a novel starvation response. To confirm that the downregulation of TAT2 is indeed a starvation response, we examined if nutrient deprivation causes a similar downregulation. Wild-type cells grown to early logarithmic phase in medium containing glucose, ammonium, and required amino acids (SC medium) were harvested by filtration and resuspended in normal SC medium and in nutrient-modified SC medium. TAT2 protein levels were then examined after 60 min of incubation in the new media. First, the medium was modified by increasing, decreasing, or removing tryptophan. Whereas increasing (20×; 800 μg/ml) and decreasing (0.1×; 4 μg/ml) the tryptophan concentration had little or no effect, tryptophan starvation resulted in an almost complete loss of TAT2 protein . Second, cells were shifted to medium modified in the nitrogen source, i.e., medium containing a reduced amount of ammonium (0.1×; 1 mM), no ammonium, or proline instead of ammonium. As shown in Fig. 1 B (middle panel), nitrogen limitation correlated with a decrease in the level of TAT2 protein, up to a complete loss of the permease in ammonium-free and proline media. In contrast to TAT2, GAP1 is activated upon shift from ammonium to the poor nitrogen source proline, and inactivated (degraded) upon shift from proline to ammonium , suggesting that TAT2 and GAP1 are inversely regulated in response to the nitrogen source. Third, cells were shifted to media with reduced (0.1×; 0.2%) or no glucose. The levels of TAT2 protein again correlated with nutrient availability, dropping to undetectable levels in the absence of glucose . Thus, the downregulation of TAT2 protein is a novel starvation response, caused by either rapamycin treatment or amino acid, nitrogen or carbon deprivation. To determine whether the nutrient regulation observed for TAT2 also occurs for other high affinity constitutive permeases, we measured histidine import in rapamycin-treated cells. The effect of rapamycin on histidine uptake was similar to that observed for tryptophan uptake (not shown). We also examined the abundance of myc-tagged histidine permease HIP1 in rapamycin-treated cells. Like TAT2, HIP1 almost completely disappeared within 60 min of rapamycin treatment . Thus, starvation-induced downregulation is not unique to TAT2, but also applies to another, and possibly to all, so-called constitutive amino acid permeases. The starvation program in yeast may involve a global downregulation of high specificity permeases. In contrast to TAT2 and HIP1, rapamycin treatment caused a significant increase in GAP1 protein, even in the presence of nutrients . The abundance of SHR3, an ER-resident amino acid permease chaperone , was not significantly affected by rapamycin treatment . Thus, the rapamycin-sensitive TOR proteins appear to regulate inversely the high specificity permeases, such as TAT2 and HIP1, and the broad specificity permease GAP1, in response to nutrient availability. Rapamycin also causes a downregulation in translation initiation . Because the downregulation of translation is earlier than the observed downregulation of TAT2, the decrease in TAT2 levels could reflect a loss of de novo protein synthesis in combination with a normally short half-life of the permease. We performed a pulse–chase experiment to determine whether the loss of TAT2 resulted from increased turnover or solely from reduced synthesis of the protein. Cells expressing HA-tagged TAT2 were grown to early logarithmic phase in the presence of radiolabeled methionine, and chased with an excess of cold methionine in the presence or absence of rapamycin. Culture aliquots were taken at 15-min intervals and processed for immunoprecipitation of the permease. As shown in Fig. 2 , the half-life of TAT2 in the rapamycin-treated cells was ∼30 min, whereas in the rapamycin-untreated cells the half-life was >90 min. Thus, the TAT2 protein is significantly more stable in exponentially growing cells than in starved cells, indicating that starvation induces degradation of TAT2. Degradation of TAT2 is likely to occur in a manner similar to that of other permeases such as FUR4, GAP1, MAL61, and GAL2. Upon NPI1/RSP5-dependent ubiquitination, these proteins are internalized, transported to the vacuole, and degraded by vacuolar hydrolases . To investigate the requirements of TAT2 degradation, we analyzed the steady state levels of HA-TAT2 in mutant and wild-type cells treated with rapamycin or drug vehicle alone . In wild-type cells, TAT2 was almost completely degraded within 60 min after addition of rapamycin, as described above. In an npi1/rsp5 mutant, defective in the ubiquitin-protein ligase NPI1/RSP5 , the amount of TAT2 protein was significantly increased in logarithmically growing cells, as compared with a wild-type strain ( NPI1 ), and the permease was still present in rapamycin-treated cells. To confirm the involvement of ubiquitin in the rapamycin-induced degradation of TAT2, we examined another ubiquitination-deficient mutant. The doa4/npi2 mutant, defective in a ubiquitin hydrolase , has a ubiqitination deficiency , possibly due to a defect in replenishing the pool of free ubiquitin . Like in npi1/rsp5 cells, TAT2 was resistant to rapamycin-induced degradation in the doa4/npi2 mutant. In an end4 mutant , defective for endocytosis of several plasma membrane proteins, TAT2 was weakly (∼10%) resistant to rapamycin-induced degradation. In a pep4 mutant lacking vacuolar proteases, TAT2 was completely resistant to rapamycin-induced degradation. To further examine the requirements of TAT2 degradation, we analyzed the cellular distribution of TAT2 in the above mutants and wild-type cells treated or untreated with rapamycin . Visualization of HA-TAT2 in rapamycin-untreated wild-type cells by immunofluorescence revealed a strong ER signal, a punctate signal reminiscent of Golgi-localized proteins, and only weak plasma membrane staining. This localization of TAT2 differs from that of the uracil permease FUR4, which is found mainly in the plasma membrane , but is similar to that of GAP1 which can be found mainly in the ER and Golgi . Furthermore, the distribution of TAT2 was the same, although the signal differed in intensity, when HA-TAT2 was expressed from a single copy plasmid (pTB287) or a multicopy plasmid (pAS55 or pAS64) (data not shown). In rapamycin-treated wild-type cells, the TAT2 signal became very faint, as expected, due to degradation of the TAT2 protein, but did not change significantly in pattern (data not shown). In rapamycin-treated npi1/rsp5 cells, TAT2 accumulated in the plasma membrane and the vacuolar membrane. The TAT2 protein behaved similarly in the doa4/npi2 mutant as in the npi1/rsp5 mutant (data not shown). The accumulation of TAT2 in the vacuolar membrane of the doa4/npi2 and npi1/rsp5 mutants is possibly due to a constitutive vacuolar protease defect in these nonconditional mutants . In rapamycin-treated end4 cells (shifted to nonpermissive temperature before drug treatment), the TAT2 signal was faint and corresponded mainly to the plasma membrane, indicating that an endocytosis defect protects plasma membrane TAT2 but not internal TAT2 from rapamycin-induced degradation . The stabilization of only the plasma membrane pool of TAT2, a small portion of the total pool, by an endocytosis defect accounts for why TAT2 was only weakly resistant to degradation in the end4 mutant (see above). In rapamycin-treated pep4 cells, TAT2 accumulated mainly in the vacuolar membrane. The cellular distribution of TAT2 in rapamycin-untreated doa4/npi2 , npi1/rsp5 , and pep4 cells was the same as described above for these cells when rapamycin treated. This may reflect a basal level of TAT2 turnover in exponentially growing (rapamycin-untreated) cells. Because the doa4/npi2 , npi1/rsp5 , and pep4 mutations are constitutive, a defect in basal internalization and degradation of TAT2 may result in an aberrant accumulation of the permease even in growing cells. To investigate further the involvement of ubiquitination in TAT2 degradation, we examined if TAT2 itself is ubiquitinated. The permease was immunoprecipitated from rapamycin-treated cells expressing HA-TAT2 alone or coexpressing HA-TAT2 and myc-tagged ubiquitin (myc-Ub) . Probing with an anti-HA antibody revealed the signal for TAT2 (∼50 kD) in both immunoprecipitates, whereas probing with an anti-myc antibody revealed only >50 kD proteins solely in the immunoprecipitate derived from cells coexpressing myc-Ub . Neither signal was detected in immunoprecipitates from cells expressing an untagged version of TAT2. This suggests that TAT2 is ubiquitinated. Taken together, the above results indicate that TAT2 degradation involves ubiquitination, vacuolar proteases, and, in part, endocytosis. Ligand-induced ubiquitination and internalization of the pheromone receptor STE2 involves the sequence SINDAKSS within the cytosolic tail of the receptor . Replacement of the aspartic acid to alanine or the lysine residue to an arginine in the DAKS core of this motif impairs the internalization of a truncated form of the receptor, whereas mutation of the aspartic acid to glutamic acid, or substitution of the first serine by an alanine, did not interfere with endocytosis. According to a structure prediction by the TOP PRED algorithm, both the NH 2 -terminal and COOH-terminal sequences of TAT2 face the cytoplasm. In the extreme NH 2 terminus of TAT2, two ExKS motifs, similar to the DxKS core of the STE2 SINDAKSS motif, are present . To determine whether the NH 2 terminus of TAT2 is required for the degradation of the permease, we expressed several NH 2 -terminal deletion variants of the permease, and assayed their stability in rapamycin-treated cells . Whereas deletion of up to amino acid 29 (TAT2Δ29) had no effect, deletion of the NH 2 -terminal 31 amino acids (TAT2Δ31) completely stabilized the protein . To further characterize the sequence required for degradation, a smaller deletion that completely removed the two ExKS motifs within the NH 2 -terminal 31 amino acids was constructed . However, this deletion did not result in a degradation-resistant variant of TAT2, suggesting that the ExKS motifs are not essential for TAT2 degradation. Ubiquitin is attached to lysine residues in target proteins. There are five lysine residues in the NH 2 -terminal 31 amino acids of TAT2, and the above analysis suggests that all five lysines must be deleted to prevent TAT2 degradation. To determine if one or more of the lysines is required for degradation, possibly as an acceptor for ubiquitin, all five lysines were changed to arginines. Single lysine mutants and several combinations, including a combination of all five substitutions, were examined for stability upon rapamycin treatment . Stabilization of TAT2 was observed only when all five lysine residues were exchanged for arginines. Furthermore, the TAT2 5K>R protein was approximately threefold more abundant (in rapamycin-treated and untreated cells) than wild-type TAT2 in rapamycin-untreated cells (data not shown). This increase in the level of TAT2 5K>R was similar to the increase of wild-type TAT2 caused by a defect in the ubiquitination machinery, as described above, and may again reflect a loss of basal turnover of the permease. Immunofluorescence on rapamycin-treated and -untreated cells revealed a pronounced accumulation of TAT2 5K>R in the plasma membrane, with no detectable signal elsewhere in the cell . As observed for wild-type TAT2 in ubiquitination mutants, the similar pattern for rapamycin-treated and -untreated cells may reflect an aberrantly high accumulation of the stabilized permease in the plasma membrane even in growing cells. The TAT2Δ29 K31R and TAT2Δ31 proteins behaved identically to TAT2 5K>R with regard to both abundance and localization (data not shown). Finally, probing immunoprecipitated TAT2 5K>R for ubiquitin failed to reveal any ubiquitinated forms of TAT2 5K>R . Taken together, these results suggest that at least one lysine residue within the NH 2 -terminal 31 amino acids of TAT2 is necessary for ubiquitination and degradation of at least the plasma membrane pool of the permease. If internal TAT2 is transported to the vacuole via the plasma membrane, TAT2 should be completely stabilized in an end4 mutant, due to a block in plasma membrane internalization. However, results presented above indicate that the large intracellular pool of TAT2 is degraded in an end4 mutant, suggesting that internal TAT2 is sorted to the vacuole independently of the plasma membrane. To investigate if TAT2 is targeted from the ER and the Golgi to the vacuole without passing through the plasma membrane, we examined the stability of TAT2 in sec4 , sec18 , and sec23 mutants. SEC4 is a small GTPase required for fusion of Golgi-derived vesicles with the plasma membrane. SEC23 is a subunit of the COPII complex required for ER to Golgi transport. SEC18 is the yeast NSF homologue and is generally required for fusion of vesicles with acceptor membranes . The amounts of TAT2 in extracts of rapamycin-treated wild-type cells and the sec mutants were compared by Western analysis . In the sec4 mutant, TAT2 was degraded like in wild-type cells. In contrast, TAT2 was completely stable in the sec18 mutant and mostly stable in the sec23 mutant. Taken together, the above results suggest that TAT2 is targeted from the ER and the Golgi to the vacuole independently of the plasma membrane. Thus, upon starvation, TAT2 appears to be diverted from the secretory pathway to the vacuolar pathway. Recently, TOR has been implicated in the control of autophagy, a starvation-induced process in which bulk cytoplasm, including organelles, is membrane enclosed and transported to the vacuole . Since the effect of rapamycin on TAT2 is a starvation response, and rapamycin exerts its effects by inhibiting the TOR proteins, we asked whether the degradation of TAT2 is an autophagic event. TAT2 stability was examined in an apg1 mutant defective for autophagy . In the presence of rapamycin, HA-TAT2 was completely turned over in the apg1 strain, indicating that autophagy is not required for the degradation of TAT2 in response to starvation . Because starvation-induced degradation of intracellular TAT2 does not rely on passage through the cell surface or on autophagy, a likely transport route to the vacuole is via the PEP12-, VPS45-, and VPS27-dependent pathway . In this VPS pathway, proteins are delivered from the Golgi to the vacuole via a prevacuolar/endosomal compartment. If TAT2 is transported to the vacuole via the VPS pathway, the permease should be stable in a rapamycin-treated mutant defective in this pathway. Indeed, TAT2 was still present in extracts of rapamycin-treated pep12 , vps27 , and vps45 cells . Taken together, the above results suggest that upon nutrient depletion, TAT2 is transported from the ER and Golgi to the vacuole via the VPS pathway, without passing through the cell surface. Mutation of the five NH 2 -terminal lysine residues in TAT2 results in an apparently complete stabilization of the permease , suggesting that the intracellular pool of TAT2, in addition to the plasma membrane pool (see above), requires ubiquitination for rapamycin-induced targeting to the vacuole and degradation. To investigate whether the NH 2 -terminal lysine residues are required for degradation of the internal pool of TAT2, temperature-sensitive sec4 cells expressing either HA-TAT2 or HA-TAT2 5K>R were pulse labeled for 3 min at permissive temperature, shifted to nonpermissive temperature, and chased in the presence of rapamycin (see Materials and Methods). This experiment was designed to investigate the fate of newly made intracellular TAT2 and TAT2 5K>R proteins that are unable to reach the plasma membrane due to the sec4 mutation. Whereas newly made HA-TAT2 was rapidly degraded in rapamycin-treated cells, HA-TAT2 5K>R was stable over a 90-min chase period , suggesting that the NH 2 -terminal lysine residues of TAT2 and ubiquitination are also required for rapamycin-induced degradation of the internal pool of TAT2. We have shown that the tryptophan permease TAT2, a constitutive amino acid permease, is indeed regulated, at the level of protein sorting and stability. In starved cells, rapamycin-treated or nutrient-deprived, TAT2 is ubiquitinated, targeted from the plasma membrane, the ER, and the Golgi to the vacuole, and then degraded. The internal TAT2 is routed to the vacuole independently of the plasma membrane. The regulation of the specific amino acid permease TAT2 is inverse to that of the broad-range permease GAP1. GAP1 is routed to the vacuole and degraded in nonstarved cells . Rapamycin inhibits the TOR proteins, and induces both the downregulation of TAT2 and the upregulation of GAP1. Thus, TOR and presumably the TOR nutrient-signaling pathway mediate the inversely regulated sorting and stability of the two types of permeases. A model summarizing our findings is shown in Fig. 8 . In growing cells, TAT2 follows the secretory pathway to the plasma membrane. In starved cells, TAT2 is targeted to the vacuole. How is this regulated sorting of TAT2 controlled? Ubiquitination is important in targeting TAT2 from the plasma membrane to the vacuole, as TAT2 accumulates in the plasma membrane in ubiquitination mutants. Furthermore, mutant TAT2 (TAT2 5K>R ) altered in five NH 2 -terminal lysine residues required for ubiquitination accumulates in the plasma membrane in wild-type cells. Ubiquitination may also be required for diverting intracellular TAT2 to the vacuole, as newly made intracellular TAT2 5K>R is not degraded upon rapamycin treatment . Jenness et al. 1997 have suggested that ubiquitination is required for delivery of a mutant STE2 pheromone receptor from an intracellular site to the vacuole. The mechanism by which ubiquitination may divert the internal pool of TAT2 from the secretory pathway to the vacuolar pathway upon nutrient deprivation remains to be determined. The alternative routing of GAP1 to the cell surface or the vacuole appears to be controlled at the level of GAP1 packaging into specialized vesicles leaving the Golgi . TAT2, and at least the histidine permease HIP1, are turned over upon starvation. This is a novel aspect of the starvation response in yeast cells. Why are specific permeases turned over upon starvation? The specific permease TAT2 and the broad-range permease GAP1 are regulated inversely, at least in response to the quality and quantity of the nitrogen source. There are several specific permeases but only two known broad-range permeases, GAP1 and AGP1, in yeast . We suggest that specific amino acid permeases are expressed and functionally maintained under nutrient-rich conditions, and are probably fine-regulated by the availability of their substrates. Once nutrients become limiting, cells may express and maintain only a couple broad-range permeases, instead of the several specific permeases, as a means to reduce energy consumption. The inverse regulation of specific and broad-range permeases provides a mechanism to optimize import with regard to the quality and quantity of nutrients. Upon rapamycin treatment or upon shift from a good nitrogen source (ammonium) to a poor nitrogen source (proline), GAP1 is stabilized and TAT2 is degraded. How is this inverse regulation of TAT2 and GAP1 achieved? To date, two posttranslational regulators of GAP1 are known, NPI1 and NPR1. NPI1, a ubiquitin ligase, presumably ubiquitinates GAP1 and thereby triggers the internalization and degradation of the permease . NPR1, a Ser/Thr protein kinase, is a positive regulator of GAP1 . The mechanism by which NPR1 activates GAP1 is unknown, but the finding that GAP1 is phosphorylated when active suggests that NPR1 directly phosphorylates GAP1 and thereby protects it from NPI1-dependent degradation . NPI1 is also responsible for inactivation of TAT2. Thus, we consider an inverse regulation via this ubiquitin ligase unlikely. However, the inverse regulation may be achieved through NPR1. Indeed, we have found that NPR1 mediates the destruction of TAT2 under conditions when the kinase protects GAP1 . Furthermore, we have shown that NPR1 is controlled by TOR and the TOR downstream effector TAP42 . Because TAT2 is regulated in response to nitrogen, carbon and amino acid availability, it is of interest to determine whether GAP1 is also regulated in response to nutrients other than nitrogen. Furthermore, it remains to be determined how the TOR nutrient–signaling pathway senses the availability of nutrients. | Study | biomedical | en | 0.999998 |
10491388 | Anti– Aquorea victoria GFP rabbit polyclonal antibody, Texas red–conjugated goat anti–rabbit and anti–mouse antibodies, and AMCA-S–conjugated goat anti–mouse antibodies were purchased from Molecular Probes. Anti-p115 polyclonal antibody was as described previously . Anti-Hsc70, anti-Hdj1, anti-Hdj2, anti-TCP1, and polyclonal anti-ubiquitin were a generous gift from Dr. Douglas Cyr (University of Alabama at Birmingham). Anti-giantin antibody was a gift from Dr. Hans P. Hauri (University of Basel, Switzerland). Anti-ubiquitin monoclonal antibody and anti-20S proteasome (α-subunit) polyclonal antibody were purchased from Calbiochem-Novabiochem. Anti-β-tubulin monoclonal antibody and nocodazole were purchased from Sigma Chemical Co. Monoclonal anti-VSV-G was a gift from Dr. Kathryn Howell (University of Colorado). Nocodazole was used at a concentration of 10 μg/μl. Clasto -lactacystin β-lactone (Calbiochem-Novabiochem) was used at a concentration of 10 μM. Monoclonal anti-p50 (mAb 50.1) was a gift from Dr. Richard B. Vallee (University of Massachusetts Medical School). Anti-vimentin monoclonal antibody was a gift from Dr. Bill Britt (University of Alabama at Birmingham). To make the different GFP chimeras used in this study, polymerase chain reaction (PCR) was used to introduce restriction sites at the NH 2 terminus (XhoI) and at different sites downstream (BamHI) of the p115 cDNA previously described . The PCR products were then digested and subcloned in pEGFP-C2 vector (Clontech). pcDNA3.1 (Invitrogen) containing wild-type CFTR was used for CFTR expression in mammalian cells. The plasmid used for p50 expression (pCMVH50myc) was a gift from Dr. Richard B. Vallee (University of Massachusetts Medical School). COS-7 cells were grown in DME supplemented with 10% FBS and 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in 5% CO 2 . Cells growing on 12-mm coverslips were transiently transfected using a calcium phosphate transfection system (GIBCO BRL). After 24 h, cells were washed once with PBS and then incubated in complete medium for an additional 24 h. Cells were imaged live on a temperature-controlled microscope at 37°C. pcDNA-wtCFTR was transfected into COS-7 cells using Lipofectamine-Plus (GIBCO BRL) according to the manufacturer's instructions. COS-7 cells were either mock transfected or transfected with GFP-250. 48 h after transfection, cells were washed with ice-cold PBS and collected by trypsinization. Each pellet was washed three times with PBS and resuspended with PBS supplemented with a mammalian protease inhibitor cocktail (Sigma). Cell pellets were then washed twice with PBS and lysed for 30 min on ice with 200 μl of either 2% Triton X-100 in PBS, IPB (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% NP-40, 0.5% deoxycholate, and 150 mM NaCl), or RIPA buffer (50 mM Tris-HCl, pH 8, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, and 150 mM NaCl), all supplemented with protease inhibitor cocktail. Lysates were then passed 10 times through a 27-gauge needle. Insoluble material was recovered by centrifugation at 13,000 g for 15 min at 4°C. Pellets were then resuspended in 200 μl of 1% SDS in PBS and sonicated for 20 s with a microtip sonicator. Equal volumes of each pellet and supernatants were boiled for 5 min in SDS-PAGE sample buffer and analyzed by SDS-PAGE. COS-7 cells grown on coverslips were infected with the temperature sensitive strain of the vesicular stomatitis virus (tsO45 VSV) at 32°C for 30 min. Cells were then shifted to 42°C for 3 h to accumulate the misfolded G protein in the ER. Transport of G protein was initiated by incubating the cells at 32°C. After 1 h, cells were fixed and processed for indirect immunofluorescence. Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with the indicated antibodies. To remove primary and secondary antibodies, membranes were incubated for 30 min in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 50°C and washed twice with PBS; 0.2% Tween 20. Membranes were then incubated with the detection reagent (ECL; Amersham Life Science Ltd.) to ensure the removal of antibodies, washed, and reprobed with the antibody indicated. COS-7 cells were transfected with GFP-250 for 12 h. Cells were then washed in PBS and incubated in methionine-free DMEM for 1 h. One well of a six-well tissue culture plate (∼2 × 10 6 cells) was used per sample. Cells were labeled with 200 μCi [ 35 S]methionine (NEN, Life Science Products) for 60 min. Incorporation was terminated by washing the cells with PBS and replacing the media with nonradioactive DMEM (methionine). Direct immunoprecipitation was carried out after lysing the cells in RIPA buffer. The cells were scraped, lysed for 30 min on ice, and then centrifuged at 14,000 rpm for 10 min. Immunoprecipitation was for 2 h at 4°C using anti-p115 polyclonal antibody and protein A agarose. Proteins were washed and separated by SDS-PAGE on a 10% gel. Gels were vacuum dried and labeled proteins were detected by fluorography and analyzed using IPLab Spectrum software (Signal Analytics). Cells grown on coverslips were washed three times in PBS and fixed in 3% paraformaldehyde in PBS for 10 min at room temperature. Paraformaldehyde was quenched with 10 mM ammonium chloride. For chaperones staining, cells were fixed for 10 min with methanol at –20°C as previously described . Cells were permeabilized with 0.1% Triton X-100 in PBS for 7 min at room temperature. The coverslips were then washed three times for 2 min each with PBS and then blocked in PBS, 2.5% goat serum, and 0.2% Tween 20 for 5 min followed blocking in PBS, 0.4% fish skin gelatin, and 0.2% Tween 20. Cells were incubated with primary antibody diluted in PBS, 0.4% fish skin gelatin, 0.2% Tween 20 for 45 min at 37°C. Coverslips were washed five times for 5 min each with PBS and 0.2% Tween 20. Secondary antibodies were diluted in PBS, 2.5% goat serum, and 0.2% Tween 20, and incubated on coverslips for 30 min at 37°C. Coverslips were washed as above and mounted on slides in 9:1 glycerol/PBS with 0.1% p-phenylenediamine. COS-7 cells were transfected with GFP-250. After 48 h, cells were washed with PBS, collected by trypsinization and pelleted in a microfuge at 4°C. The pellet was washed twice with PBS and then fixed for 90 min with 1.5% glutaraldehyde in 0.1 M sodium cacodylate pH 7.4. Cells were washed three times with sodium cacodylate and postfixed in OsO 4 in 0.1 M sodium cacodylate pH 7.4 for 60 min on ice. After washing three times with sodium cacodylate 0.1 M, cells were subjected to a series of graded ethanol dehydration (30, 50, 70, 90, 95, 100%) followed by 1 h incubation in 1:1 100% ethanol/Polybed resin (Polysciences, Inc.). After two changes of fresh 100% resin, the cell pellets were transfected to gelatin molds and polymerized in fresh resin overnight at 60°C. Gold epoxy sections (100 nm thick) were generated with a Reichert Ultracut ultramicrotome and collected on 200 mesh copper grids. The grid specimens were then stained for 20 min with saturated aqueous uranyl acetate (3.5%) diluted 1:1 with ethanol just before use, followed by staining with lead citrate for 10 min. Stained samples were subsequently examined on a JEOL 100CX electron microscope. COS-7 cells grown on glass coverslips were sealed into a silicon rubber chamber placed on a glass slide and containing buffered medium with Hepes 25 mM, pH 7.5. Images were acquired in a Zeiss Axiovert 30 inverted microscope (Carl Zeiss Inc.). The microscope was equipped with a 100×, 1.4 NA objective (Carl Zeiss Inc.) and a cooled charge-coupled device (Photometrics) for 12 bit detection. IpLab Spectrum software (Signal Analytics) was used to control image acquisition and manipulation. Deconvoluted images were obtained by collecting images on an OlympusIX70 inverted microscope with a 40×/1.35 NA objective and deconvoluted using IPLab software (Signal Analytics). 4 μm of optical sections 0.4 μm thick was analyzed. Figure 8 A: Dynamics of aggresome formation in vivo. Individual frames collected every 60 s for a period of 2 h. Frames corresponding to time points 60–120 min were exported to a movie file using QuickTime™ 3.0 software. Video available at http://www.jcb.org/cgi/content/full/146/6/1239/F8/DC1 GFP has been extensively and successfully used as a fusion partner to host proteins to monitor their intracellular localization and fate . The ideal chimera is a protein that preserves all the targeting and physiological functions and localization of the host protein but is now fluorescent. GFP-tagged chimeric proteins have been targeted successfully to practically every major organelle of the cell . Because each GFP represents one fluorophore, relatively high levels of expression are required to give bright signals. Several GFP constructs have been expressed at high levels without affecting proper localization and function . However, nonsuccessful fusions are almost never published, making it very difficult to know if overexpression of a specific chimera can trigger an abnormal cellular response . We have shown previously that the wild-type peripherally membrane-associated transport factor p115 localizes to the Golgi complex and to VTCs in interphase cells . Similarly, when a GFP chimera containing full-length p115 fused to the COOH terminus of GFP was expressed in COS-7 cells, the fusion protein localized to the Golgi complex as shown by a high degree of colocalization with the Golgi marker giantin . p115 contains 959 amino acids organized into a globular head containing two domains (H1 and H2) with strong homology to a yeast protein Uso1p and a tail domain containing four coiled-coil (C1-C4) segments . At the COOH terminus, p115 contains a conserved acidic domain (AD). Eliminating the COOH-terminal region of the GFP full-length p115 chimera by removing the COOH-terminal acidic domain, coil 4, and a portion of coil 3 of p115, generated a construct with a normal Golgi localization . In contrast, when COS-7 cells were transfected with a GFP chimera consisting of GFP fused to the NH 2 -terminal first 252 amino acids of p115 (GFP-250), the construct did not colocalize with the Golgi marker giantin . After 48 h of transfection, ∼50% of transfected cells showed majority of the GFP signal concentrated in a single large circular structure localized close to one side of the nucleus. In addition, in most cells, GFP fluorescence was also detected in small punctate structures scattered throughout the periphery of the cells. The finding that normal Golgi morphology appears disrupted in cells containing this perinuclear structure will be discussed later. To examine if the targeting of the GFP-250 chimera to the perinuclear structure was dependent on the position of the GFP within the chimera, we tested the localization of a construct containing the same fragment of p115 as GFP-250, but with the GFP fused to its COOH terminus (250-GFP). Interestingly, overexpression of 250-GFP construct did not cause the formation of the perinuclear structure , nor did overexpression of the vector alone . Overexpression of either 250-GFP or GFP gave a diffuse pattern suggesting cytosolic and nuclear localization as previously shown for GFP . Therefore, it appears that only a specific arrangement of protein sequences results in a protein that is deposited in the perinuclear structures. In data to be presented, we show that GFP-250 is present in insoluble aggregates. However, it is important to note that even though misfolding is likely to be involved in the mechanism of GFP-250 aggregation, the GFP moiety of the chimera must be properly folded, since green fluorescence is detected in these studies. The large perinuclear structure containing GFP-250 showed striking similarity to a recently described novel subcellular compartment, the aggresome . As previously reported, overexpression of the polytopic membrane protein CFTR or inhibition of proteasome activity in cells expressing CFTR leads to the accumulation of stable CFTR aggregates in a large structure close to the MTOC region. To further characterize the structures formed by GFP-250, we examined GFP-250 transfected cells by electron microscopy. The efficiency of transfection was usually close to 40% and a significant proportion, usually >50% of transfected cells, contained a single large perinuclear structure when analyzed by fluorescence . At the ultrastructural level, transfected cells contained a relatively large (∼4 μm) rounded structure of electron-dense particles adjoining the nucleus . The structure was composed of individual dense particles, ∼50–90 nm in size , analogous to the 60–80-nm particles reported to contain misfolded CFTR . Membranous organelles were detected within the particulate structure and surrounding the aggregate . The aggregate shown in Fig. 3 is surrounding one of the centrioles , indicating that it formed around the MTOC. At higher magnification, long filaments can be seen surrounding the aggregate . Aggresome formation by CFTR caused a redistribution of the intermediate filament protein vimentin to the aggresomal region forming a cage-like structure . Overexpression of GFP-250 elicited the same response as determined by indirect immunofluorescence using anti-vimentin antibodies. As shown in Fig. 4 , vimentin forms a ring around the aggregated GFP-250 , that surrounds the aggregated protein core . These findings suggest that overexpression of a cytosolic protein can also trigger aggresome formation. Molecular chaperones bind to and stabilize aggregation-sensitive proteins and facilitate their ultimate fate, be it folding, assembly, transport to a particular subcellular compartment, or disposal by degradation . In a recent report, Hsp70 and Hsp90 have been shown to be recruited to the centrosomal region upon inhibition of the proteasome . Since aggresomes contain misfolded proteins destined for degradation, it might be expected that such polypeptides could be complexed with specific chaperones. To determine if distinct classes of molecular chaperones are associated with the aggresome, indirect immunofluorescence was used to determine the localization of four different cytosolic chaperones in GFP-250–transfected cells. Localization of three chaperones of the Hsp70 system (Hsc70, and two Hsp40 members, Hdj1 and Hdj2) and of one chaperonin (TCP1) was examined. As shown in Fig. 5 , of the four molecules tested, three (Hdj1, Hdj2, and TCP1) showed a high degree of colocalization with GFP-250 within the aggresome. Interestingly, Hsc70 was restricted to the periphery of the aggresome forming a ring around the GFP-250 signal. The restricted localization of Hsc70 to the periphery of the aggresome is perplexing since Hsc70 participates in protein folding events in conjunction with a matching member of the Hsp40 family, Hdj1 or Hdj2 . The differential localization might suggest that Hsc70 chaperone is recruited later in the process of the aggresome formation, or that it is associated with vimentin surrounding the aggresome, but not with the aggregated particles. Supporting the latter possibility, Hsc70 colocalized with vimentin in a ring structure around the aggresome . The proteasome has been implicated in the degradation of incompletely folded or misfolded proteins . In the case of CFTR, both the mutant forms and up to 70% of the wild-type protein fail to fold correctly and are eliminated by an ATP-dependent process that requires covalent modification with ubiquitin and degradation by the proteasome . Similarly, Wigley et al. 1999 reported the recruitment of the proteasomal machinery to the centrosome in cells overexpressing CFTR. To determine if the GFP-250 aggresome also recruited the proteasome, we analyzed the distribution of the α-subunit of the 20S proteasome in GFP-250–expressing cells. As shown in Fig. 6 A, the proteasome is localized to the GFP-250 aggresome, suggesting that it may be involved in the degradation of GFP-250. To examine this directly, COS-7 cells were transfected with the GFP-250 chimera and after 36 h were incubated with the proteasome inhibitor clasto -lactacystin β-lactone for 11 h. Cells were then fixed and examined for green fluorescence to determine the percentage of transfected cells that formed an aggresome. A parallel experiment was performed in the absence of the proteasome inhibitor. As shown in Fig. 6 B, ∼53% of control cells (700 cells counted) formed an aggresome, whereas ∼88% of cells treated with the proteasome inhibitor (700 cells counted) formed an aggresome. To confirm that the proteasome is involved in the degradation of GFP-250, COS-7 cells transfected with GFP-250 were subjected to pulse–chase analysis in the presence or absence of clasto -lactacystin β-lactone. Fig. 6 C shows that the stability of the radiolabeled GFP-250 increases approximately twofold in the presence of the inhibitor. These results suggest that the proteasome is recruited to the GFP-250 aggresome and plays a key role in the degradation of GFP-250. It has been shown previously that inhibiting the activity of the 20S proteasome and/or overexpressing of CFTR or CFTR mutants induce the accumulation of multiubiquitinated detergent-insoluble forms of the proteins . To examine the solubility of GFP-250, increasingly stringent solubilization methods were used. As shown in Fig. 7 A, even under the most stringent conditions using Triton X-100 and SDS (RIPA buffer), GFP-250 was only detected in the insoluble fraction. In contrast, β-tubulin was detected only in the soluble fraction under all solubilization conditions, suggesting that the extraction methods efficiently disrupted the microtubule cytoskeleton. The usual characteristic of polyubiquitination is a ladder of bands spaced by ∼7-kD intervals or the appearance of a high molecular mass smear in an SDS gel, both of which reflect the covalent attachment of multiple ubiquitin chains to the target protein . The ubiquitination serves as a proteasome degradation signal for the target protein . The aggresomes formed in cells with inhibited proteasome contained CFTR conjugated to ubiquitin . To examine if aggresomes formed by overexpression of GFP-250 contained polyubiquitinated GFP-250, transfected COS-7 cells were separated into Triton X-100–soluble and –insoluble fractions, and immunoblotted with an antibody raised against GFP. As shown in Fig. 7 B, a single band with the molecular mass (∼55 kD) predicted for GFP-250 was detected in the detergent-insoluble fraction. Higher molecular mass bands or a high molecular mass smear were not detected, suggesting that the aggregated GFP-250 chimera was not ubiquitinated. To confirm this finding, the nitrocellulose membrane was stripped and reprobed with polyclonal antibodies raised against ubiquitin. As shown in the ubiquitin-probed panel, multiple bands were detected in the soluble and the insoluble fractions. A major band of ∼65 kD, few minor bands as well as a smear comprising a wide range of molecular masses is detected in the soluble fraction. A single major band of ∼35 kD is detected in the insoluble fraction, but does not correspond to the molecular mass of GFP-250. These results suggest that GFP-250 is not ubiquitinated to a detectable extend. Ubiquitination of some substrates appears labile and to confirm that GFP-250 present within the aggresome is not ubiquitinated, we examined COS-7 cells transfected with GFP-250 and processed by immunofluorescence with antibodies to ubiquitin. As shown in Fig. 7 C, the GFP-250 containing aggresome showed no significant colocalization with ubiquitin, which showed a dispersed punctate staining throughout the cell. To ensure that the anti-ubiquitin antibodies can react with ubiquitin in this assay, we transfected COS-7 cells with CFTR since ubiquitin has been shown to colocalize with CFTR in the aggresomal region in proteasome-inhibited cells . After 36 h of transfection with CFTR, cells were treated overnight with clasto -lactacystin β-lactone to induce aggresome formation. The cells were then fixed and processed by immunofluorescence with antibodies to CFTR and ubiquitin. As previously reported , a high degree of colocalization of CFTR and ubiquitin was observed in the aggresome region . Taken together, these results suggest that the proteasome is involved in the degradation of GFP-250 in an ubiquitin-independent manner. Although it has been shown that most proteins degraded by the proteasome are multiubiquitinated , ubiquitin-independent proteasome degradation has been demonstrated for some proteins . Previous work has shown that the aggresome localizes to the MTOC and that nocodazole inhibits the process, but the dynamics of aggresome formation have not been studied. Using time-lapse imaging techniques in living cells, we followed the formation of aggresomes in COS-7 cells expressing GFP-250. Images were captured every 5–120 s for up to 130 min. A representative series of images showing aggresomal growth is shown in Fig. 8 A . Small aggregates usually formed in the periphery of the cell and moved toward the MTOC in straight or curvilinear tracks. Occasionally, some of the particles moved in reverse directions. The speed of the particles was not constant over time. Particles usually moved in a discontinuous, stop and go manner. Fig. 8 B shows a tracing of a path taken by a representative particle moving towards the aggresome. The particle moved a total distance of 14.5 μm in 45 min, which represents an average speed of 0.32 μ/min, before merging with the aggresome. To estimate the rate of aggresome growth, fluorescence intensity was measured in a region of interest (ROI) containing the juxtanuclear aggresomal compartment and normalized against total fluorescence. The total fluorescence remained constant over the time course of the experiment, suggesting that photobleaching was not significant during this interval. Each individual image was measured and plotted as a change of fluorescence intensity over time . The increment in fluorescence intensity of the aggresome was linear over time, and after 120 min, the fluorescence intensity of the aggresome was doubled. Regression analysis ( r 2 = 0.98) suggests that the process of aggresome formation follows a first order linear kinetics. As previously shown, the microtubule depolymerizing drug nocodazole prevents the formation of perinuclear aggresomes and causes the formation of smaller protein aggregates dispersed in the periphery of the cells . To show directly that the speed and path of particle delivery to the aggresome is affected by microtubule disruption, we incubated GFP-250–transfected COS-7 cells in the presence of nocodazole for 1 h at 4°C to depolymerize the microtubules. The cells were then shifted to 37°C and the aggresome dynamics were imaged. The behavior of a single representative particle over 360 s was analyzed in nocodazole-treated and control cells. As shown in Fig. 8 D, in untreated cells, the particle velocity oscillated between 0 and 0.25 μm/s. In cells treated with nocodazole, the particle velocity was dramatically decreased, oscillating between 0 and 0.03 μm/s, directly showing that particle movement responsible for aggresome growth is dependent on presence of intact microtubules. At any given time point, a significant percentage of the particles were either moving very slowly or not moving at all. A population analysis of 10 particles over 35 time points (total ∼350 events) indicated a tailed distribution of movement speeds, with ∼50% of the events occurring between 0 and 0.025 μm/s . This distribution was dramatically altered in cells incubated with nocodazole. After 1 h of nocodazole incubation, 99% of the movement events occurred at speeds between 0 and 0.025 μm/s . Although some of the particles moved at speeds comparable to those in control cells, the movement was never directional, and none of the particles analyzed moved >0.5 μm over a period of 30 min . Taken together, these results indicate that perinuclear aggresomes form by the movement of peripherally nucleated smaller particles towards the MTOC. The loss of motility of peripheral aggregates after nocodazole treatment suggests that a microtubule-dependent motor might be involved in their minus-end–directed movement to the MTOC. The vast majority of minus-end–directed transport processes in interphase cells require dynein, which is typically associated with dynactin. Cytoplasmic dynein has been implicated in the distribution of late endosomes and lysosomes, and the centrosomal localization of the Golgi complex, as well as the retrograde transport of membranous organelles in axons, vesicular transport from early to late endosomes, and traffic to the Golgi of pre-Golgi intermediates . Although molecular motors have been clearly implicated in transport of membrane bound organelles, much less is known about the microtubule-dependent interaction of motors with membrane free particles. Dynein/dynactin-associated minus-end motor activity can be experimentally inhibited by overexpressing the p50/dynamitin component of the dynactin complex . To test if the dynactin complex is involved in the transport of peripheral aggregates to the MTOC, we cotransfected COS-7 cells with GFP-250 and p50/dynamitin and examined the formation of aggresomes in cells expressing both proteins. As shown in Fig. 9 A, ∼58% of control cells (302 cells counted) transfected only with GFP-250 formed an aggresome, but only ∼18% of cells cotransfected with GFP-250 and p50/dynamitin and expressing high levels of p50/dynamitin (143 cells counted) showed aggresome formation. In most of the cotransfected cells, small GFP-250 aggregates were detected, but remained in the periphery of the cell . Aggresomes did form in a proportion of cells expressing p50/dynamitin, and this is most likely due to incomplete inactivation of the dynactin complex. Alternatively, since aggresome formation can take <2 h, some cells could form an aggresome before a significant level of dynactin inactivation was achieved. However, the significant inhibitory effect of p50/dynamitin overexpression suggests that the translocation of peripheral GFP-250 aggregates to the MTOC is powered by the microtubule motor complex of dynein/dynactin. The aggresome is localized to the region of the cell usually occupied by the Golgi complex and as already shown in Fig. 1 F, aggresome formation interferes with proper Golgi localization. We investigated this phenomenon in more detail, and found that aggresome size is inversely correlated with proper Golgi localization, that is, as the aggresome grows, the Golgi becomes more disorganized and distorted around the aggresome . It appears that the aggresome forms a physical barrier between the Golgi cisternal elements and the MTOC around which they are usually arranged. Similarly, microtubules, which are normally assembled and radiate from the central MTOC , are distorted in cells containing large aggresomes . Instead of the normal astral distribution, a central hole containing the aggresome is visible in most cells. More peripherally distributed MTs appear normal. Many studies have shown that Golgi disruption or changes in MT organization can cause defects in protein transport . To determine if protein transport was affected by the cellular changes accompanying aggresome formation, we analyzed the transport of a marker protein, the G protein of the vesicular stomatitis virus (VSV-G), in aggresome containing cells. COS-7 cells were first transfected with GFP-250 for 48 h to promote aggresome formation. Cells were then infected with a temperature-sensitive strain (VSVts045) of the virus at the nonpermissive temperature of 42°C, and incubated at this temperature for 3 h. This leads to the synthesis and accumulation of the VSV-G protein in the ER but does not allow further entry of the VSV-G protein into the secretory pathway . To allow transport of VSV-G protein out of the ER, cells are shifted to the permissive temperature of 32°C. The movement of the VSV-G protein from the ER into the Golgi and later to the cell surface can be followed by immunofluorescence at different time points after the temperature shift. As shown in Fig. 10 C, an untransfected cell and a transfected cell containing a relatively large GFP-250 aggresome have been infected at the nonpermissive temperature and contain VSV-G protein within the ER. This represents the starting point of the transport. Such cells were then shifted to the permissive temperature of 32°C for 1 h. In both the untransfected cell and in the aggresome containing cell, the VSV-G protein is transported from the ER to the Golgi. Interestingly, although the Golgi is displaced around the aggresome, it appears functionally competent for protein transport in a manner analogous to that in infected cells not containing an aggresome. Even though changes in transport kinetics can not be excluded at this point, the data suggest that ER to Golgi transport of cargo proteins is not overtly inhibited by formation of the aggresome. A recent report has described the initial characterization of a novel subcellular structure, the aggresome . Aggresomes form by the deposition of misfolded proteins in a large structure surrounding the MTOC. The initial report documented the formation of aggresomes in cells expressing CFTR or PS1, two integral membrane proteins that span the membrane multiple times. In both cases, treating cells with compounds that inhibit proteasome activity significantly increased the formation of aggresomes. Based on these results, it was proposed that aggresome formation represents a general cellular response to aggregated proteins. To examine the generality of this cellular response, we analyzed whether cells expressing various chimeric proteins encoding the entire sequence of the soluble protein GFP fused in frame to various coding regions of the cytosolic protein p115 form aggresomes. GFP chimeras containing the entire coding region of p115 or the NH 2 -terminal 3/4 portion of p115 did not form aggresomes and were targeted to the expected cellular localization, the Golgi. In contrast, a chimera containing only the NH 2 -terminal 250 amino acids of p115 aggregated into insoluble particles and was deposited within an aggresomal structure. Interestingly, the location of the 250 amino acid p115 sequence relative to the GFP was critical for aggresome formation: aggresomes formed only when GFP preceded the p115 sequence, whereas the reverse construct was not misfolded and did not lead to aggresome deposition. It must be stressed that GFP-250 was at least partially folded since the GFP moiety was fluorescent, suggesting that the p115 sequence was misfolded to expose hydrophobic domains capable of aggregation. Hydropathy plot of the NH 2 -terminal 250–amino acid region of p115 shows many hydrophobic side chains and it is likely that synthesis of only a portion of the globular head region of p115 prevents correct intramolecular folding and leads to aggregation. The GFP-250 aggresome was morphologically indistinguishable from that formed by CFTR and PS1, suggesting that cytosolic proteins can also form aggresomes and supporting the hypothesis that formation of aggresomes is a general cellular response when the degradative capacity of a cell is overtaxed. The previous report documented that aggresomes localize to the MTOC and that treatment of cells with nocodazole prevents aggresome formation . We used time-lapse imaging to obtain quantitative measurements of aggresome formation. Aggresome precursors seem to nucleate throughout the cells, most likely during translation of nascent chains on polysomes. As described previously, the high effective concentration of nascent polypeptides during biosynthesis due to macromolecular crowding could stimulate aggregation . Aggregate particles move towards the MTOC at speeds exceeding those of simple diffusion. The movement is directly correlated with intact microtubules, and particles do not move in nocodazole-treated cells. The speed of particles appears comparable to that measured for different motor families involved in a variety of cellular functions . The nature of the motor activity involved in aggresome formation was investigated by overexpressing the p50/dynamatin subunit of the dynein/dynactin complex. The observed inhibition in aggresome formation suggests that dynein/dynactin plays a key role in the translocation of peripheral aggregates to the MTOC where they form the aggresome. Ultrastructural examination of aggresomes indicates a complex architecture of individual particles interspersed with subcellular organelles and filaments, and surrounded by membranous organelles and vimentin fibers. The organelles within the aggresome appear to be predominantly mitochondria and lysosomes. Surrounding the aggresome are other membranes, including the Golgi. At the molecular level, the aggregated protein appears to recruit cellular chaperones to the aggresome. This is not unexpected since chaperones are known to be associated with misfolded proteins and aid in their presentation for degradation . It is possible that members of the Hsp70 and the chaperonin family may play a role in GFP-250 degradative pathway. Interestingly, whereas two members of the Hsp40 family (Hdj1 and Hdj2) and a chaperonin (TCP1) were associated with particles throughout the aggresome, Hsc70 was localized in a ring structure around the aggresome but was not concentrated within the aggresome interior. The rationale for this differential distribution is currently unknown but might reflect sequential participation of the different chaperones. One of the most effective ways to prevent aggregation is to rapidly target the misfolded protein to degradation. In agreement, our data show that the proteasome is recruited to the GFP-250 aggresome. Similar results were obtained during CFTR overexpression and aggresome formation in a recently published manuscript . Degradation of most proteins by the proteasome requires the covalent conjugation of ubiquitin chains, which target the protein to degradation . Interestingly, our data suggest that although the proteasome is responsible for GFP-250 degradation, GFP-250 is not ubiquitinated. Previous studies have reported ubiquitin-independent proteasomal degradation . Formation of aggregates of misfolded protein within specialized cells has been linked to a number of pathological states . However, whether the deposition of a protein causes secondary cellular problems is less well understood. We have examined the overall distribution of a number of subcellular organelles and found that the Golgi complex and the arrangement of microtubules are disrupted by aggresome formation. Instead of the normally compact Golgi structure surrounding the MTOC, the Golgi elements were dispersed around the aggresome. Similarly, microtubular networks were partially disorganized, with microtubules surrounding the aggresome, instead of originating from the MTOC. However, despite these changes, the cells exhibited many normal processes. Specifically, viral infection and subsequent viral protein synthesis and targeting appeared normal, as did the retention of the viral transmembrane G protein within the ER at nonpermissive temperature. The temperature-sensitive VSV-G protein can not fold properly and is retained within the ER membrane 42°C. When the temperature is shifted to 32°C the G protein folds correctly, exits the ER, and is transported to the Golgi. All the processes of protein synthesis and quality control operational within normal cells were also operational in cells containing large aggresomes. Similarly, the traffic from the ER to the Golgi appeared relatively normal in aggresome-containing cells. Whereas we can not currently exclude changes in the rate or efficiency of transport, it appears that presence of a large aggresomal structure within a cell does not negatively influence at least some of their physiological processes. It seems that the segregation of the aggregated proteins into a single structure might, in fact, sequester problematic proteins and thereby maintain relatively normal cellular milieu. That the cells retain capacity for normal life is also suggested by the finding that the life spans of cells containing aggresomes were not fundamentally different from normal cells (data not shown). These results suggest that pathology in cells containing aggregated proteins is probably not due to having an aggresome that inhibits vital cellular functions, but is more related to the functional unavailability of the aggregated protein. | Other | biomedical | en | 0.999997 |
10491389 | Established mouse embryo fibroblasts (MEFs) from wild-type, p21 cip1 -null, and p53-null C57B6J mice were generous gifts from Jim Roberts (Fred Hutchinson Cancer Research Center, Seattle, WA) and Tyler Jacks (MIT, Cambridge, MA). The cells had been maintained on a standard 3T3-like protocol which resulted in establishment of MEF cell lines. For G0-synchronization, MEFs were brought to confluence, washed, and then cultured in serum-free DME or defined medium (see below) for 2 d. To stimulate entry into the cell cycle, G0-synchronized cells were trypsinized, suspended in 10% FCS-DME, and reseeded on tissue culture dishes (monolayer) or agarose-coated petri dishes (suspension) using 2 × 10 6 cells per 100-mm dish similarly to the procedure described in Böhmer et al. 1996 . To study the relative effects of growth factors and fibronectin, trypsinized quiescent cells (2 × 10 6 cells) were suspended in 10 ml defined medium (1:1 DME: Hams F-12, 15 mM Hepes, pH 7.4, 3 mM histidine, 4 mM glutamine, 8 mM sodium bicarbonate, 10 μM ethanolamine, 10 μg/ml transferrin, 0.1 μM sodium selenite, 0.1 μM MgCl 2 , and 1 mg/ml BSA). Some of the cells were treated with a cocktail of purified growth factors (10 ng/ml PDGF, 1 μM insulin, and 2 nM EGF) that allowed for optimal induction of p21 cip1 . Cells were then plated on 100-mm dishes that had been coated (16 h at 4°C) with fibronectin (100 μg) or 2 mg/ml fatty acid–free, heat-inactivated BSA in PBS (which blocks cell adhesion in serum-free culture). Coating with fibronectin or BSA was performed as described . In experiments with UO126 (Promega), G0-synchronized cells in 100-mm dishes were directly stimulated with purified growth factors in defined medium. For all experiments, cells were washed 2–3 times with PBS, collected by scraping (monolayer cultures) or low-speed centrifugation (suspension cultures), and extracted for Northern blotting, luciferase assays, and immunoblotting, or immunoprecipitation and in vitro kinase assays. In studies using serum as the mitogenic stimulus, the extracts of quiescent cells were prepared before incubation of the cells in monolayer or suspension. For studies in defined medium, extracts of quiescent cells (in growth factor-free defined medium) were prepared both before and after incubation of the cells in monolayer and suspension; similar results were obtained from both controls. Note that MEFs attached to fibronectin and cultured with growth factors in defined medium remained well spread for up to 12 h. To study the consequence of ERK activation in suspended cells, we used NIH-3T3 cells stably transfected with a constitutively active MEK-1 (S218D/S222D) in a tetracycline-repressible expression system . The transfectants were maintained at <50% confluence in DME, 10% calf serum, 0.5 mg/ml G418, and 0.4 mg/ml hygromycin. Tetracycline (2 μg/ml) was added daily. MEFs (1.5 × 10 5 in 2 ml DME, 10% FCS) were plated in 35-mm dishes and incubated overnight. The resulting monolayers (∼80% confluent) were washed with DME before transient cotransfection with 1 μg of p21 cip1 promoter-luciferase plasmid (0-luc; gift of Wafik El-Deiry), 0.1 μg of the renilla luciferase expression plasmid, pRL-SV40 (Promega), and 5 μl lipofectamine (Life Technologies) in a total volume of 1 ml serum-free DME without antibiotics. After a 5-h incubation, 1 ml of 20% FCS-DME was added to each well and the cultures were incubated overnight. 18 h after transfection, the cells were G0-synchronized by 2-d incubation in serum-free DME. After trypsinization, an aliquot of the cells was removed for determination of p21 cip1 -promoter activity at quiescence; the remainder was resuspended in 10% FCS-DME and then replated in 100-mm dishes (0.5–1 × 10 5 cells/10 ml) in monolayer and suspension. At selected times, cells were washed, collected, and extracted in 50 μl of Passive Lysis Buffer (Promega) before analysis of firefly luciferase and renilla luciferase activity using the Dual-Luciferase reporter assay system (Promega). p21 cip1 promoter activity was normalized to a constant amount of renilla-based luminescence to correct for differences in transfection efficiency. Northern blotting, immunoprecipitations, and in vitro kinase assays were performed as described . Immunoblotting for pRb, cyclins, MEK, and ERKs was performed as described after fractionation of cell lysates on reducing SDS gels containing 7.5% acrylamide. Immunoblotting for CKIs was performed similarly except that the gels contained 15% acrylamide. The protein concentration of cell lysates destined for immunoblot analysis was determined by Coomassie binding (BioRad Protein Assay), and equal amounts (100 μg for the analysis of cyclins, cdks, and CKIs, and 25 μg for the analysis of MEK* and ERK) were fractionated on SDS gels. Cells destined for immunoblot analysis with anti-ERK or anti-phospho-ERK antibodies were extracted as described . Rabbit polyclonal antiserum against cyclin A was prepared in this laboratory, and the antiserum to p21 cip1 was the generous gift of Claudio Schneider (AREA Science Park, Trieste, Italy). All other antisera were purchased: anti-cyclin E (sc-481) and anti-cdk4 (sc-260) from Santa Cruz Biotechnology; anti-cyclin D1 (06-137), and anti-cdk2 (06-505) from Upstate Biotechnology; anti-MEK-1 , anti-p27 kip1 , and anti-ERK from Transduction Laboratories, anti-pRb from Ciba-Corning; and anti-phospho-ERK from New England BioLabs. Our initial studies compared the temporal expression pattern of p21 cip1 when adherent and nonadherent MEFs were exposed to mitogens . Consistent with studies by others (see above), we find that the level of p21 cip1 is low in quiescent MEFs, strongly induced within a few hours after exposure of adherent MEFs to mitogens (refer to 3 h, monolayer), and then declines to a barely detectable level (refer to 12 h, monolayer) as the cells progress into mid-late G1 phase. However, we also observed that if MEFs are stimulated with mitogens in the absence of substratum, the initial induction of p21 cip1 is preserved (3 h, suspension) while the subsequent downregulation is incomplete (compare monolayer and suspension cells at 12 h). Addition of purified growth factors to quiescent MEFs in serum-free medium also resulted in a strong induction of p21 cip1 (similar to that seen with serum), and the degree of induction was similar whether cells were cultured on a substratum or in suspension (compare Mn to Sp and Fn+GFs to BSA+GFs). Moreover, p21 cip1 was not induced when MEFs were cultured on fibronectin-coated dishes in the absence of growth factors (Fn-GFs). These results strongly argue that growth factors, and not adhesion factors in serum, are responsible for the early G1 phase induction of p21 cip1 . Together, the results of Fig. 1A and Fig. B , indicate that G1 phase regulation of p21 cip1 can be divided into two discrete phases: an initial induction that is mediated by mitogens and a subsequent downregulation that is enhanced by cell adhesion to substratum. Consistent with studies implicating ERK activation in the induction of p21 cip1 (see above), we found that the new generation MEK inhibitor, U0126 completely blocked the induction of p21 cip1 when quiescent MEFs were treated with purified growth factors . This effect was associated with the inhibition of ERK activation (determined by direct detection of phospho-ERK and ERK2 gel-shift). Treatment with rapamycin or LY294002 did not inhibit the activation of ERK or the induction of p21 cip1 . Thus, activation of the MEK/ERK pathway is necessary for the induction of p21 cip1 by growth factors. NIH-3T3 transfectants expressing a constitutively active MEK-1 (MEK*) under control of a tetracycline-repressible promoter (tet-MEK*-3T3 cells) were then used to determine if activation of the MEK/ERK pathway would be sufficient to induce p21 cip1 . Suspended MEFs were incubated in the presence and absence of tetracycline and a minimal mitogenic stimulus (0.5% serum, which permitted the synthesis of MEK* in the absence of tetracycline). In the presence of tetracycline, 0.5% serum had a small effect on ERK activation (assessed by direct detection of phospho-ERK) and little effect on the expression of p21 cip1 . In the absence of tetracycline, MEK* was induced, resulting in a strong phosphorylation of ERKs and an induction of p21 cip1 . Thus, MEK* is sufficient to induce p21 cip1 in the absence of both cell anchorage and a strong mitogenic stimulus. This result supports other studies showing that expression of constitutively active raf results in the induction of p21 cip1 . Next we examined the relationship between the duration of an ERK signal and the induction of p21 cip1 . Addition of purified growth factors to MEFs in serum-free culture induced a complete activation of ERKs (determined by gel-shift and direct measurement of phospho-ERK) in both nonadherent and adherent cells . However, ERK activity declined in suspended cells while it was sustained in adherent cells . p21 cip1 was induced to the same degree in both the suspended and adherent cells (at 1–3 h in several independent experiments). In several experiments, the analysis of ERK activity by gel-shift, immunoblotting with an anti-phospho-ERK (specific for the dually phosphorylated form) and in vitro kinase assays (not shown) all demonstrated that transient ERK activity can be induced by growth factors in suspended cells while sustained ERK activity requires both growth factors and cell anchorage. Our interpretation of these results is that a transient ERK activation is sufficient to induce p21 cip1 . These results also indicate that the induction of p21 cip1 is anchorage-independent because transient ERK activation does not require cell adhesion to substratum (see below). Note that the induction of cyclin D1, which requires a sustained ERK activation , does not occur when suspended MEFs , NIH-3T3 cells , or normal human fibroblasts are stimulated with mitogens. Interestingly, a mitogen dose response curve showed that when MEFs are stimulated with our standard growth factor cocktail, fibronectin was not necessary for efficient activation of ERK . However, lower growth factor concentrations that partially activated ERK in suspended cells did activate ERK completely when the cells were attached to fibronectin . Fibronectin alone minimally activated ERK under these conditions (data not shown). These results confirm other studies showing that fibronectin and growth factors can synergize to regulate ERK activity (see Discussion), but also show that growth factors alone can fully activate ERK if cells are provided with a sufficiently strong mitogenic stimulus. We emphasize, however, that this strong mitogenic stimulus allows for transient, but not sustained, ERK activation in suspended cells . To assess directly the duration of the ERK signal that is required for the induction of p21 cip1 , we activated ERK by stimulating quiescent MEFs with purified growth factors in defined medium and then treated the cultures with UO126 at 10, 20, and 40 min before collection at 1 h. Immunoblot analysis showed that (a) growth factors induced a complete activation of ERK by 10 min , (b) this effect persisted for at least 60 min , and (c) UO126 rapidly inhibited growth factor–dependent ERK activation . Importantly, the induction of p21 cip1 was completely blocked when UO126 was added either 10 or 20 min after stimulation with growth factors, but it was easily detected when UO126 was added after 40 min . Considering that the inhibitory effect of UO126 on ERK activation is complete within 20 min, we conclude that an ERK signal of 40–60 min is sufficient to induce p21 cip1 . The same experimental approach was applied to the analysis of cyclin D1 expression, and the results showed that cyclin D1 was not induced even when UO126 was added 60 min after growth factor stimulation. These data support and extend the results of Fig. 3 , directly demonstrating that the ERK signal required for induction of p21 cip1 is transient relative to that required for the induction of cyclin D1. After its initial induction, p21 cip1 levels are strongly downregulated in mid-late G1 phase, and the completeness of this effect requires cell adhesion to substratum . To identify the basis by which adhesion affects p21 cip1 downregulation, we compared the stability of p21 cip1 protein by stimulating adherent and nonadherent MEFs with 10% FCS (to induce p21 cip1 ) before the addition of cycloheximide. Immunoblot analysis was performed on lysates of cells collected 0–120 min after addition of cycloheximide. The results showed that the half-life of p21 cip1 was ∼30 min under both culture conditions . We were unable to find antibodies suitable for confirmation of this result by immunoprecipitation of p21 cip1 from pulse–chase-labeled cells, but our results with cycloheximide indicate that the turnover of p21 cip1 is not strongly affected by cell adhesion to substratum. Consistent with the lack of a detectable effect on p21 cip1 stability, both mRNA and promoter analyses showed that cell adhesion significantly enhances repression of p21 cip1 gene expression. Northern blotting showed an initial induction of p21 cip1 mRNA in both adherent and nonadherent MEFs . p21 cip1 mRNA levels then declined, but the decline was much more pronounced in adherent cells . p21 cip1 promoter-luciferase assays gave similar results except that there was essentially no decline in promoter activity when cells were cultured in suspension. This difference in p21 cip1 promoter activity vs. p21 cip1 mRNA or protein levels in suspended MEFs suggests that a constitutive, anchorage-independent turnover of p21 cip1 mRNA may also play a role in setting the steady state level of p21 cip1 protein. Nevertheless, the combined results of Fig. 5 and Fig. 6 strongly indicate that changes in gene expression play a major role in the adhesion-dependent repression (as well as the growth factor–dependent induction) of p21 cip1 . We examined the potential contribution of p53 in G1 phase regulation of p21 cip1 gene expression by performing Northern blots with MEFs derived from p53-null mice. Consistent with many studies implicating p53 in the induction of the p21 cip1 gene, we found that the levels of p21 cip1 mRNA were generally reduced about three- to fivefold when compared with those observed in wild-type MEFs (not shown). However, the pattern of mitogen-dependent induction and adhesion-enhanced repression was retained in the p53-null MEFs . Although this result does not exclude a potential role for p53 in the ECM-dependent downregulation of p21 cip1 gene expression, it does show that p53 is not required. Since the induction of p21 cip1 requires ERK activity, we considered the possibility that the decay of ERK activity was responsible for downregulating p21 cip1 in mid-late G1 phase of adherent cells. However, this potential mechanism is not compatible with the fact that p21 cip1 is poorly downregulated in suspended cells where the ERK signal decays quickly. We then considered the possibility that sustained ERK activity might be phosphorylating a repressor of p21 cip1 gene expression. To address this potential mechanism, we forced sustained ERK activity in suspended cells and asked if p21 cip1 expression was repressed, as it would be in monolayer cells. tet-MEK*-3T3 cells were cultured in monolayer and suspension in the presence and absence of tetracycline. We found that downregulation of p21 cip1 failed to occur in the suspended cells, even when a sustained ERK signal had been enforced . We conclude that the downregulation of p21 cip1 resulting from cell adhesion to ECM is independent of ERK. Cyclin E–cdk2 activity is not induced when fibroblasts are treated with mitogens in the absence of a substratum, and the lack of kinase activity correlates with an increased expression of p21 cip1 and p27 kip1 . To determine if this increased expression of p21 cip1 is causally related to the inhibition of cyclin E–cdk2 activity, we asked if cyclin E–cdk2 activity would be anchorage independent in suspended cells lacking p21 cip1 . G0-synchronized MEFs derived from wild-type and p21 cip1 -null mice were mitogen-stimulated in monolayer and suspension before collection and analysis of cyclin E–cdk2 kinase activity in vitro. As expected, the activation of cyclin E–cdk2 was completely blocked when wild-type MEFs were cultured in suspension . In contrast, cyclin E–cdk2 activity was readily detectable in lysates from suspended p21 cip1 -null cells, at ∼50% of the value seen in lysates from the adherent p21 cip1 -null cells. (Because the p21 cip1 -null and wild-type MEFs are independent isolates, comparisons of cyclin E–cdk2 activity in lysates of quiescent, adherent, and suspended cells should only be made within each established line.) Note that other established markers of G1 phase cell cycle progression (induction of cyclin D1, complete downregulation of p27 kip1 , phosphorylation of pRb, and expression of cyclin A) were adhesion dependent in both wild-type and p21 cip1 -null MEFs, consistent with our previous studies . The expression of cyclin E and cdk2 was also similar in the wild-type and p21 cip1 -null cells. Thus, the cyclin E–cdk2 kinase activity detected in suspended p21 cip1 -null MEFs can be attributed specifically to the loss of p21 cip1 . In turn, this result indicates that downregulation of p21 cip1 participates in the activation of cyclin E–cdk2. The incomplete rescue of cyclin E–cdk2 activity in suspended p21 cip1 -null MEFs supports previous studies by us and others which indicate that adhesion-dependent downregulation of p27 kip1 also contributes to the adhesion dependency of cyclin E–cdk2 activity. Several studies have shown that growth factors and the ECM cooperate to regulate cell cycle progression. Phosphorylation/activation of the ERKs and induction of cyclin D1 are two well-established examples of this cooperation . These events are causally related because cyclin D1 expression is induced by sustained ERK activity . Although the exact mechanisms by which growth factors and ECM signals cooperate to regulate ERK activity are still under investigation, it is generally thought that the regulation of G1 phase ERK activity by growth factors and the ECM reflects a convergence of RTK and integrin signals upstream of ERK . This convergence is important for expression of cyclin D1 . In contrast, our data with p21 cip1 show that a strong mitogenic stimulus is sufficient to induce p21 cip1 in early G1 phase and that cell anchorage subsequently allows for full repression of p21 cip1 expression in mid-late G1 phase. These results show that growth factor/ECM cooperation involves parallel as well as convergent signaling. Our results with pharmacologic inhibitors and conditional expression of constitutively active MEK show that the activation of ERKs plays a major role in the induction of p21 cip1 by growth factors. This effect probably contributes to the assembly of cyclin D–cdk4/6 complexes . Indeed, a p21 cip1 -mediated assembly of cyclin-cdk complexes could explain the results of Cheng et al. 1998 , which indicate that activation of the MEK/ERK pathway is sufficient to override the mitogen requirement for assembly of cyclin D–cdk4/6 complexes. In contrast to the induction of cyclin D1, growth factors can induce p21 cip1 whether or not cells are attached to a substratum. This anchorage independency of p21 cip1 induction reflects the fact that a transient activation of ERK is sufficient to induce p21 cip1 . In our studies and some others , transient ERK activation occurred in response to growth factors and in the absence of cell adhesion. However, others have also reported that growth factors poorly activate ERK when cells are cultured in the absence of a substratum . The different results may reflect how long cells are kept in suspension before growth factor stimulation or whether the cells have received a sufficiently strong mitogenic stimulus as described in Fig. 3 C. Others have shown that the induction of p21 cip1 requires a stronger ERK signal than does the induction of cyclin D1 . In agreement with these studies, our results do show that the persistent ERK signal seen throughout G1 phase and associated with cyclin D1 expression is not as strong as the transient, early G1 ERK signal associated with the induction of p21 cip1 . Some studies also suggest that sustained ERK activity mediates the induction of p21 cip1 , while our results show that a transient activation is sufficient. A likely explanation for these different results is that most of the studies by others have relied on overexpression of activated rafs, and the nature of this experimental approach precludes an analysis of effects mediated by transient ERK activity. Our data also suggest that the effect of cell anchorage on mid-late G1 repression of p21 cip1 gene expression is important for cell cycle progression. We and others have previously reported that p21 cip1 and p27 kip1 levels increase when fibroblasts are cultured with mitogens in suspension, and this increase in CKIs is associated with inactivity of the cyclin E–cdk2 complex. Cyclin E–cdk2 is partially anchorage independent in cells lacking p21 cip1 , implying that downregulation of p21 cip1 in normal cells contributes to the activation of cyclin E–cdk2. The increased expression of p21 cip1 in suspended cells has typically been interpreted as an induction, but the results shown here indicate that impaired downregulation of p21 cip1 is the proper explanation. Brugarolas et al. 1998 have also examined the effect of p21 cip1 on the anchorage dependency of cyclin E–cdk2 activity. Consistent with our results, they found that cyclin E–cdk2 activity in suspended pRb/p21 cip1 -null MEFs was higher than that in wild-type MEFs. However, we found that cyclin E–cdk2 activity in suspended p21 cip1 -null MEFs was half of that seen in the adherent p21 cip1 -null MEFs while they reported that the cyclin E–cdk2 activity of suspended pRb/p21 cip1 -null cells was only 20% of that observed with adherent pRb/p21 cip1 -null cells. The difference between their results and ours may reflect the fact that our analysis was performed with cells in the first G1 phase while their studies used cells incubated in suspension for three days before analysis. The cell cycle blocks in their system and ours may be of a different nature and not directly comparable. Moreover, we can not exclude the possibility that the pRb-null phenotype of the MEFs used by Brugarolas et al. 1998 may also contribute to the different results. There is precedent for negative regulation of p21 cip1 protein by rho . Negative regulatory motifs have also been mapped to the 3′ untranslated region in the p21 cip1 mRNA . Downregulation of the p21 cip1 promoter may also be specific to positional cues in the cell cycle because the p21 cip1 promoter is downregulated in mid-late G1; cells treated with mitogens in suspension fail to reach mid-late G1 . The effect of cell anchorage on these different modes of regulation remains to be elucidated. Importantly, the downregulation of p21 cip1 observed in response to cell adhesion is independent of ERK activity and can even occur in the presence of a sustained ERK signal. This allows the cell to fully downregulate p21 cip1 expression in the presence of the sustained ERK signal needed for the induction of cyclin D1. Several laboratories have shown that high-intensity raf signals induce p21 cip1 and also result in a p21 cip1 -dependent cell cycle arrest . As discussed above, the induction of p21 cip1 by raf is probably the result of ERK activation and is consistent with our studies. The persistently elevated expression of p21 cip1 characteristic of cell cycle arrest in high intensity raf transformants suggests that overexpression of constitutively activated raf can override the normal ERK-independent signaling mechanism(s) that control the mid-late G1 phase downregulation of p21 cip1 by the extracellular matrix. While others have shown that sustained ERK activation is required for cell cycle progression, our results indicate that transient ERK activation is not without effect. Rather, we propose that transient ERK activity results in the induction of p21 cip1 while sustained ERK activity, mediated by growth factor/ECM cooperation, and results in the induction of cyclin D1 . The induction of p21 cip1 and cyclin D1 are both important for the assembly of cyclin D–cdk4/6 complexes . In addition, p21 cip1 inhibits cyclin E–cdk2; our results indicate that full downregulation of p21 cip1 gene expression requires cell adhesion to ECM and that this effect contributes to the control of cyclin E–cdk2 activity. The importance of p21 cip1 in regulating adhesion-dependent G1 phase progression is highlighted by the study of Brugarolas et al. 1998 which showed that MEFs null for pRb and p21 cip1 are anchorage-independent for growth. Nevertheless, several studies do indicate that ECM-dependent regulation of p27 kip1 also plays a role in regulating cyclin E–cdk2 activity. p27 kip1 levels are typically regulated posttranscriptionally, and ubiquitin-mediated degradation is thought to play a critical role in this process (see introduction). Thus, the mechanism by which the ECM controls the steady state expression of p27 kip1 is likely to be very different from that of p21 cip1 and an interesting matter for investigation. | Study | biomedical | en | 0.999998 |
10491390 | Immortalized Xenopus melanophores were cultured as described previously . Immunofluorescent localization of myosin V was performed using a clonal nonpigmented cell line, clone 47, or gray cells, derived from the original melanophore cell line . Melanophores containing a lower melanin content were selected by freezing the original cell line in 95% FCS and 5% DMSO, according to standard protocols. Approximately 5% of the cells survived thawing and reculturing, many of them possessing large vesicles containing small (∼0.2 μm) particles of melanin. This cycle of freezing and thawing was repeated once again and pigment-deficient cells were cloned twice on 10-cm tissue culture plates using the cloning ring technique. A morphologically stable clone was selected and expanded. Over 50% of the cells in this population contained numerous unmelanized vesicles ∼1-μm diam. These vesicles responded to hormone treatment by aggregating and dispersing as normal melanosomes and were, therefore, considered pigment-free melanosomes. Further melanin production was inhibited by treating the cells for 2 wk with 1 mM phenylthiourea. Cultures isolated in this manner were grown in standard growth medium, supplemented with 1 mM phenylthiourea. To preserve cellular morphology for microscopy, cells were transfected using the FuGENE 6 transfection reagent (Boehringer Mannheim Corp.) following the vendor's protocols. To prepare the plasmid pcDNA3-Myc-MST, which contains the COOH-terminal 601 amino acids of the mouse myosin Va gene fused to the COOH terminus of the myc epitope tag , the following PCR primers were constructed: 5′-AAA AAG CTT AAA CCA TGG AGC AAA AGC TCA TTT CTG AAG AGG ACC TGG GGA TCC AAG CTG-3′ and 5′-AAA CTC GAG TCA GAC CCG TGC GAT GAA-3′ (GIBCO BRL), and used to amplify the myosin short tail DNA from the construct pCMV2-FLAG-MC-ST . The product of this PCR was digested with XhoI and HindIII, and cloned into the vector pcDNA3 (Invitrogen Corp.). Melanophores were plated on acid-washed polylysine-coated glass coverslips and cultured for 24 h. Cells were then briefly rinsed in 0.7× PBS and fixed for 20 min in a solution of freshly prepared 3% paraformaldehyde in 0.7× PBS. A solution of 0.1% Triton X-100 was used to permeabilize cells for 15 min. For immunofluorescent staining, cells were blocked using 3% BSA in the same solution for 10 min. Myc epitope-tagged proteins were stained using the 1-9E10.2 mAb diluted 1:1,000 into the BSA/Triton buffer for 60 min. Myosin V distribution was visualized with the DIL2 polyclonal antibody (see below) at a dilution of 1:8,000. The cells were then washed with PBS, stained using FITC-conjugated goat anti–mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:100 for 60 min, washed with 0.7× PBS, and mounted in 80% glycerol in 0.1 M sodium borate, pH 8, supplemented with N -propyl gallate. For fluorescent actin staining, rhodamine-conjugated phalloidin (Molecular Probes, Inc.) was diluted to 0.33 nM and included with the secondary antibodies. Images were obtained using a Zeiss Axioskop equipped with a CH250 cooled CCD camera (Princeton Instruments). The gray-scale histograms of the images were stretched to utilize their full dynamic ranges using Adobe Photoshop. Interphase and metaphase-arrested Xenopus egg extracts were prepared as described , with the following modifications. CSF-arrested frog eggs were activated by treatment with 10 μM A23187 for 5 min and allowed to progress to interphase in the presence of 100 μg/ml cycloheximide before extract preparation. Metaphase-arrested extracts were prepared by treating interphase extracts with 0.13 mg/ml bacterially expressed Δ90, a nondegradable cyclin B construct, for 45 min at room temperature . All extracts were supplemented with 5 μM latrunculin A (BIOLMOL) to depolymerize actin . Histone H1 kinase assays were used to monitor activity of cdc2/p34 kinase activity during every experiment . High-speed supernatants were prepared by centrifuging egg extracts at 150,000 g in a TLA 100.3 rotor (Beckman Instruments, Inc.) for 30 min at 4°C. Endogenous egg organelles were isolated by floatation as described in Lane and Allan 1999 . Melanosomes were isolated from cultured melanophores, essentially as described in Rogers et al. 1998 . In brief, melanophores were grown in 10-cm tissue culture plates to confluency. Cells were rinsed with IMB50 (50 mM imidazole, pH 7.4, 1 mM magnesium acetate, 1 mM EGTA, 150 mM sucrose, 0.5 mM EDTA, 1 mM dithiothreitol, 150 μg/ml casein, 1 mM ATP) then scraped into 2 ml of the same buffer supplemented with protease inhibitors (10 μg/ml each chymostatin, leupeptin, and pepstatin, and 1 mM PMSF). All further manipulations were performed on ice. Cells were lysed by 5 to 10 passes through a 26-gauge needle attached to a 1 ml hypodermic syringe and diluted to 10 ml. The lysate was centrifuged at 600 g in an HB-6 rotor (Sorvall) for 5 min to remove nuclei and unbroken cells. The supernatant was recovered and centrifuged again for 5 min at 2,000 g in an HB-6 rotor to pellet melanosomes. Melanosome pellets were then gently resuspended either in Xenopus egg extracts (melanosomes from two plates per 150 μl extract) or IMB50 supplemented with 1 mM ATP as a control. After a 30 min incubation at room temperature, melanosome/extract mixtures were diluted to 1 mL with IMB50 and layered atop a 5-ml cushion of 80% Percoll in IMB50 and purified by centrifugation at 2,000 g for 10 min in an HB-6 rotor. Melanosomes to be used in motility assays were resuspended in 100 μl IMB50 supplemented with 1 mM ATP. Those destined for SDS-PAGE and immunoblotting were dissolved in 20 μl of sample buffer. Egg extracts were immunodepleted using either affinity-purified DIL2 (see below) or preimmune serum obtained from the same rabbits, following a published protocol described in Desai et al. 1998 . Electrophoretic separation of proteins was routinely performed using discontinuous 7.5% SDS-PAGE gels . In experiments comparing protein composition between different treatments of melanosomes, purified pigment granules were resuspended in sample buffer, boiled for 5 min, and equivalent volumes of organelles were diluted into 1% SDS. The absorbance of melanin at 550 nm was determined for each sample and this measurement was used to normalize the load in each lane to an equivalent number of organelles. Immunoblotting was performed using the protocol of Towbin et al. and bound antibody was detected by chemiluminescence using SuperSignal (Pierce Chemical Co.). Myosin V was detected using a polyclonal antibody, DIL2, raised against bacterially expressed neck domain of the dilute isoform of mouse myosin V, and affinity-purified as described . Kinesin-II was detected using an mAb cross-reactive with the motor's 85-kD subunit, K2.4 . The intermediate chain of cytoplasmic dynein was probed with an mAb, m74-1 . The Nitella -based motility assay was performed essentially as described , with the following modifications. Before dissection, each Nitella internodal cell was treated with 2 mM N -ethyl maleimide for 5 min to poison the plant's endogenous cytoplasmic streaming and ensure that all motility observed was due to the activity of melanosome-associated myosin. Nitella cells were then dissected into buffer containing 2 mM dithiothreotol, 2 mM ATP, and 10 μM phalloidin to stabilize filamentous actin. Melanosomes or protein A beads were then pipetted onto dissected Nitella filets and observed using time-lapse video-enhanced bright-field microscopy with a 40× long-working distance objective mounted on a Diaphot 300 inverted microscope (Nikon, Inc.). Images were captured using a Newvicon camera and an Argus-10 video processor (Hamamatsu Phototonics) and recorded onto s-VHS tapes with a time-lapse video recorder (Panasonic). To examine the activity of myosin V in egg extracts, protein A–agarose beads (Sigma Chemical Co.) were incubated with affinity-purified DIL2 polyclonal antibody at a protein concentration of 25 μg/ml, or with preimmune serum for 1 h at 4°C. The beads were then washed in IMB50 by resuspension and centrifugation five times to remove unbound antibody. The preimmune-bound bead pellets (∼50 μl vol) were then resuspended in 200 μl of interphase or metaphase egg extracts clarified of membranes by centrifugation at 150,000 g in a TLA 100.3 rotor (Beckman Instruments, Inc.) to preclear for 1 h at 4°C. The beads were pelleted, and each aliquot of extract was then incubated either with DIL2- or preimmune-conjugated beads for 1 h at 4°C. After washing five times in IMB50 supplemented with 2 mM ATP, these beads were used in the Nitella assay, as described above. Xenopus egg extracts arrested in interphase or metaphase were labeled with 200 μCi 32 P i in 20 μl reactions and incubated for 30 min at room temperature. Reactions were stopped by the addition of 1 ml ice-cold IP buffer (10 mM Tris, 80 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, pH 7.5) supplemented with 1% Triton X-100, and a protease inhibitor cocktail (10 μg/ml each leupeptin, pepstatin, and chymostatin, and 1 mM PMSF) and incubated on ice for 10 min. The samples were clarified by centrifugation at 16,000 g for 15 min and precleared for 90 min by incubation with normal rabbit serum, prebound to 25 μl of a 50% protein A–agarose bead suspension. After centrifugation, 25 μl of 0.5 mg/ml affinity-purified DIL2 antibody was added and incubated for 4 h at 4°C. The samples were then incubated with 30 μl of protein A beads for 30 min, and the beads were collected by centrifugation at 10,000 g for 10 min. The following regimen of washes was then performed: four times with ice-cold IP buffer, one time with IP buffer supplemented with 500 mM sodium chloride, and once with 50 mM Tris, pH 8. Pellets were resuspended in 20 μl sample buffer and analyzed by SDS-PAGE and autoradiography. Using an in vitro assay, we previously demonstrated the presence of an actin-based motor on the surface of melanosomes purified from Xenopus melanophores . Tentative identification of this motor as myosin V was based on the observation that this motor is enriched in purified melanosome fractions relative to whole cell extracts, while members of several other myosin classes were absent. To determine conclusively whether myosin V was actually involved in melanosome transport in this system, we sought to disrupt its function in melanophores and to observe the effects on pigment granule transport. Since latrunculin A-induced disruption of the actin cytoskeleton in Xenopus melanophores results in microtubule-dependent aggregation of the pigment to the cell center , we hypothesized that inhibition of the melanosome-associated myosin would produce the same effect. Melanophores were, therefore, transfected with a construct encoding an epitope-tagged fragment of mouse myosin Va . This fragment, lacking the NH 2 -terminal motor domain, consists of the COOH-terminal 601 amino acids of myosin V, and includes part of the central stalk domain believed to be important in homodimerization of myosin V heavy chains by coiled-coil interaction, and the globular tail domain, thought to be the cargo-binding site. This construct, termed myosin V short tail (MST), has been shown to act as a dominant-negative inhibitor of myosin V in mouse melanocytes; its expression mimicking the naturally occurring genetic-null phenotype in this cell type . Melanophores were transfected with the MST construct; 48 h later cultures were fixed and transfected cells identified by immunofluorescent staining for the myc epitope-tag present at the NH 2 terminus of the mutant construct. Xenopus melanophores usually grow in culture with their melanosomes dispersed throughout their cytoplasm unless induced to aggregate to a tight, central mass by treatment with melatonin. Without exception, every cell expressing MST aggregated its pigment to the cell center . Immunolocalization of expressed myc -tagged MST revealed that the protein was present throughout the entire volume of the cell. Melanosome aggregation was not due to a disruption of the actin cytoskeleton, as staining with fluorescent phalloidin revealed a filamentous actin distribution in these cells, which was similar to nontransfected cells (data not shown). Control cells transfected with green fluorescent protein (GFP) underwent cycles of pigment aggregation and dispersion, indicating that pigment aggregation was an effect specifically caused by MST . To verify the involvement of myosin V in melanosome transport, we sought to determine whether or not the motor was present on melanosomes in situ by immunofluorescent staining. The chemical properties of melanin make this approach problematic, however, for two reasons. First, melanin is a polymer composed of modified tyrosine residues and is rife with charged carboxyl and nonpolar aromatic side chains . Previous studies have documented the high affinity of melanin for various compounds, and it has been our experience that melanin avidly binds many proteins present in cell extracts . Second, as melanin has been evolved to act as a light-absorbing pigment, it interferes with fluorescence microscopy, especially in aggregated cells where the pigment mass often occludes staining. To circumvent these problems, we developed a melanin-free clonal melanophore subline. The original Xenopus melanophore cell line was subjected to two freeze–thaw cycles to select for melanophores possessing a low melanin-content. After two rounds of cloning, a morphologically stable cell line was isolated. Any residual melanin was eliminated by culturing the cells in 1 mM phenylthiourea, a potent tyrosinase inhibitor, for two weeks. This cell line, designated clone 47 or gray cells, possesses numerous vesicles which are ∼1 μm in diameter, and respond to treatment with MSH and melatonin by dispersing to the cell periphery or aggregating to the cell center . Therefore, we believe that these vesicles are melanosomes devoid of pigment. Gray cells were fixed and immunofluorescently stained with an antibody specific for myosin V. The antibody was highly enriched on punctate vesicular structures that aggregated to the cell center upon treatment with melatonin and dispersed throughout the cells after exposure to MSH . We conclude that myosin V is present on melanosomes in vivo in Xenopus melanophores and remains bound to these organelles, at least to some degree, during aggregation and dispersion. Previous studies examining Xenopus melanophores in situ have shown that these cells fail to transport their pigment in response to MSH or melatonin during mitosis . Work from our lab has established that melanosome transport occurs along both the microtubule and actin cytoskeletons in a coordinated manner . We considered the possibility that the melanosome-associated motors are differentially regulated throughout the cell cycle. Since nothing is known about whether actin-mediated organelle transport is regulated during cell division, we chose to focus upon this question by treating melanosomes with Xenopus egg extracts arrested in metaphase or interphase and examining their motility in vitro. We initially prepared Xenopus egg extracts arrested in interphase or metaphase essentially according to the protocols of Murray 1991 , as modified by Allan 1993 . Frog eggs were activated using calcium ionophore in the presence of cytochalasin D to prevent actin polymerization. Cycloheximide was included to arrest them in interphase, and extracts were obtained by centrifugal crushing. Extracts at this stage were found to remain stably in interphase. Metaphase arrested-extracts were prepared by further supplementing these extracts with the nondegradable cyclin derivative, Δ90 . Mitotic extracts were found to stably maintain high MPF kinase activity (data not shown). Both interphase and metaphase extracts prepared this way completely inhibited actin-based motility of melanosomes in vitro using the Nitella assay, as compared with untreated control organelles. Biochemical studies of purified myosin V demonstrated that this motor binds to actin with high affinity, even in the presence of ATP . Therefore, we speculated that cytochalasin-capped fragments of actin filaments bound to myosin V present on the melanosomes, effectively blocking the motor from interacting with exogenous Nitella actin filaments. To circumvent this possibility, latrunculin A, a drug which binds to monomeric actin and, unlike cytochalasin, induces complete depolymerization of filamentous actin, was included during extract preparation . To establish a basal level of melanosome motility, organelles were purified from melanophores and scored for their ability to move in vitro using the Nitella assay. In agreement with our previous results , we found that ∼90% of the total number of pigment granules exhibited unidirectional motility . When isolated, melanosomes were preincubated in interphase frog egg extracts supplemented with latrunculin and purified by density gradient centrifugation. The fraction of organelles transported along actin filaments was ∼85%, virtually indistinguishable from the untreated control. However, treatment with metaphase-arrested extracts dramatically inhibited actin-based motility; only ∼10% of the melanosomes exhibited motility in vitro. Incubation of the melanosomes with metaphase extracts, therefore, decreased motility nearly eightfold, compared with interphase extracts or untreated organelles. We hypothesized that the inhibition of motility observed following metaphase extract treatment could be due to one of two possible mechanisms: myosin V may dissociate from melanosomes or the motor could be rendered inactive during mitosis. To test this first possibility, melanosomes were isolated, treated with either interphase- or metaphase-arrested extracts, and examined by electrophoresis and immunoblotting for myosin V . The number of melanosomes in each sample was normalized by optical density to ensure that an equal number of organelles were analyzed for each treatment. In addition, Coomassie blue staining of gels run in parallel also verified the amount of protein loaded per treatment . Immunoblotting revealed that untreated melanosomes contained the same amount of myosin V as interphase extract-treated organelles. However, in melanosome fractions treated with metaphase extracts, myosin V was undetectable. To verify that the source of the myosin V we were detecting was from melanosomes, and not from endogenous egg organelles that might fuse or aggregate with pigment granules, extracts were clarified by high-speed centrifugation before melanosome treatment. High-speed mitotic supernatants were as effective in release of myosin V from melanosomes as extracts prepared by low-speed centrifugation . Immunoblots for myosin V revealed that the motor was present in both mitotic- and interphase-arrested egg extracts in approximately equal amounts . This indicated that the protein was not subjected to proteolytic degradation in a cell cycle-dependent manner. Endogenous egg organelles from both types of extracts were purified by flotation through a sucrose gradient to exclude soluble proteins and analyzed for the presence of myosin V . Interestingly, the motor was found to remain associated to these organelles in both interphase and metaphase extracts. This result suggests that the dissociation of myosin V may be an organelle-specific phenomenon. Furthermore, it conclusively rules out the possibility that melanosome fractions became contaminated with endogenous organelles during treatment with the egg extracts. We noted that the mobility of myosin V bound to organelles did not exhibit a shift in molecular weight in either population of organelles or as compared with soluble myosin V from either type of extract. If melanosome-bound myosin V was able to exchange with the soluble pool of egg-derived motor, then our observations might also reflect a cell cycle-dependent association of egg myosin V with melanosomes. To test whether this was the case, we immunodepleted myosin V from both interphase- and metaphase-arrested extracts before melanosome addition. We reasoned that if there were an exchange between the organelles and the extracts, then we would detect a net release from interphase-treated melanosomes in myosin V-depleted extracts. Immunoblots of organelles treated with mitotic extract depleted of myosin V lacked the motor, whereas immunodepleted interphase extracts retained myosin V (data not shown). However, when we compared the relative amount of myosin V bound to melanosomes after treatment with motor-depleted extracts with organelles treated with extracts immunodepleted using preimmune serum, we noted a quantitative difference. In myosin V-depleted extracts, melanosomes retained less motor, indicating that egg-derived myosin V was able to exchange with the melanosome-bound protein in interphase-arrested extracts (data not shown). These results indicate that melanosomal myosin V is able to exchange with myosin V derived from interphase extracts, but neither the egg or melanosomal myosin can bind to organelles in metaphase-arrested cytosol. Previous work from our lab has demonstrated that the bidirectional transport of pigment granules along microtubules in Xenopus melanophores is due to the activities of the plus-end directed motor, kinesin-II, and the minus end directed motor, cytoplasmic dynein . Niclas et al. 1996 demonstrated that dynein-driven motility of Golgi membranes and ER membranes exhibits cell cycle-dependent regulation in Xenopus mitotic egg extracts, and this inhibition is due to dissociation of the motor. Our system allowed us to test if this is a general phenomenon for dynein-driven motility. Consistent with the results of Niclas et al. 1996 , quantitative immunoblotting for dynein using an antibody raised against the intermediate chain demonstrated that this motor is released from melanosomes in metaphase extracts . Interestingly, dynein intermediate chain in extract-treated melanosome fractions exhibits a mobility shift of ∼10 kD, compared with its apparent molecular weight of 83 kD on untreated melanosomes. This mobility shift has been observed by other groups who have attributed it to represent posttranslational modification or the recruitment of an egg-specific isoform of the motor . In an effort to distinguish between these two possibilities, we treated egg extracts with alkaline phosphatase to determine whether this mobility shift was due to phosphorylation. The migration of dynein intermediate chain remained unaffected by this treatment (data not shown), indicating that melanosomes likely recruited an egg-specific isoform. Kinesin-II was found to remain associated with metaphase-extract–treated pigment granules by immunoblotting with an antibody that recognizes the 85-kD subunit of the motor . Our experiments with the dominant-negative myosin V construct demonstrated that the activity of this motor is necessary for proper dispersion of melanosomes in melanophores. It is, therefore, possible that modulation of myosin V's activity is a key regulatory event during the cycles of aggregation and dispersion within these cells. Since elevation of intracellular cAMP and the subsequent activation of protein kinase A (PKA) plays a key role in triggering pigment dispersion, we considered the possibility that the activity of this kinase may play a role in the association of myosin V with melanosomes throughout progression of the cell cycle. The activity of PKA in cycling Xenopus egg extracts has been extensively studied, and has been found to play an important role during the transition of mitosis to interphase . The basal level of cAMP production and PKA activity was found to decrease during the transition from interphase to mitosis, and to exhibit a peak in activity just before the transition to interphase . If PKA activity were essential for myosin V attachment to melanosomes, then the diminished activity of the kinase in metaphase-arrested extracts might account for the motor's dissociation. To test this hypothesis, we incubated purified melanosomes in metaphase egg extracts in the presence of 50 μM cAMP and 1 mM 1-isobutyl 3-methyl xanthine to inhibit phosphodiesterases. Comparison of immunoblots of treated versus untreated metaphase extracts showed no difference in the amount of myosin V present on melanosomes; the motor dissociated from the organelles in both cases (data not shown). Addition of the PKA catalytic subunit to metaphase extracts, likewise, did not prevent this dissociation (data not shown). Mitotic release of myosin V from melanosomes was, therefore, not due to the inactivity of PKA. In addition to dissociation from its cargo, another potential regulatory mechanism for myosin V could be inhibition of its motor activity. To test this possibility, affinity-purified myosin V antibody was bound to protein A-conjugated agarose beads. The beads were then used to isolate myosin V from interphase- and metaphase-arrested egg extracts that had been clarified of membranes by ultra-centrifugation. Beads with attached myosin V were analyzed in the Nitella motility assay. Myosin V immunoisolated on beads from interphase and mitotic extracts exhibited vigorous motility in the Nitella assay. Virtually every bead from both samples exhibited motility, with average velocities of 84.2 ± 19 nm/s for interphase beads and 52.8 ± 17 nm/s for metaphase beads. These velocities are somewhat faster than those observed for untreated melanosomes in vitro (41 ± 20 nm/s). Melanosomes treated with interphase extracts exhibited average velocities of 32.8 ± 7.8 nm/s, while metaphase treated melanosomes traveled at 27.8 ± 10.5 nm/s. Beads conjugated with preimmune serum from the same rabbit in which the DIL2 antibodies were generated exhibited no motility, indicating that the movements we observed were due to immunoadsorbed myosin V. We conclude that the motor activity of myosin V is not inhibited in mitotic extracts. Since many cellular processes, including dynein-mediated transport, are modulated in a cell cycle-dependent manner by phosphorylation, we tested the possibility that myosin V might be regulated similarly. Interphase- and metaphase-arrested egg extracts were treated with [ 32 P]orthophosphate to produce an endogenous pool of labeled ATP to act as a substrate for kinases present in the extracts. Myosin V was then immunoprecipitated and equal amounts of protein were analyzed by electrophoresis and autoradiography. In both interphase and metaphase extracts, a 200-kD myosin V heavy chain was immunoprecipitated and found to be phosphorylated . Quantitation of the amount of radioactive phosphate incorporated into the protein in each treatment revealed that in mitotic cytosol it was labeled approximately fivefold greater than in interphase extract. Myosin V is, therefore, more highly phosphorylated in metaphase, compared with interphase. Interestingly, several additional phosphoproteins coimmunoprecipitated with myosin V. In interphase extracts, proteins with molecular weights of 70 and 20 kD exhibited labeling, while in metaphase extracts, species of 85, 100, 120, and 140 kD remained associated . The molecular weights of these proteins do not correlate with any known subunits of this motor, and the significance of these possible associations is unknown. The metaphase-arrested extracts that we used were prepared by treatment of interphase extracts with Δ90, a sea urchin cyclin B construct modified to delete its ubiquitinization sequence . Δ90 associates with and activates cdc2/p34 kinase, driving the extracts into metaphase, but since the cyclin's degradation sequence has been removed, the kinase activity of the cyclin B/p34 complex remains constitutively active and the extracts cannot progress further. We speculated that myosin V might be phosphorylated directly by cdc2/p34 mitotic kinase. To test this hypothesis, we purified melanosomes and treated them in vitro with commercially available recombinant cyclin B/cdc2 kinase. After the melanosomes were repurified by density gradient centrifugation, immunoblotting for myosin V showed that the motor continued to remain associated with the organelles in amounts similar to untreated controls (data not shown). Furthermore, when phosphorylated in the presence of 32 P, no significant amount of isotope was observed to incorporate into any melanosome proteins in the molecular weight range expected of myosin V (data not shown). Finally, phosphorylation by cyclin B/cdc2 kinase did not affect the amount of melanosome motility in the Nitella assay, as compared with untreated control organelles. Histone H1 was employed as a control substrate in these experiments to show that cdc2 was active under our experimental conditions in the presence of melanosomes. We conclude that metaphase-induced phosphorylation of myosin V is not directly due to cdc2/p34, but rather to the activity or activities of a different kinase or phosphatase, or both, which exhibit differential activities in metaphase extracts. It has become evident over the past several years that cells partition membrane-bound organelles to their daughters by precisely regulated, yet unique, mechanisms. Intracellular membranes undergo a specific choreography within the spindles of living PtK 2 cells, first collecting along the microtubules to gather at the poles through prometaphase, followed by an abrupt exclusion from the spindle at metaphase . Using a GFP-labeled resident Golgi apparatus protein, Shima et al. documented the fragmentation of the Golgi apparatus in living HeLa cells during mitosis and observed that the resultant vesicles segregate to each daughter cell via a static association with each pole of the spindle in a microtubule-dependent process . GFP-labeled peroxisomes, which are transported vectorally along microtubules during interphase, lose their association with the cytoskeleton during cell division and appear to be segregated randomly . In frog melanophores, melanosomes are excluded from the mitotic spindle and fail to respond to the hormonal stimuli that induce them to aggregate or disperse during interphase . One common feature between these examples is the apparent downregulation of microtubule-based transport during metaphase. This hypothesis is borne out by the work of the Allan and Vale laboratories, which demonstrated that dynein-mediated microtubule minus end-directed transport, along with plus end transport, of the ER and Golgi membranes are inhibited in metaphase-arrested Xenopus egg extracts . In the present study, we examined whether myosin V was subjected to cell cycle-dependent regulation. Our previous work demonstrated that melanosomes purified from Xenopus melanophores exhibit actin-based motility in vitro . Furthermore, the unconventional myosin, myosin V, was found to be enriched in melanosome fractions, compared with whole melanophore extracts, suggesting that this motor is responsible for movement along actin filaments. In this study we have confirmed this hypothesis both in vivo and in vitro by using a dominant-negative approach to block myosin V function and by immunolocalizing myosin V to melanosomes in unpigmented melanophores. To test cell cycle-governed regulation of myosin V bound to melanosomes, we prepared Xenopus egg extracts arrested either in interphase or metaphase. Melanosomes incubated in interphase extracts and untreated organelles exhibited vigorous motility in the Nitella assay, but mitotic-treated melanosomes showed an eightfold decrease in their in vitro movement. Furthermore, this mitotic inhibition was caused by dissociation of myosin V from melanosomes without an accompanying inhibition of its motor activity. We used a polyclonal antibody raised against a 27-kD fragment of myosin V for detection of this motor and do not believe that our inability to detect it on blots of metaphase-treated melanosomes is due to masking of the epitope by posttranslational modification. Membranous organelles purified from egg extracts did not exhibit cell cycle-induced dissociation, however, suggesting that mitotic release of myosin V may be specific for certain organelles. Interestingly, we did observe a slight difference in the velocities of soluble myosin V between interphase and mitotic extracts, which may indicate a second level of regulation of this motor. Dissociation of myosin V was accompanied by increased phosphorylation of its heavy chain in mitotic extracts, relative to interphase extracts. Therefore, we propose a mechanism whereby myosin V-driven organelle transport is inactivated during mitosis by phosphorylation-induced dissociation from melanosomes. Although myosin V is one of the best characterized unconventional myosins, its regulation is poorly understood. To date, only one other study has addressed this issue directly. Prekeris and Terrian 1997 demonstrated a calcium-induced release of myosin V from synaptic vesicles in vitro, as well as in isolated synaptosomes . They showed that this dissociation occurred in the absence of ATP and was, therefore, not due to phosphorylation of the motor. We believe that the metaphase-induced release of myosin V from melanosomes occurs through a different regulatory mechanism for two reasons. First, in our study, melanosomes and Xenopus egg extracts were prepared in the presence of the calcium chelator, EGTA. Second, treatment of purified melanosomes with exogenous calcium failed to affect the amount of myosin V bound to the organelles (data not shown). Tissue-specific isoforms of myosin V are produced as the result of differential RNA splicing; brain and epidermal myosin V each possess different protein domains as a result . It is possible that this difference in calcium sensitivity may be specific to the neuronal isoform. Alternatively, it may be that organelle receptor proteins for the motor respond to different signals; those on synaptic vesicles, synaptophysin and synaptotagmin II, release myosin V upon exposure to calcium, whereas the unidentified receptor on melanosomes does not . It is interesting to compare the regulatory mechanisms that govern cytoplasmic dynein with those of myosin V. Although both transport membrane-bound organelles along different cytoskeletal filaments during interphase, they dissociate from their cargo during mitosis. Cytoplasmic dynein has been implicated in other processes during cell division, such as spindle formation, chromosome transport, and spindle orientation . It is possible that dynein dissociates from its membrane-bound organelle cargo so that it may be recruited to perform these other tasks during mitosis. Alternatively, it may also be that the motor is subjected to identical regulatory mechanisms during interphase and mitosis, but specificity of the cargo transported by dynein changes during the cell cycle, and this differential targeting is modulated during mitosis. A recent study of the distribution of myosin V during mitosis has shown that this motor is present in the spindle and midbody of dividing cells, suggesting that it too may play a role during mitosis . However, the fact that dilute myosin V null mice do not exhibit gross mitotic defects suggests that if it plays a role in cell division, it is either nonessential or it is compensated by other factors . The best understood example of unconventional myosin regulation has resulted from the study of ameboid myosin I. Myosins IA, IB, and IC from Acanthamoeba all exhibit increased actin-based motility and ATPase activity upon phosphorylation of a conserved serine or threonine present in an actin-binding loop in the motors' heads . Bement and Mooseker, after subjecting all known myosin sequences to a comprehensive sequence comparison, noted that in all other known myosins, except for myosin VI, this residue is replaced with either glutamate or aspartate . This observation led them to postulate the TEDS rule, in which they hypothesized that the requirement for phosphorylation on this residue in the majority of myosins may have been evolutionarily relieved by the substitution of an acidic amino acid residue . If this is true, then it is logical to assume that differential regulation of other unconventional myosins, such as myosin V, could be achieved by differential attachment the motors to their cargoes. This argument is borne out in the case of myosin V by the results of our study, as well as those of Prekeris and Terrian 1997 . Other mechanisms should not be excluded, however. Myosin V is a phosphoprotein in nervous tissue and is a substrate for calcium–calmodulin activated kinase II in vitro . We have shown here that myosin V is phosphorylated in interphase egg extracts, albeit at lower levels than in metaphase-arrested extracts, without affecting the amount of motor bound to melanosomes. This observation suggests the possibility that the motor possesses multiple phosphorylation sites, which may be modified differentially throughout the cell cycle. The significance of this phosphorylated state is unknown, however, and the physiological relevance of these observations has yet to be established. In addition to the dissociation of myosin V from synaptic vesicles, in vitro studies of the motor have demonstrated that its motility is inhibited by calcium and this inhibition is likely due to loss of myosin V-associated calmodulin light chains . Paradoxically, calcium treatment also increases the motor's actin-stimulated ATPase activity . Intracellular modulation of calcium levels also may be a potential mechanism of myosin V function. | Study | biomedical | en | 0.999998 |
10491391 | A 129 isogenic genomic library (obtained from Dr. A. Nagy, Samuel Lunenfeld Research Institute, Toronto, Canada) was screened with the full-length KLC1 cDNA . Various genomic clones were characterized by restriction digestion followed by Southern analysis. One clone, g5.1, was determined to contain the translational start site. This clone was further characterized by extensive restriction digests and partial sequencing. A 2.5-kb HindIII/PstI genomic fragment was subcloned into pBS II KS (−; Stratagene) as the 3′ flanking arm (pBS-3′). A 1.4-kb BglII/NotI subcloned fragment was digested with EcoRV and Eco0109, and the smaller 1.2-kB fragment was blunted with Klenow and subsequently subcloned into the XhoI site of pBS-3′. The resulting targeting construct (pBS-5′+3′) had both the 5′ and 3′ flanking arms and unique SalI and NotI sites. The exon that was removed encoded a 72-amino acid sequence starting at QHSDSSA and ending at NILALVY. This region contained genomic sequences encoding the 10 amino acids before the beginning of the first TPR (tetratrico peptide repeat) domain, the entire first TPR domain, and 20 amino acids of the second TPR domain . Removal of this segment of genomic DNA should result in out of frame translation of the remainder of the KLC1 gene . The 6-kB SalI fragment of pGT-IRES β-geo , containing the IRES β-geo cassette, was subcloned into pBS-5′+3′ to complete the targeting construct . The targeting vector was linearized using the unique NotI site, and 20 μg of the linearized DNA was electroporated into R1 embryonic stem (ES) cells as described by Wurst and Joyner 1993 . The ES cells were grown for 2 d before selection with 125 μg/ml of G418 (active weight; GIBCO BRL) for an additional 10 d. 94 ES cell colonies were isolated and the fastest growing 67 colonies were checked by PCR for a homologous recombination event. The 5′ PCR primer (CTAATTTTGGACTTCCAGCAAAGAC) encoded KLC1 genomic DNA sequences residing outside the targeting vector. The 3′ primer (TACACCTGGCCAGTGAGGCTTCTA), used for PCR, encoded sequences within the en-2 region of the IRES β-geo cassette. The resulting PCR product is ∼1.4 kb. Of the initial 67 clones checked, 6 gave PCR products of the expected size. These clones (A1, A2, E2, F1, F11, and H4) were verified as homologous recombinants by Southern analysis of SacI digested genomic DNA. The probe used was a 240-bp SacI/BglII fragment adjacent to the targeted sequences. Clones that tested positive for recombination events were trypsinized to single cell state and microinjected into 3.5-d C57BL/6 embryos to produce chimeric mice. Tips of tails were isolated from mice between 2–3 wk old. The tail tips were digested overnight at 55°C in tail lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 0.5% SDS, and 0.25 mg/ml proteinase K). DNA was purified by a single phenol/chloroform extraction, ethanol precipitated, and resuspended in 100 μl of 10 mM Tris, pH 8.0, 1 mM EDTA. Genotyping was done by PCR using two sets of primers within one reaction. The first set of PCR primers amplified an 800-bp fragment from the LacZ region of the targeted gene. The sequences for these primers are as follows: 5′-GCATCGAGCTGGGTAATAAGCGTTGGCAAT, 3′-GACACCAGACCAACTGGTAATGGTAGCGAC. The second set of primers amplified a region (210 bp) across the deleted exon of the KLC1 gene. The 5′ primer (CGGGCTGTTTCTCTTGGCTTGCTC) encoded sequences within the 5′ short flanking arm of the targeting vector. The 3′ primer (GGAGCGTGCGCAGCCTTGCAGGGA) encoded sequences in the deleted exon of the KLC1 gene. PCR conditions were as follows: denaturation at 94°C for 10 min, 35 cycles of denaturation at 94°C for 1 min, annealing and elongation at 72°C for 4 min, and a 10-min final elongation at 72°C. All 98 offspring from the first 12 litters of heterozygous × heterozygous matings were weighed at 10, 14, 18, and 21 d after birth. The pups were ear punched at day 10 to distinguish them from each other. Genotyping was done as described. 25 sets of adult mice that were caged together were also weighed after ∼1 yr of age as a final data point. 15 sets of mice (wild-type, heterozygous, and homozygous) were also tested for sensorimotor defects. Essentially, mice were allowed to hang upside down from chicken wire (1-cm gauge) attached to a bell jar ∼1.5–2 ft above a surface. They were subsequently timed until they fell off. Timing was cut off at 2 min of hanging upside down. Western analysis was done as described by Rahman et al. 1998 . Essentially, a whole brain from either a 6-wk-old wild-type, heterozygous, or homozygous mouse was homogenized in PBS (140 mM NaCl, 2.5 mM KCl, 10 mM Na-phosphate dibasic, 2 mM K-phosphate monobasic, pH 7.4) with protease inhibitors. The crude lysate was centrifuged at 3,000 g and the supernatant quantitated for total amount of protein by Bradford analysis (BioRad). Equivalent amounts of total protein were loaded per lane on 10% polyacrylamide gels. Western analysis was done with mAb 63-90 , polyclonal antisera directed against KLC1 and KLC2 , polyclonal antisera directed against KIF5A (1:100 dilution), polyclonal sera directed against KIF5B , or an mAb to actin (1:5,000 dilution; Boehringer Mannheim Catalog #1 378 996). Bands were visualized by incubating with either HRP-conjugated goat anti–rabbit IgG (Zymed Labs, Inc.) or goat anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies, and subsequent processing with ECL (Nycomed Amersham Inc.). For quantitative Western blot analysis of protein amounts, the antibodies were first calibrated to give linear detection of signals over a linear dilution range (10–50 μg of the brain supernatant/lane). Equal amounts (40 μg) of brain supernatant were loaded for the wild-type, heterozygous, and mutant KLC1 samples and probed with the above antibodies. Band intensities were quantitated using NIH Image and relative ratios of the intensities were calculated. For sucrose gradient analysis of kinesin-I, brain from either a 6-wk-old wild-type, heterozygous, or homozygous mouse was homogenized in buffer A (10 mM Hepes, pH 7.3, 0.5 mM EGTA, 0.5 mM MgCl 2 , 50 μM ATP) in the presence of protease inhibitors. The crude lysate was centrifuged at 3,000 g to remove unbroken cells and nuclei, and the postnuclear supernatant subsequently centrifuged in a Sorval T-1270 rotor at 35,000 rpm for 30 min at 4°C. The resulting high-speed supernatant (0.4 ml) was then top-loaded onto a 5–20% linear sucrose gradient (10.4 ml in buffer A), and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 14 h. 16 fractions (∼0.6 ml each) were collected from the top of the gradient using a fraction collector. Equal volumes of each fraction were loaded onto 10% SDS polyacrylamide gels, and quantitative Western analysis done as described. Control protein markers, alcohol dehydrogenase (7.4 S), catalase (11.3 S), and β-galactosidase (16 S) were solubilized and centrifuged in parallel sucrose gradients, and the enzymatic activities measured in each fraction to determine the peak of each marker. Immunoprecipitation reactions were carried out as described by Rahman et al. 1998 . Whole brains from 6-wk-old wild-type, heterozygous, and homozygous mice were homogenized in 1 ml of RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) with protease inhibitors. The homogenates from brain from each type of mouse were spun at 100,000 g for 30 min to yield lysate. 100 μl of lysate was precleared with preblocked protein A–Sepharose beads (Zymed Labs, Inc.) and subsequently used for immunoprecipitation reactions. Sufficient quantities of either anti-KLC1 and anti-KLC2 , or anti-KIF5A and anti-KIF5B antibodies were added to the precleared lysate so as to completely immunoprecipitate the antigens in the lysate. The antibody–protein complex was precipitated with protein A–Sepharose beads (Zymed Labs, Inc.). The beads were washed several times with RIPA buffer, once with 50 mM Tris, pH 6.8, and resuspended in 2× SDS loading buffer. Equivalent volumes of lysates and immunoprecipitates, and supernatants from immunoprecipitates were loaded in each lane and subsequently analyzed by Western blotting as described. Immunofluorescence was done as described by Rahman et al. 1998 . 6-wk and older wild-type, heterozygous, and homozygous sets of mice were perfused with 4% paraformaldehyde and the spinal cord and dorsal root ganglion (DRG) were surgically removed. All tissues processed for immunofluorescence were postfixed for 2 h and then transferred to 30% sucrose in PBS overnight for cryoprotection. 10–14-μm cryosections were prepared for immunostaining. Primary antibodies were added to a buffer containing 1× PBS, detergent (either 0.6% Triton X, 1% Triton X, or 0.15% saponin), and 5% BSA for overnight incubations. Antibodies against KLC1 and KLC2 were used at a final concentration of 100 μg/ml. KIF5A and KIF5B antibodies were used at 1:50 and 1:20 dilutions, respectively. 63-90 was used at a 1:250 dilution of the ascites fluid. Giantin was used at a 1:50 dilution of supernatant. Monoclonal β′-COP antibody (CMIA10) was used at a dilution of 1:2,000 . Fluorescein-conjugated sheep anti–rabbit IgG (2 μg/ml) and Texas red-labeled goat anti–mouse IgG (2.5 μg/ml), or fluorescein-conjugated sheep anti–mouse IgG (2 μg/ml) and Cy5-conjugated goat anti–rabbit (2 μg/ml) secondary antibodies were used for visualizing staining. A BioRad MRC-1024 confocal laser scanning microscope was used to collect images. The sciatic nerves of anesthetized mice were ligated as described in Smith 1980 , Hirokawa et al. 1990 , and Hanlon et al. 1997 . After a 6-h ligation, the mice were transcardially perfused with 4% paraformaldehyde and both the ligated and unligated sciatic nerves were removed from the mouse. The tissue was subsequently processed for immunofluorescence as described above. All mice used for these ligation experiments were ∼6-wk-old. To analyze KLC1 function, we generated mice with a gene-targeted mutation in the KLC1 gene. The construction of a true null mutation in this gene was complicated by instability of the 5′ region of the KLC1 gene in plasmid vectors. Thus, a KLC1 targeting construct that removed the entire gene could not be made. Instead, we made a construct that removed the majority of the gene, including the region encoding the highly conserved TPR domain , and left only a small piece encoding the alpha-helical coiled coil needed for interaction with KHC . Although a small amount of the encoded KLC1 fragment could be detected under some conditions, several lines of evidence discussed here suggest that this mutant is functionally null. The promoterless IRES-β-geo targeting vector was used to enrich for homologous recombination at the correct locus. The targeting frequency observed with KLC1 was ∼10%. Six clones were obtained that tested positive for recombination by PCR and Southern analysis . Of these six clones, E2 and F11 produced mice that were highly chimeric (>50% coat color contribution from ES cell lineage). All highly chimeric male mice obtained from the E2 and F11 lines were able to transmit through the germline, as observed by agouti coat color of the offspring from C57BL/6 matings. However, two of these male chimeric mice consistently transmitted to offspring at >90% efficiency. Since homologous recombination only affects one copy of the targeted gene, agouti coat color by itself is not an indication of a heterozygous mouse. Therefore, all agouti offspring from chimera and C57BL/6 or 129 SvEv matings were genotyped by PCR . The first 100 genotypings were also done in tandem with dot blots using a β-galactosidase probe to verify reproducibility of the PCR reactions (data not shown). Heterozygous mice did not display any discernible abnormalities and were indistinguishable from their wild-type littermates. Heterozygous mutant mice were interbred to produce homozygous mice. All offspring from matings of heterozygous mice were genotyped by PCR . PCR genotyping of mouse tails from offspring of matings of heterozygous mice showed that homozygous KLC1 mutant mice are viable . These mice are, however, significantly smaller than their wild-type or heterozygous littermates . All offspring from the first 12 litters (98 mice total) were sexed, genotyped, and weighed periodically until weaning at 21 d after birth. In a mixed 129/BL6 background, the predicted Mendelian ratio of 1:2:1 was observed of wild-type (KLC1 +/+; n = 25/98), heterozygous mutant (KLC1 +/−; n = 54/98), and homozygous mutant (KLC1 −/−; n = 19/98) animals. A 1:1 ratio of male (n = 51/98) to female (n = 47/98) mice was also observed. Although mice were first weighed, genotyped, and ear punched for tracking purposes at day 10 after birth, mice of noticeably small size were evident at birth and presumed to be homozygous mutant, although they were too young to be genotyped. The size difference between wild-type or heterozygous and homozygous mice was also noticeable in adults . Besides the size difference, KLC1 mutant mice also exhibited visible motor defects. One such deficit was quantitated by testing the ability of the mice to hang upside down from chicken wire . Heterozygous mutant mice were normal for every metric we tested, indicating that the KLC1 mutation is recessive. To verify the absence of normal KLC1 in gene-targeted mice, and to assess the effects of removing normal KLC1 on the behavior of KLC2, KIF5A, and KIF5B, Western analysis of brain and sciatic nerve tissue homogenates was done. The abundance of each of these proteins was compared between littermates that were wild-type, heterozygous mutant, and homozygous mutant. Careful quantitation using actin as an internal control standard revealed no discernible difference in the levels of either KIF5A or KIF5B in crude cytoplasmic lysates of brain from each of the three genotypes . KLC1 and KLC2 levels in brain were assessed with mAb 63-90 , which is an mAb that recognizes a shared epitope in the coiled-coil region of both KLC1 and KLC2. Hence, it should recognize both full-length and truncated KLC1 with comparable affinity. The levels of KLC1, but not KLC2, were dramatically reduced in crude cytoplasmic brain extracts from KLC1 −/− mice . A very small amount of the truncated KLC1 protein product could also be detected. The lack of any measurable change in the amounts of KIF5A and KLC2 in KLC1 mutants suggests that there is a pool of KIF5A that is not associated with any KLC in KLC1 mutants; this supposition is confirmed by the sucrose gradient analysis described below. In sciatic nerve extracts, no obvious changes were observed in the levels of KIF5A, KIF5B, or KLC2 (data not shown). Similar to what was observed with brain extracts, only a small amount of the KLC1 fragment could be detected in sciatic nerve extracts from mutant animals. Interestingly, immunofluorescence with both anti-KLC2 and mAb 63-90 suggest that KLC2 levels are reduced in the sensory neuron cell bodies of the DRG from mutant animals. However, levels of KLC2 did not seem to be affected much in the cell bodies of the motor neurons in the ventral horn of the spinal cord (data not shown). As described previously, KLC3 could not be reliably detected . To assess interactions of wild-type and mutant KLC1 with KIF5A and KIF5B, to test directly for a pool of KIF5A free of KLC, and to determine if there was obvious compensation of KLC1 function by KLC2, immunoprecipitation studies combined with Western analyses and sucrose gradient analyses of brain extracts were conducted. Immunoprecipitations with anti-KIF5A and KIF5B antibodies from brain lysates indicate that KLC2 is still capable of forming complexes with either KIF5A or KIF5B in KLC1 mutant mice . There was no obvious difference in the amount of KLC2 that coprecipitated with KIF5A or KIF5B between wild-type, heterozygous, and homozygous KLC1 mutant mice; normal KLC1 and the KLC1 fragment were not detected in homozygous mutant immunoprecipitations. Although the level of truncated KLC1 is greatly reduced in mutant brain, this observation suggests that only a small amount of KIF5A or KIF5B, if any, is associated with the KLC1 fragment. Sucrose gradient analysis was used to test directly the supposition that KLC1 mutants have a pool of KIF5A lacking KLC that is not ordinarily found in wild-type. Quantitative Western blotting of sucrose gradient fractions of mutant and wild-type brain extracts revealed that in wild-type, all KIF5A sedimented in fractions containing KLC1 and KLC2. The KLC1 mutant, however, showed a consistent shift in sedimentation of KIF5A to lighter fractions with significantly less KLC present. Similar, though not as extreme, behavior of KIF5B was also seen. This difference presumably arises because KIF5B is expressed in nonneural cells where KLC1 is ordinarily less abundant, whereas KIF5A is expressed primarily in neural cells in which KLC1 expression is enhanced. Finally, we observed some KIF5A and KIF5B that sedimented at much higher S values in KLC1 mutants. The origin of this species is unknown at present, although it could represent an aggregated component. To examine the distribution of KLC and KHC in neuronal cell bodies, and to evaluate whether loss of normal KLC1 led to changes in the behavior of the KIF5A and KIF5B subunits of kinesin-I, immunofluorescence studies were carried out. Motor neuron cell bodies in the ventral horn of the spinal cord and sensory neuron cell bodies in the DRG were examined. In KLC1 mutant homozygotes, KIF5A was seen to accumulate abnormally in tubular structures in both the motor neuron cell bodies and the sensory neuron cell bodies in the DRG . To determine if the aberrant localizations of KIF5A corresponded to known membrane compartments, motor neuron cell bodies were double labeled with KIF5A antibodies and a variety of other antibody markers for the ER, Golgi apparatus, lysosomes, and mitochondria. Interestingly, the aberrant KIF5A staining in the motor neuron cell bodies of the ventral horn was seen to colocalize only with the cis-Golgi marker giantin in KLC1 homozygous mice and not with other markers tested . Similar colocalization of KIF5A and giantin was seen in sensory neuron cell bodies of the DRG . To confirm that giantin probes were labeling cis-Golgi compartment as expected, double labeling with antibodies directed against the well characterized Golgi marker mannosidase II (MannII) were done; precise colocalization of MannII and giantin was observed . Finally, KLC1 mutant neuronal cells did not exhibit any obvious changes in either mitochondrial or steady-state lysosome distribution visualized by immunofluorescence (data not shown). Mice that were heterozygous for the KLC1 mutation showed a primarily diffuse staining pattern of KIF5A in both motor and sensory neuron cell bodies , suggesting that the small amount of KLC1 fragment does not actively induce abnormal behavior of KIF5A. Similar to wild-type animals, only faint staining by KIF5A antibodies in or near the Golgi apparatus was seen in the motor and sensory neurons of heterozygous mutant animals. KLC2 staining levels were consistently reduced in the sensory neuron cell bodies of KLC1 mutant mice. In addition, the strong and diffuse KLC1 staining ordinarily seen in wild-type was not observed in the cell bodies of homozygous mutant sensory and motor neurons (data not shown), confirming that normal KLC1 was gone and that the KLC1 fragment was virtually absent from the cell bodies. The combined Western and staining data thus suggest that the truncated KLC1 protein levels are dramatically reduced in the cell bodies, compared with wild-type levels of KLC1. Neither mAb 63-90 or polyclonal KLC1 antibodies showed any costaining of the KLC1 fragment and giantin or KIF5A in homozygous KLC1 mutant cells. KIF5B staining was also altered in homozygous KLC1 mutant sensory, but not in motor neuron cell bodies; the normal diffuse pattern was altered to a more punctate distribution that did not colocalize with known ER, mitochondrial, or Golgi markers (data not shown). Intriguingly, COPI distribution detected by mAb CM1A10 staining of β′-COP in sensory neuron cell bodies was also altered by the lack of functional KLC1. The staining pattern changed from one that resembled Golgi apparatus staining in wild-type sensory cells to a more punctate staining pattern in KLC1 mutant cells . The altered CM1A10 staining largely colocalized with the aberrant KIF5B staining . To gain a qualitative assessment of KIF5A and KIF5B behavior in axonal transport, we used sciatic nerve ligation experiments. Sciatic nerve ligation is a unique way of visualizing directional movement along microtubules. Microtubules within the axons of the sciatic nerve are arranged such that their minus end is toward the cell body and the plus end is towards the synaptic terminal. Hence, accumulations of motor proteins proximal to the site of ligature indicate plus end directed movement, whereas accumulations on both sides of the ligature usually indicate minus end directed movement . Ligation experiments indicate that KIF5A and KIF5B are able to move in a plus end directed fashion in KLC1 mutant mice . There were no discernible differences between accumulations in wild-type, heterozygous, and homozygous mutant KLC1 mice. Mice lacking wild-type KLC1 are abnormal. These mice exhibit overt movement defects, small size, and in particular, their sensory and motor neurons have obvious alterations in the intracellular localization of kinesin-I and COP-I components. Together, these data demonstrate that KLC1 has crucial functions, even in the presence of the potentially redundant KLC2 and KLC3. To understand fully the phenotype of the KLC1 mutant that we generated, and its implications for the intracellular function of KLC1, it is necessary to evaluate the nature of the KLC1 mutation that we made. There are several lines of evidence that support the view that the KLC1 mutation we made is functionally null with respect to the phenotypes observed, in spite of the presence of a KLC1 fragment of low abundance. First, Western blots of brain tissue from mutant animals suggest that only a very small amount of the KLC1 fragment relative to wild-type KLC1 is present. Second, immunofluorescence of sensory and motor neuron cell bodies in DRG and spinal cord reveals only faintly detectable staining of the KLC1 fragment relative to the robust staining ordinarily seen in wild-type. Third, immunoprecipitation experiments from whole brain provide no evidence for the association of the KLC1 fragment with KIF5A or KIF5B. Fourth, the sites of aberrant accumulation of KIF5A or KIF5B in DRG or spinal cord do not exhibit costaining with KLC antibodies in mutants, suggesting that the aberrantly behaving KHC chains do so as a result of the absence of KLC, rather than an active influence of the KLC1 fragment. Fifth, the overall abundance of KIF5A and KLC2 are unchanged, even though KLC1 has been removed. Thus, there appears to be a larger than normal pool of KIF5A lacking associated KLC, a suggestion that was confirmed by sucrose gradient analysis. Sixth, heterozygotes are normal with respect to all phenotypes we observed, suggesting that the fragment does not actively exert cellular effects. It remains formally possible that the low abundance KLC1 fragment does have some residual function, but if so, we have failed to detect it. Together, the data on the KLC1 mutant mice provide new insight into the potential functions of KLC. In particular, they provide new information about organismal requirements for KLC function, and provide new insights into the role of KLC in kinesin-I function. Kinesin-I isolated from most species has a 1:1 stoichiometric ratio of KHC and KLC. However, studies in Neurospora crassa show that KLC proteins do not copurify with KHC in biochemical preparations. Studies in sea urchin also suggest that stoichiometric amounts of KLC do not always copurify with KHC . These results raise the possibility that KLC function may be most important in a subset of kinesin-I processes in larger organisms or large cells. This view seems plausible, given that flies , worms , sea urchin , and squid have a single gene encoding KLC, whereas mammals have at least three genes encoding KLC. Nonetheless, KLC1 mutants have a constellation of visible phenotypes, even though KLC2 is clearly present. The KLC gene products in mammals may have different roles, which partially overlap, in intracellular transport in neuronal cells. Given the strong neuronal phenotype and ultimate lethality of mutants lacking KHC or KLC in Drosophila and mutants lacking KHC in C. elegans , one might expect severe neuronal abnormalities in animals bearing mutant KLC1. Although KLC1 mutant mice were significantly smaller than their heterozygous or wild-type siblings and showed some neuronal deficits, they did not exhibit neuronal problems as severe as those seen in Drosophila . One explanation for this apparent discrepancy is that although KLC1 is predominantly expressed in tissues of neuronal origin, these tissues also express KLC2, and possibly KLC3. Thus, loss of KLC1 may reduce the overall pool of functional KLC below some necessary threshold, but otherwise KLC2, or perhaps KLC3, can carry out the specific functions that KLC1 ordinarily has. A second explanation is that Drosophila axons are of much smaller caliber than the mouse axons, and hence, a defect leading to diminished cargo transport in a smaller axon might actually cause significant blockage of any type of fast transport within that axon. In fact, both KLC and KHC mutants in Drosophila exhibit membranous clogs in the segmental nerves . A variety of fast transport motors and cargoes are seen to colocalize in the clogs, suggesting that they may impair multiple pathways of axonal transport. A mouse axon may be able to overcome this blockage problem by having more volume to accommodate membranous accumulations. Hence, lethality due to null mutations of KHC and KLC in Drosophila may be a secondary phenotype due to the physiology of the organism, rather than because of a crucial requirement for certain cargoes to be transported by kinesin-I. A third explanation is that KLC1 may be required for a subset of kinesin-I molecules in the cell to transport unique cargoes, but the transport of these cargoes is not essential for neuronal viability. Further work is needed to distinguish among these possibilities. The most plausible explanation for the cellular phenotype observed in KLC1 mutant mice is that a subset of kinesin-I holoenzyme function is disturbed in KLC1 mutant mice. The abnormal accumulations of KIF5A and KIF5B may reflect either blocks in some nonessential transport processes that require KLC1, kinetic accumulations resulting from reduced rates of some events, or accumulations at abnormal sites. Although KIF5C was not analyzed owing to a lack of appropriate reagents at the time these experiments were conducted, we presume that future work will find that its behavior is altered as well. It is unclear why the cellular distribution of KIF5A is perturbed in both sensory and motor neurons whereas the cellular distribution of KIF5B is only detectably perturbed in sensory neurons. Whether these cell-type specific differences reflect divergent roles of KIF5B in motor and sensory neurons or a different balance between active and inactive states is unknown at present. Previous studies have indicated that kinesin-I may play roles in a variety of different cellular processes, e.g., trafficking between the Golgi apparatus and the ER , mitochondrial placement , lysosomal movement , and perhaps late events in the secretory pathway. Double labeling experiments indicated that accumulations of KIF5A in sensory and motor neuron cell bodies colocalized with the cis Golgi marker giantin. This observation is consistent with the hypothesis that kinesin-I plays some role in transport events involving, or initiating, at the Golgi apparatus. Interestingly, β′-COP staining was also radically redistributed in sensory neuron cell bodies in KLC1 mutant animals. Therefore, the redistribution of β′-COP in the mutant DRG cells may be caused by some disturbance in the rate or character of transport between Golgi apparatus and ER, or perhaps interference by the aberrant accumulations of KIF5A or KIF5B. It is unlikely, however, that there is an absolute block in these events, since the mice are viable and the Golgi apparatus has a grossly normal organization and distribution in these cells. KLC1 mutant neuronal cells also did not exhibit any obvious changes in either mitochondria or steady state lysosome distribution by immunofluorescence studies (data not shown), although KIF5B localization was abnormal. These differing observations suggest that neuronal and ubiquitous forms of kinesin-I may have distinct pathways of activity that do not fully overlap, and may diverge in different cell types, depending on the cargo or other burden in each particular cell. Formally, there are four possible models for KLC function. The first, the KLC inhibition model, is that KLC negatively regulates KHC function. This model is supported by indirect work suggesting that the enzymatic ATPase activity of KHC is enhanced upon removal of its tail or KLC . This model is also supported by recent studies indicating that KLC inhibits KHC from binding microtubules at physiologic pH, however, shift of pH to 6.8 releases this inhibition . Interestingly, in a transient transfection system in COS cells, the coiled-coil domain of KLC by itself was capable of binding KHC and causing this inhibitory effect. These studies also suggested that excess KHC by itself, in the absence of coexpressed KLC, would translocate along microtubules to the periphery of the cell. Our observations are not entirely consistent with this study and do not readily support the KLC inhibition model. We do not observe accumulation of the KIF5A or KIF5B KHC chains at the cellular periphery or on filamentous elements in the cytoplasm of KLC1 mutant neurons. Instead, we see accumulations in or near the Golgi apparatus of KIF5A and punctate accumulations of KIF5B associated with COPI. It is possible that these observational differences can be explained by the fact that COS cells are not neurons; further work is needed to uncover the source of this discrepancy. The KLC inhibition model is also inconsistent with previous data suggesting that kinesin-I in Neurospora and sea urchin may not require stoichiometric amounts of KLC. The second model of KLC function, the KLC attachment model, suggests that KLC is required for general attachment to cargo. This model is supported by recent work showing that perfusion of squid axoplasm with an mAb directed against the TPR domain of KLC significantly inhibits transport of vesicles and organelles. Therefore, one would expect that removal of the TPR domain in KLC1 would prevent KHC from interacting properly with all potential cargoes. In this view, KLC1 mutant mice would be expected to exhibit cellular defects suggestive of the absence of cargo binding. In fact, KLC1 mutant mice show accumulations of KIF5A (and possibly KIF5B) on putative cargoes in the cell bodies of the motor and sensory neurons. The third model, the KLC specific targeting model, suggests that KLC is required for targeting of KHC to some, but not all cargoes. This model is supported by recent work indicating that a particular splice form of KLC is preferentially localized on mitochondria, but not on other potential kinesin-I cargoes . In this view, our observation of abnormal accumulations of KIF5A on or near the Golgi apparatus in KLC1 mutants would result from absence of proper targeting, leading to improper targeting to the Golgi apparatus. However, the observation that faint but detectable KIF5A is seen in or near the Golgi apparatus in wild-type neurons, coupled to many previous observations of kinesin-I in association with Golgi elements in a variety of cell types , suggests that the Golgi region is a normal site of KIF5A binding. The fourth and final model, which is the model we favor at present, is the KLC activation model. This model suggests that KLC is required to activate KHC after proper cargo binding. This model is consistent with data in Drosophila demonstrating that KLC is essential for kinesin-I function , and is also supported by our observation that in the absence of functional KLC1, KIF5A accumulates at a site of presumed transport initiation in or near the Golgi apparatus. In fact, several previous experiments suggest that there is a normal association of some kinesin-I with elements of the Golgi apparatus, perhaps because kinesin-I plays some role in transport initiating at the Golgi apparatus . Under this view, KIF5B is also accumulating at a presently unidentified site of transport initiation in KLC1 mutants. This model can also accommodate the suggestion that cargo binding is required for activation of the kinesin-I holoenzyme by either causing pH shift or small conformation changes. Further work on cells cultured from these mutant animals and permeabilized cell systems may help to test this model, and to understand the details of the proposed activation that is revealed by the KLC1 mutant. | Study | biomedical | en | 0.999996 |
10491392 | Human Op18 derivatives, with or without an 8–amino acid COOH-terminal Flag epitope, were expressed using the pET-3d expression vector and purified from Escherichia coli as described . The COOH-terminal–truncated Op18, with the sequence encoding amino acids 100–147 deleted (Op18-Δ100-147), and the NH 2 -terminal–truncated Op18, with the sequence encoding amino acids 5–55 deleted (Op18-Δ5-55), were constructed using PCR strategies . The same strategies were used to construct the Op18-Δ5-9, Op18-Δ5-16, Op18-Δ5-25, Op18-Δ5-38, Op18-Δ5-46, and Op18-Δ90-147 derivatives and primer sequences are available from the authors on request. Constructs for expression of glutathione-S-transferase (GST)–Op18 fusion proteins in E . coli were generated by inserting an NcoI-NotI fragment of wild-type and deleted Op18 derivatives into NcoI-EcoRI–digested pGEX 4T-1 (Pharmacia) together with a double stranded adapter of the two oligonucleotides: 5′-AATTCGCGGC-3′ and 5′-CATGGCCGCG-3′. The GST-Op18 fusion proteins were expressed and purified on glutathione-Sepharose 4B beads as recommended by the manufacturer (Pharmacia). For expression of Op18 deletion mutants in human cell lines, Op18 derivatives were excised from pBluescript SK+ using HindIII and BamHI, and Op18 fragments were cloned into the corresponding sites of the EBV-based shuttle vector pMEP4 . Analysis of tubulin GTPase activity was performed in PEM buffer (80 mM piperazine- N,N ′-bis[2-ethanesulfonic acid], 1 mM EGTA, 4 mM Mg 2+ , pH 6.8) containing 17% glycerol and 5 mM adenyl-5′-yl imidodiphosphate (AMP-PNP; to inhibit nonspecific ATPase activity). GTPase activity in the presence of free GTP, which allows multiple substrate turnover, was monitored by incubating tubulin (5–10 μM) with α-[ 32 P]GTP (100 μM; 2 × 10 6 dpm per 25-μl reaction) at 37°C in the absence or presence of specific Op18 derivatives. To analyze exchange independent GTP hydrolysis in a single turnover reaction, tubulin (100 μM) was preloaded with α-[ 32 P]GTP (100 μM; 50 × 10 6 dpm per 25-μl reaction) on ice for 30 min. Tubulin in complex with α-[ 32 P]GTP was recovered by centrifugation through a desalting column (P-30 Micro Bio-Spin; Bio-Rad Laboratories) and GTP hydrolysis was followed at 37°C. In the absence of tubulin, but in the presence of BSA or Op18 (15 μM), these columns retained >99.99% of all α-[ 32 P]GTP. Control experiments showed that the Op18 preparations used neither bind nor hydrolyze α-[ 32 P]GTP. Nucleotide hydrolysis was quantitated as described . In brief, aliquots were removed at the times indicated, adjusted to contain 0.1% SDS, and heated for 2 min at 80°C. Aliquots (0.6 μl) were spotted onto polyethyleneimine cellulose plates (Merck) and GDP-separated from GTP by ascending chromatography in 1.2 M NH 4 COOH acidified with 1.2 M HCl. PhosphorImager (Molecular Dynamics, Inc.) analysis of radioactive spots was used for quantification. Analyses of Op18–tubulin association in crude extracts from transfected K562 cells were performed after cell lysis (50 × 10 6 per ml, on ice) in PEM buffer, pH 6.8, containing glycerol (5%), Triton X-100 (0.5%), β-glycerophosphate (10 mM), leupeptin (20 μM), pefablock (1 mM), and benzamide (1 mM). Cell extracts were clarified by centrifugation and, thereafter used for pull-down assays as described below. For equilibrium binding experiments, COOH terminally Flag-tagged wild-type and truncated Op18 derivatives (2–10 μM) and tubulin (0.8–36 μM) were mixed and allowed to associate on ice for ∼15 min to ensure equilibrium. Independent of the Op18 derivative tested, association of Op18–F-tubulin complexes is rapid and equilibrium reached within a few minutes . Op18–Flag-tubulin mixes (48 μl) were added to agarose beads (12 μl) coupled with the Flag-epitope specific M2 antibody (Sigma Chemical Co.), and incubated for 30 min at 8°C to capture Op18-Flag-tubulin complexes. Alternatively, glutathione-Sepharose beads (Pharmacia) were used to analyze tubulin binding to NH 2 terminally GST-tagged Op18 derivatives. To allow rapid separation of Op18–tubulin complexes bound to M2 or glutathione beads, the bead suspension was applied into the cap of a 1.5-ml Eppendorf tube containing a bottom layer of 0.4 ml of PEM complemented with 27%:17% sucrose/glycerol, pH 6.8, and a top layer of 0.2 ml of PEM with 17% glycerol, pH 6.8. The caps were closed with care, to keep the bead suspension hanging in the cap, and the samples were centrifuged (for 1 min at 21,000 g ) to separate bead-bound and free material. The supernatants and pellets were boiled in SDS–sample buffer and resolved by 10–20% gradient SDS-PAGE as described . Tubulin and Op18 content were quantified by Coomassie blue staining of protein bands followed by scanning using a personal densitometer (Molecular Dynamics). Bovine tubulin and a standard recombinant Op18 preparation, in which the protein mass had been determined by amino acid analysis, were used as internal standards. In a more sensitive binding assay, binding was analyzed after labeling of tubulin with α-[ 32 P]GTP as described above under Assays of Tubulin GTPase Activity. Control experiments confirmed that in the absence of free GTP, α-[ 32 P]GTP remained stably associated with tubulin over the time-course of the assay, both in the presence and absence of Op18. A trace of 125 I–Op18-F was also included to allow simultaneous quantification of Op18 and tubulin in the same sample. There are two major benefits with this strategy. First, the amount of Op18-F (∼30% of total Op18-F) present in the fraction of free tubulin after separation of M2-coupled beads can be compensated for in each data point and, second, only biologically active (i.e., GTP bound) tubulin was detected in the fraction of free tubulin. The contribution of nonspecific tubulin binding was generally ∼3% of the amount of free tubulin and was subtracted from presented data. Since the position of the epitope tag used for pull-down of Op18–tubulin complexes may influence binding, in particular if large deletions are introduced close to the epitope tag, tubulin binding of Op18 derivatives was analyzed using either the NH 2 -terminal GST-tag or COOH-terminal Flag-tag. The data showed that the position of the epitope tag does not significantly alter Op18–tubulin binding characteristics (data not shown). To calculate equilibrium dissociation constants, data points from equilibrium binding experiments were fitted either to a hyperbola or to a model assuming two-site positive cooperativity in binding . Written in the form of a binding curve, a two-site positive cooperativity model has the form, \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*}B=\frac{B\;{\mathrm{max}}}{2}{\times} \left {1}/{ \left \left[1+\displaystyle\frac{{\mathrm{2}}F}{K_{d}{\mathrm{1}}}+\displaystyle\frac{F^{2}}{ \left \left(K_{d}{\mathrm{1}}{\times}{\mathit{K}}_{d}2\right) \right }\right] \right } \right {\times}\frac{{\mathrm{2}}F}{K_{d}{\mathrm{1}}} \left \left(1+\frac{F}{{\mathit{K}}_{d}2}\right) \right {\mathrm{,}}\end{equation*}\end{document} where K d 1 and K d 2 are the equilibrium dissociation constants for Op18 binding of the first and second tubulin heterodimer respectively, B is the Op18/tubulin molar ratio of complex-bound proteins, and F is the free tubulin concentration. Comparison of fits was performed using the F-test provided by GraphPad Prism (GraphPad Software, Inc.). To determine dissociation rates of Op18–tubulin complexes, M2 beads coated with Flag-tagged Op18 derivatives were incubated with tubulin (20 μM) for 15 min at 8°C to attain equilibrium binding. Before centrifugation through a sucrose/glycerol cushion, bead suspension was diluted 100-fold in PEM ± glycerol (17%) and incubated for 20–900 s. Op18–tubulin complexes were quantitated by the dual isotope labeling strategy described above. Dissociation rates were calculated assuming one phase exponential decay using GraphPad Prism software. Cells were washed in PBS and disrupted by boiling in SDS–sample buffer. The cell extract was clarified by centrifugation and, thereafter, separated by 10–20% gradient SDS-PAGE, followed by transfer to nitrocellulose filters. Cellular Op18 was detected by probing filters with affinity-purified anti-Op18 . For detection of Flag epitope-tagged Op18, the anti–Flag-M2 antibody (Sigma Chemical Co.) was used. Cellular tubulin was detected by probing filters with anti–α-tubulin (clone B-5-1-2; Sigma Chemical Co.). Probing of filters, detection of specific antibodies by 125 I–protein A, and PhosphorImager analysis of radioactive bands was performed as described . Quantification of Op18 and tubulin in cell lysates was obtained by comparing signals with standard curve generated by separating graded amounts of bovine tubulin and recombinant Op18 on the same SDS-PAGE. The errors between independent determinations were routinely <20% and the protein mass of bovine tubulin and recombinant Op18 was determined by amino acid analysis. The conditions used for transfection studies and the pMEP4 shuttle vector system have been described previously . Conditional expression of various Op18 derivatives was achieved by using the hMTIIa promoter, which can be suppressed by low concentrations of EDTA (50 μM) and induced by Cd 2+ . The amount of pMEP-Op18 constructs per electroporation was adjusted to obtain comparable expression levels of all Op18 derivatives. 5 μg DNA was used of pMEP-Op18-wt-F and pMEP-Op18-Δ5-9-F, whereas 12.5 μg was used of pMEP-Op18-Δ5-25-F and Op18-Δ100-147-F. The cellular content of MT polymers was determined by extracting soluble tubulin in an MT-stabilizing buffer followed by quantification of tubulin in the particulate and soluble fraction as described . For flow cytometric analysis, cells were extracted, fixed, and stained with anti–α-tubulin (clone B-5-1-2) as described . Analysis of 150,000 cells per transfected Op18 derivative was performed using a FACs-calibur together with the Cell Quest software (Becton Dickinson & Co.). Primary cultures of newt lung cells were grown on coverslips in chambers at room temperature (23°C) as previously described . For microinjection studies, cells were typically used 4–6 d after each culture had begun. Newt lung cells at the peripheral regions of the cell sheet were microinjected with various Op18 protein derivatives in a 2-mM phosphate buffer. Injected volumes are likely consistent with previous estimates of 5–10% of cell volume , resulting in final intracellular concentrations of 8–16 μM of various Op18 derivatives. Cells were injected on an Olympus CK2 inverted microscope with attached Narishige MN-151 manipulator, using a capillary holder and prepulled glass microtips (ID 0.5 μm; World Precision Instruments, Inc.). Cells were microinjected, incubated for 3 h at room temperature, and then lysed and fixed for antitubulin immunofluorescence as described . Microinjected cells were identified based on their position relative to the tissue explant. Cells were examined using a 60× 1.4 NA Plan Apo objective on an Olympus BH-2 microscope. Images were projected to an MTI CCD-72 camera, integrated (32 frames), and stored using NIH Image software (version 1.57). The final figure was composed using Canvas 6.0 software (Deneba Software). Complex formation with tubulin is a likely minimal requirement for tubulin-directed signals of Op18. To analyze tubulin complex formation of the series of NH 2 - and COOH-terminal truncations of Op18 depicted in Fig. 1 A, Flag epitope-tagged Op18 (Op18-F) and tubulin were mixed at an equimolar ratio (10 μM each). Op18-F-tubulin complexes were recovered using anti–Flag antibody coupled beads together with a rapid one-step gradient protocol. The bead-bound Op18-F-tubulin complexes were resolved and quantified by SDS-PAGE followed by densitometric scanning of stained gels. The result shows that at a 1:1 molar ratio, essentially all tubulin was in complex with Op18-wt . A partial loss of tubulin binding was evident after truncations of the NH 2 -terminal region covering amino acids 5–9 to amino acids 5–46. Further truncation into the functionally important heptad repeats results in a major loss of tubulin binding. It is also shown that large COOH-terminal truncations, represented by the Op18-Δ100-147 and Δ90-147 derivatives, result in a major loss of tubulin binding. The next step in the analysis was to determine the effect of Op18 truncations on stimulation of tubulin GTPase activity. This analysis was performed under identical buffer conditions and tubulin concentrations as in Fig. 1 B. Under these conditions, Op18-wt causes a modest 1.7-fold increase in the basal tubulin GTPase activity . Interestingly, despite decreased tubulin binding, truncations in the NH 2 -terminal region up to amino acid 46 result in enhanced GTPase activity. Decreased tubulin binding of the COOH-terminal–truncated derivatives was associated with the opposite activity, namely significant suppression of the basal tubulin GTPase activity. Consistent with its lack of tubulin binding, Op18-Δ5-55 showed no detectable modulation of GTPase activity. Fig. 1 D shows Op18-mediated stimulation of tubulin GTPase activity in the presence of nocodazole, an MT-disrupting agent that by itself stimulates tubulin GTPase activity. Nocodazole, under these conditions, mediates a fivefold enhancement of both basal and Op18-wt–stimulated GTPase activity. Interestingly, albeit that there is a general fivefold increase, we observed the same pattern of GTPase stimulatory/suppressive activities of the truncated Op18 derivatives as in the absence of nocodazole. Since truncations of an NH 2 -terminal region of Op18 results in enhanced stimulation of tubulin GTPase activity, and truncation of a COOH-terminal region results in suppression of both the basal- and nocodazole-induced GTPase activity, it appears that the native Op18 contains tubulin binding determinants with multiple regulatory effects on tubulin GTP metabolism. In the experiments shown in Fig. 1 , the tubulin concentration is too low to support detectable polymerization (data not shown). In the assays performed in the presence of the antipolymerizing drug nocodazole, it is even less likely that the observed Op18-specific effects are caused by a mechanism involving tubulin polymerization. Furthermore, all the Op18 derivatives tested, except Op18-Δ5-55, mediate varying degrees of antipolymerizing activity under the present conditions (data not shown). Hence, we conclude that the activity profiles of the truncated Op18 derivatives reflect modulation of the tubulin GTPase activity under nonassembly conditions. Op18 may modulate tubulin GTP metabolism by altering the rate of nucleotide exchange and/or hydrolysis. To specifically analyze Op18 regulation of the GTP hydrolysis rate, selected Op18 derivatives were mixed in the presence of nocodazole with tubulin prebound to α-[ 32 P]GTP (note that a threefold molar excess of Op18 was used to increase the fraction of tubulin in complex with mutated Op18). The assay was performed in the absence of free GTP, and hydrolysis of tubulin-bound α-[ 32 P]GTP (i.e., a single turnover event) was followed over time. As shown in Fig. 2 A, in contrast to the result obtained in the presence of free α-[ 32 P]GTP in the reaction mix , the data show that Op18-wt and NH 2 -terminal–truncated derivatives were indistinguishable in their stimulation of GTP hydrolysis in a single turnover assay. It is also shown that the COOH-terminal–truncated derivative (Δ100-147) has a potent suppressive effect on the rate of GTP hydrolysis. Since nocodazole was included in the reaction mix, which results in a fivefold increase in both the basal- and Op18-stimulated level of GTP hydrolysis , it appears that this derivative exerts a general suppression of tubulin GTP hydrolysis. In the presence of free α-[ 32 P]GTP in the reaction mix, which allows multiple turnovers of GTP, truncation of the NH 2 -terminal region of Op18 enhances stimulation of tubulin GTPase activity both in the absence and presence of nocodazole . However, in a single turnover reaction, these same derivatives were indistinguishable from Op18-wt . This suggests that Op18-wt may contain an NH 2 -terminal region that inhibits GTP exchange. To address this, selected Op18 derivatives and prebound α-[ 32 P]GTP-tubulin were first mixed, and GTP hydrolysis in the absence of nocodazole was followed for 20 min at 37°C. As shown in Fig. 2 B, Op18-wt and NH 2 -terminal–truncated derivatives show the same stimulation of tubulin α-[ 32 P]GTP hydrolysis, as measured in a single turnover assay, whereas the COOH-terminal–truncated derivative suppresses the basal hydrolysis obtained with tubulin alone, which agrees with the data obtained in the presence of nocodazole . To assess if Op18 blocks GTP exchange, the reaction was chased with cold GTP after 21 min and subsequent alterations in the rate of α-[ 32 P]GTP hydrolysis were monitored . As expected from the rapid GTP exchange rate of free tubulin , the data show that a chase with cold GTP results in an immediate attenuation of the basal α-[ 32 P]GTP hydrolysis. In the presence of Op18-wt, however, the result of a chase with cold GTP was modest and the rate of α-[ 32 P]GTP hydrolysis shows only a slow decline with time, which is contrasted by a more profound effect of the chase in the presence of NH 2 -terminal–truncated derivatives. This indicates that Op18 exerts a potent tubulin GTP exchange inhibitory activity that involves the NH 2 -terminal region. To further address the importance of the NH 2 -terminal region for regulating GTP exchange of tubulin, tubulin and cold GTP were mixed and allowed to reach steady state GTP hydrolysis at 37°C. Thereafter, α-[ 32 P]GTP was added and the generation of α-[ 32 P]GDP was monitored. This protocol provides selective detection of GTP hydrolysis after exchange to a labeled nucleotide. Given the indistinguishable GTP hydrolysis rates in the presence of wt and NH 2 -terminal–truncated derivatives , inhibition of nucleotide exchange would be manifested as decreased generation of α-[ 32 P]GDP in the presence of Op18-wt. As predicted, the data in Fig. 2 C shows that NH 2 -terminal truncation of Op18 results in a twofold increase of α-[ 32 P]GTP hydrolysis as compared with the native Op18 protein. Thus, Op18-mediated inhibition of GTP exchange via its NH 2 -terminal region provides a mechanism that explains the observation that NH 2 -terminal truncation enhances stimulation of tubulin GTPase activity under conditions allowing multiple turnovers of GTP . Inhibition of GTP exchange together with the demonstrated positive and negative modulation of the rate of GTP hydrolysis, as schematically outlined in Fig. 2 D, indicates that Op18 binding to tubulin results in multiple tubulin-directed activities that can be genetically dissected and assigned to specific regions of the protein. Since the COOH-terminal is essential for stimulating hydrolysis, whereas deletion of this region results in a derivative with the opposite activity, namely suppression of the GTP hydrolysis rate, it appears that there exist regions in the remaining polypeptide that must counteract the stimulatory activity of the COOH-terminal. Since Op18-Δ5-46 stimulates GTP hydrolysis to the same extent as Op18-wt, it follows that the region with suppressive potential would be located between amino acids 46 and 100 as depicted in Fig. 2 D. However, it is not possible to test this prediction by deletion analysis since Op18-Δ5-46 represent the largest possible NH 2 -terminal truncation that does not cause termination of tubulin binding and, thereby, modulation of GTPase activity . It has been proposed that the Op18–tubulin complex is relatively stable since it resists gel filtration and analytical ultracentrifugation . To evaluate if the tubulin-directed activities described above is the result of a stable complex between Op18–tubulin or, alternatively, if Op18 mediates tubulin-directed activities in a dynamic complex, we determined the dissociation rates of selected Op18 derivatives in the presence or absence of glycerol as described in Materials and Methods. The data in Table show that Op18-wt-tubulin complexes have a rapid dissociation rate that decreases about fourfold in the presence of glycerol. The dissociation rate constant in the absence of glycerol (0.010 s −1 ) is similar to what has been reported using plasmon resonance measurement . NH 2 -terminal truncations result in faster dissociation that is slowed down ∼10-fold by glycerol. The Op18-Δ100-147 derivative formed the most unstable complex with tubulin and glycerol has only a minor stabilizing effect. In conclusion, Op18 forms dynamic complexes with tubulin and, in the case of Op18-Δ100-147, the complex has a half-life of ∼35 s in the buffer conditions used throughout this study (i.e., in the presence of glycerol).This indicates that Op18 modulates GTP hydrolysis by multiple transient tubulin interactions during the time of the assay and not by forming stable complexes with tubulin. The dynamic nature of the Op18–tubulin complex may appear contradictory to a previous report by us in which we employed a similar experimental approach . Thus, we noted that the estimated Op18/tubulin ratio was ∼1:3 at equilibrium, and upon a 20-fold dilution the ratio rapidly changed to an apparently stable 1:2 ratio. Therefore, we suggested that one Op18 complexed with three tubulins rapidly changed upon dilution to a stable complex containing two tubulins. However, the apparent stability was due to formation of a new equilibrium after the dilution step. This misinterpretation, together with a 33% error in our previous estimation of Op18/tubulin molar ratio, was the cause of this misconception. As shown above, Op18 deletion mutants are useful analytical tools to dissect modulation of tubulin GTP metabolism, and the results suggest that physically separate tubulin binding motifs of Op18 transmit distinct tubulin-directed signals. Assuming that separate tubulin binding motifs are all involved in the overall binding affinity between Op18 and tubulin, specific truncations would be anticipated to result in a step-wise loss of affinity. To approach these questions, we performed detailed analyses of Op18–tubulin interactions. Op18–tubulin association previously has been shown to be rapid, and equilibrium is reached in <5 min using either wild-type or truncated derivatives . Selected COOH terminally Flag-tagged Op18 derivatives, and their NH 2 terminally GST-tagged counterparts (see Materials and Methods) were analyzed for their ability to bind tubulin over a range of concentrations by the strategy described under Fig. 1 . Scatchard analysis of these data showed that all Op18 derivatives tested, bind close to 2 mol tubulin per mol (Op18-wt, 2.3 ± 0.07; Op18-Δ5-9, 2.1 ± 0.05; Op18-Δ5-25, 2.1 ± 0.08; and Op18-Δ100-147, 2.0 ± 1.3; three independent determinations in PEM, pH 6.8). However, because of difficulties to quantify low tubulin concentrations by scanning Coomassie blue–stained gels, the independence or cooperativity in Op18–tubulin binding was difficult to assess. Therefore, we adopted a strategy involving labeling of tubulin with α-[ 32 P]GTP. Control experiments showed that in the absence of free GTP, α-[ 32 P]GTP remained stably associated with tubulin over the time-course of the assay both in the presence and absence of Op18 (for 30 min at 8°C). To allow simultaneous quantification of Op18 and tubulin in the same sample, a trace of 125 I-Op18-F was added. Using this dual labeling approach, we obtained bindings curves of sufficient quality for detailed analysis of affinities . It is clear from the binding curves for Op18-wt that half saturation of tubulin binding to Op18-wt is reached at 1.2 μM at pH 6.8 (A) and 4.3 μM at pH 7.4 (B). A previous study has also noted pH regulation of affinity , and, in this study, an equilibrium dissociation constant ( K d ) of 0.5 μM at pH 6.5 was determined by plasmon resonance measurement, which is in reasonable agreement with our data obtained at pH 6.8. However, Scatchard analysis of both pHs (C and D), shows that tubulin binding to Op18 is a complex process with observed nonlinear data point distribution typical for positive cooperativity in binding . Analysis of tubulin binding curves of selected Op18 mutants showed that half saturation of NH 2 -terminal truncation derivatives requires two to fourfold higher tubulin concentrations, as compared with Op18-wt, and that the COOH terminally truncated protein is most severely affected in its tubulin affinity. Since the position of the epitope tag used for pull-down may influence the effect of specific truncations, we also analyzed tubulin binding of selected Op18 derivatives tagged in the NH 2 terminus with GST (see Materials and Methods). The results obtained using GST-tagged derivatives were essentially the same as using the COOH-terminal Flag-tag (data not shown). Hence, the binding appears independent of the position of the epitope tag. It is notable that tubulin binding to all Op18 derivatives tested is a cooperative process as indicated by the sigmoidal binding curves and nonlinear Scatchard plots. Glycerol, which is commonly used in tubulin buffers, slows down Op18–tubulin dissociation ( Table ). In agreement with this result, it is shown in Fig. 4 A that glycerol enhances tubulin binding of Op18-wt and NH 2 terminally truncated derivatives. The binding of Op18-Δ100-147 was essentially unaltered, consistent with the minor effect of glycerol observed on the dissociation rate of this derivative. The experiment in Fig. 4 was performed with 17% glycerol, but even 6% glycerol was sufficient for a major enhancement in tubulin binding (data not shown). The presence of glycerol shifts the curves of the nonlinear Scatchard plots but the data are still typical for positive cooperativity in the interaction. Two-site positive cooperativity implies that Op18 first binds one tubulin with a given affinity (termed K d 1), and this generates a second site with a higher affinity (termed K d 2). A two-site positive cooperativity model gives the best fit with experimental data, obtained using different buffer conditions in Fig. 3 and Fig. 4 , and was used to calculate K d 1 and K d 2. The results are summarized in Table . Deletion analysis shows that both the NH 2 - and COOH-terminal regions are important for binding of the first tubulin (defined by K d 1), and this is particularly evident in the presence of glycerol, which primarily enhances K d 1. It is also clear that COOH-terminal truncation, as defined by Op18-Δ100-147, results in a major decrease in binding of the second tubulin (defined by K d 2), whereas NH 2 -terminal truncations have only a minor effect. Finally, comparison of Op18-wt with deletion mutants reveals a stepwise decrease in affinity as reflected by K d 1 and K d 2 as well as the estimated free tubulin concentration required for half saturation of Op18. This indicates that Op18 binds tubulin via multiple nonessential contact points distributed over a major part of the Op18 sequence. This is consistent with the above dissection of tubulin-directed activities of Op18, which reveals multiple tubulin-directed activities that can be separated by deletion analysis and assigned to distinct regions spanning a major part of the Op18 peptide. Hence, the data support a model where physically separate tubulin binding motifs of Op18 transmit distinct tubulin-directed signals. The significance of Op18 mutant phenotypes observed in vitro was evaluated by transfection experiments using a shuttle vector directing inducible expression of Op18-wt and the deletion derivatives . The amount of transfected DNA was adjusted to obtain comparable expression levels of the ectopic proteins in the human K562 cell line. Estimated levels of endogenous tubulin and Op18, together with the induced level of each of the ectopic Op18 derivatives, are shown in Table . The estimated tubulin concentration (23 μM) is similar to that reported for frog extracts , and assuming that each Op18 binds two tubulin heterodimers, it is notable that the molar concentration of endogenous Op18 (10 μM) is sufficient for complex formation with essentially all cellular tubulin, even under conditions of complete tubulin depolymerization. Since Op18-wt has higher affinity for tubulin than the truncated protein, it seems likely that endogenous Op18 levels are sufficient to outcompete tubulin binding of overexpressed deletion derivatives. To test this prediction, we performed an in vitro experiment using components at a similar molar ratio as observed in intact cells. As shown in Fig. 5 A, pull-down assays using GST-fused Op18-wt in the presence of a fivefold molar excess of the indicated Op18 derivatives show the expected 80% competition of tubulin binding by Op18-wt, whereas Op18-Δ5-25 competes with only 20% of the binding and competition by Op18-Δ100-147 is essentially undetectable. In light of this finding, it seems likely that endogenous Op18 will inhibit most of the complex formation of ectopic Op18 deletion mutants with cellular tubulin in transfected cells. This was evaluated in crude cell extracts of K562 cells transfected with Flag-epitope–tagged Op18 (treated exactly as the cells analyzed in Table ). Complex formation was evaluated and as shown in Fig. 5 B, about half of all cellular tubulin is in complex with Op18-wt-F. As predicted, tubulin complex formation by truncated derivatives was much lower. This data predict that the modest five to eightfold overexpression of Op18 deletion mutants would not result in significant tubulin sequestering, and that potential MT destabilization is likely to be attributable to more specific mechanisms. To search for a phenotype of Op18 deletion mutants, transfected K562 cells were induced to express wild-type and truncated Op18 derivatives for 4 and 6 h. As shown in Fig. 6 A, all the Op18 derivatives analyzed are expressed at comparable levels, and expression of all derivatives results in dramatic MT destabilization . Thus, as shown by both biochemical determination (B) and flow cytometric analysis (C and D, note the log scale), all the truncated Op18 derivatives retain at least 50% of wild-type MT destabilizing activity. The specificity of destabilization by the deletion mutants analyzed in Fig. 6 is indicated by previous studies of Op18-Δ5-55, which by several criteria is functionally inactive in vivo . Since truncated Op18 proteins in the presence of endogenous Op18 have only a minor potential to complex with cellular tubulin , formation of stable tubulin sequestering complexes cannot explain the phenotype. Hence, Op18 mutants destabilize MTs by more specific mechanisms, possibly involving tubulin/MT–directed signaling. Fig. 6 shows that both the NH 2 -and COOH-terminal–truncated derivatives have a similar gross phenotype with respect to the MT-destabilizing activities, whereas the in vitro analysis shows that these two classes of deletion derivatives have opposite effects on the rates of tubulin GTP hydrolysis. Assuming that the tubulin-directed activities are linked to the phenotype mediated by Op18 derivatives in intact cells, it follows that the NH 2 - and COOH-terminal–truncated mutants cause the same gross phenotype by distinct mechanisms. A prediction from the proposition above, namely that NH 2 - and COOH-terminal–truncated mutants cause MT destabilization by distinct mechanisms, is that the remaining cellular MT networks would exhibit morphological cues, reflecting the different modes of action. The spherical shape of K562 leukemia cells make them unsuitable for morphological analysis. Therefore, we analyzed MTs in newt lung cells 3 h after microinjection of purified Op18 protein derivatives. At high intracellular concentrations (40–100 μM), Op18-wt, Op18-Δ5-9, Op18-Δ5-25, and Op18-Δ100-147 all mediated a massive loss of MT polymer, whereas the inactive variant Op18-Δ5-55 did not alter MT polymers, demonstrating the specificity of Op18-mediated polymer loss (data not shown). To analyze the phenotypes at concentrations similar to that of endogenous Op18 in K562 cells ( Table ), Op18 derivatives were microinjected to an estimated intracellular concentration of 8–16 μM. Under these conditions, we could discern clear differences in MT-directed activities of Op18 derivatives. As shown in Fig. 7 , injection of Op18-wt and the COOH-terminal–truncated derivative (Op18-Δ100-147) resulted in loss of MTs from the lamella. While a few long MTs continued to extend out towards the cell surface, many MTs appeared shorter and did not enter the lamella region. In contrast, the NH 2 -terminal truncations (Op18-Δ5-9 and Op18-Δ5-25) had less effects, but some loss of MT polymer was observed. The loss of MTs from the lamellar region, which most likely reflect shortened MTs, provided a clear morphological assay to compare the phenotype of Op18 derivatives. We measured the number of cells that exhibited 25% or more of the lamella region cleared of MTs. The results are summarized in Fig. 8 and show that microinjection of Op18-wt resulted in a 12-fold increase in the fraction of cells with cleared lamella. The NH 2 terminally truncated proteins showed low activity, whereas the Op18-Δ100-147 protein was almost as active as the wild type. Hence, analysis of lamellar clearing indicates an overlapping phenotype of Op18-wt and the Op18-Δ100-147 derivative that is dissociated from that of the NH 2 -terminal–truncated derivatives. It is notable that while the Op18-Δ5-9 derivative is significantly more efficient than Op18-Δ100-147 in decreasing the total MT content of K562 cells , the same 4–amino acid NH 2 -terminal truncation attenuates most of the lamellar clearing phenotype in newt cells . These results dissociate the lamellar clearing phenotype observed in newt cells from the decrease in total MT content of K562 cells and support the idea that the NH 2 - and COOH-terminal–truncated proteins in part regulates MTs by distinct mechanisms. This in turn suggests that Op18-wt has the potential to regulate the MT system by more than one specific mechanism. Here, we have performed a deletion analysis of Op18, the prototype member of a novel class of MT regulators, and identified regions involved in differential modulation of tubulin GTP metabolism. How these in vitro activities segregate from tubulin binding properties and specific MT-regulating activities in intact cells was determined to address the mechanism by which Op18 exerts its regulatory role. These analyses were facilitated by the maintenance of protein stability by all the truncations tested, which indicated that Op18 lacks distinct domain structures. Using these truncated Op18 proteins, physically separated tubulin binding motifs of both the NH 2 - and COOH-terminal regions were found to transmit distinct tubulin-directed signals. These signals were manifested on the level of modulation of both exchange and hydrolysis of tubulin-bound GTP . By analyzing the activities of deleted Op18 derivatives in K562 cells, we obtained data most consistent with MT destabilization by a mechanism other than tubulin sequestration. Hence, K562 cells contain sufficient endogenous Op18 to sequester all unpolymerized tubulin and NH 2 - and COOH-terminal deletion mutants, with decreased tubulin binding affinity, were unable to compete for tubulin binding . However, despite a low level of association with cellular tubulin, these mutants still cause a drastic phenotype in intact cells . Moreover, a detailed analysis of tubulin binding shows that Op18-Δ100-147 has even less potential than Op18-Δ5-25 to regulate MT polymerization by a simple sequestering mechanism ( Table and Table ). Nevertheless, these two derivatives still caused a similar decrease of MT content in transfected K562 cells . Given the demonstration that tubulin sequestering is not a principal mechanism, the result in this study raises two major questions: (1) are the demonstrated region-specific in vitro activities of Op18 also manifested as distinct MT-directed activities in intact cells? (2) And if so, what is the specific role of modulation of tubulin GTP metabolism? By comparing the phenotypes mediated by Op18 derivatives in transfected K562 cells and analysis of MT morphology in microinjected newt cells , it is evident that NH 2 - and COOH-terminal–truncated Op18 do exert different MT-directed activities consistent with in vitro data. Moreover, given the demonstrated in vitro tubulin GTP modulatory activities of Op18 and the well established functional importance of GTP hydrolysis by tubulin , it seems reasonable to assume that Op18 has the potential to regulate MT assembly by its modulation of tubulin GTP status. Analysis of truncated Op18 derivatives showed distinct levels of modulation, namely inhibition of GTP exchange together with either inhibition or stimulation of GTP hydrolysis. If modulation of tubulin GTP status is central to Op18 function, it should be possible in the future to link these in vitro activities to specific MT-directed activities. However, given the observed complexity, evaluation of this possibility by segregation analysis requires design of additional mutants with a more defined spectrum of activities together with a more refined phenotypic analysis in intact cells. A central mechanistic question is whether Op18 regulates MT assembly by modulation of the GTP status of the free tubulin pool or possibly by local regulation at the tip of the MT. Op18-mediated accumulation of free GDP–tubulin would be a potential mechanism for catastrophe promotion by Op18-wt . However, the Op18-Δ100-147 derivative also promotes catastrophes , but has the opposite overall activity of Op18-wt, namely inhibition both of the basal- and nocodazole-stimulated GTP hydrolysis of free tubulin . This excludes accumulation of free GDP–tubulin as the mechanism behind catastrophe promotion. Analysis of Op18–tubulin association, in crude cell extracts from transfected cells, provides independent evidence against regulation of MT dynamics via modulation of the free tubulin pool. The data show that endogenous native Op18 successfully competes with truncated Op18 mutants for binding to cellular tubulin , which implies that these mutants exert their effect in intact cells by an alternative mechanism . Hence, some of the available data are clearly incompatible with a mechanism inferring Op18 modulation of the GTP status of the free tubulin pool. While this does not exclude a potential role of Op18 modulation of the GTP-status of free tubulin, it does suggest that Op18 exerts at least part of its regulatory activity by an alternative mechanism, such as interaction with the tips of MTs. A finding in favor to such a mechanism is that Op18 promotes catastrophes only from the plus end of MTs , the end where the E site GTP of β-tubulin is exposed , which hints to an MT end–specific activity that may involve GTP hydrolysis at the tip. As would be predicted from a requirement for GTP hydrolysis, it has been shown that Op18 does not depolymerize MTs capped with tubulin containing the nonhydrolyzable GTP analogue guanylyl (α, β)-methylene diphosphonate . Given the evidence of plus end specificity and involvement of GTP hydrolysis, it is clear that Op18 acts by a different mechanism from that demonstrated for another class of catastrophe promoters, exemplified by XKCM1 and XKIF2, which act at both ends of MTs by inducing a conformational change that does not involve GTP hydrolysis . Here, we show that Op18 retains significant tubulin binding after extensive deletions and the data demonstrate that Op18 binds tubulin heterodimers via multiple nonessential contacts that span residue 9 through the major part of the Op18 polypeptide ( Table and Table ). It is notable that all tubulin binding Op18 derivatives (i.e., all derivatives except Op18-Δ5-55) also manifest one or more levels of tubulin-directed modulation of GTP metabolism . Our data show that Op18 binds tubulin according to a two-site cooperative binding model, which implies that binding of the first tubulin creates a high affinity binding site for the second tubulin . Hence, tubulin–tubulin interactions may be important for generation of the second binding site. This raises the possibility that some of the demonstrated tubulin-directed activities of Op18 are generated by Op18-promoted tubulin dimerization. However, two lines of evidence indicate that this is not sufficient for stimulation of GTPase activity. First, such a mechanism predicts that increasing Op18 concentrations (i.e., well above K d 1; Table ), which favors Op18 binding to a single tubulin, would eventually lead to inhibition of tubulin GTPase activity. However, Op18 dose responses up to 60 μM, using 5 μM tubulin both in the presence or absence of glycerol, fail to reveal high dose inhibition of tubulin GTPase activity (data not shown). Second, we have recently reported that two classes of Op18 mutants dissociate tubulin binding from stimulation of GTPase activity . One class was mutated in heptad repeats of hydrophobic residues and the other involved substitution of phosphorylation sites to negatively charged glutamic acid residues. The resulting mutants showed a comparable decrease in their tubulin binding, but only the class of mutants with exchange of hydrophobic residues were defective in stimulation of tubulin GTPase activity. Hence, although Op18-promoted tubulin–tubulin interactions may be functionally important, it is evidently not sufficient for Op18-mediated stimulation of GTPase activity. In a previous in vitro study of MT assembly , the Op18-Δ5-25 and Op18-Δ100-147 derivatives were used to show that the catastrophe promoting activity of Op18 requires the NH 2 -terminal region of Op18, whereas a polymerization rate inhibiting activity requires the COOH-terminal region. We proposed that a tight tubulin complex formed by either Op18-wt or Op18-Δ5-25 inhibited the polymerization rate by tubulin sequestering. Under the conditions used for both derivatives, inhibition of the tubulin polymerization rate was only observed at pH 6.8 and not at 7.4. At the time, this was readily explained by the reported increase of Op18–tubulin binding at the lower pH. In this study, we determined binding affinities of Op18-wt and Op18-Δ5-25 at pH 6.8 and 7.4, using the same conditions as during MT assembly (i.e., PEM buffer in the absence of glycerol; Table ). It is evident that tubulin binding of Op18-Δ5-25 at pH 6.8 is essentially the same as Op18-wt binding at pH 7.4. Hence, assuming a simple sequestering mechanism, the present binding data predict that the polymerization inhibitory activity of Op18-wt at pH 7.4 should be similar to the activity of Op18-Δ5-25 at pH 6.8. This was clearly not the case since these two derivatives showed indistinguishable polymerization inhibitory activity at pH 6.8 and none showed inhibition at pH 7.4 . These results show that the pH sensitive Op18 inhibition of polymerization rates cannot be explained by a sequestering mechanism as proposed by us and others and that a more specific pH-regulated mechanism must be involved. The in vitro study discussed above shows that the Op18-Δ100-147 derivative promotes catastrophes at both pH 6.8 and 7.4 without a detectable inhibition of the polymerization rate. It is notable that this derivative was found here to be almost as efficient as Op18-wt in causing the observed lamellar clearing phenotype in newt cells . This phenotype is diagnostic for shortening of MTs and is the predicted phenotype of a specific catastrophe promoting factor. This supports the physiological significance of the catastrophe activity of specific Op18 derivatives observed in vitro. However, although Op18 is versatile with respect to modulation of GTP metabolism of free tubulin, more work is required to deduce the mechanism by which Op18 regulates MT dynamics. | Study | biomedical | en | 0.999995 |
10491393 | Extract preparation, immunoprecipitations (IPs), cell fractionation, and analysis of phosphorylation were as in Peifer 1993 ; anti-Arm IPs were at 1:40 and anti-dAPC2 IPs at 1:50. Samples were analyzed by 6% acrylamide SDS-PAGE, transferred to nitrocellulose, and immunoblotted with rat anti-dAPC2 (1:500), or mouse mAbs anti-Arm(7A1) at 1:500 , anti–Bicaudal D (BicD) at 1:50, and anti–β-tubulin (Amersham Pharmacia Biotech) at 1:100, followed by ECL (Amersham Pharmacia Biotech) detection. Two-hybrid experiments were as in Pai et al. 1996 . Yeast cells were transformed with plasmids encoding portions of the Arm repeat region fused to the LexA DNA binding domain and encoding fragments of dAPC or dAPC2 fused to the GAL4 activation domain; the control plasmid pCK4 expresses only the activation domain. Values are averages of duplicate β-galactosidase assays on more than six independent transformants. Anti-dAPC2 antisera were raised in rats by Pocono Rabbit Farms against a GST-dAPC2 fusion. Embryos were fixed in 37% formaldehyde/heptane (1:1) for 5 min (for anti–β-tubulin antibody, fix was 50 mM EGTA, pH 8, 33% formaldehyde). Larval tissues were fixed in 4% paraformaldehyde in PBS for 20 min. All were blocked and stained in PBS with 1% goat serum and 0.1% Triton X-100. Antibodies were used as follows: anti-dAPC2, 1:1,000; rabbit polyclonal antibody anti-Arm, 1:100; anti–β-tubulin (Amersham Pharmacia Biotech) 1:100; rhodamine phalloidin (Molecular Probes), 1:1,000; antiphosphohistone (Upstate Biotechnology), 1:200; anti-Prospero, 1:5 (kindly provided by C. Doe, University of Oregon, Eugene, OR). dAPC2 Δ S was induced by ethyl methanesulfonate (EMS) in a screen for suppressors of wg PE4 . dAPC2 Δ S also suppresses the null allele wg CX4 . Further analysis revealed that dAPC2 Δ S is a homozygous viable, temperature-sensitive maternal-effect lethal mutation mapping to 95E–F. Stocks for epistasis analysis were constructed at 18°C. Epistasis analysis was conducted at 25°C as in Table . Antibody staining and RNA in situ hybridization were as in Dierick and Bejsovec 1998 . Cuticle preparations were as in Wieschaus and Nüsslein-Volhard 1986 . Genomic DNA from dAPC2 Δ S homozygotes, from the background chromosome, and from two wild-type stocks was subjected to PCR with overlapping sets of primers. PCR products were analyzed both by direct sequencing and by cloning into TA-vectors (Promega) and sequencing at least two independent clones. 10 expressed sequence tags from the Berkeley Drosophila Genome Project correspond to dAPC2 ; we obtained sequence of several cDNAs and the corresponding genomic region . These predict a 1067 amino acid protein with striking similarity to other APC family members . All share an NH 2 -terminal conserved domain, 6 Arm repeats, and a series of βcat binding (15 and 20 amino acid repeats) and Axin binding motifs. dAPC2 is shorter at its NH 2 and COOH termini than other APCs. dAPC2 lacks the COOH-terminal basic region (the putative MT binding site) found in hAPC and dAPC , as well as the hAPC region containing binding sites for Discs-large (DLG) and EB1. Substantial alternative splicing is unlikely, as there are only two small introns in coding sequences (63 and 197 nucleotides). dAPC2 is most similar to other APC family members in the Arm repeats, where it most closely resembles dAPC; hAPC2 is more similar to hAPC (dAPC2 is 81% identical to dAPC and 57% identical to hAPC). Thus, there is no correspondence between individual human and fly proteins, even though both phyla show neural-enriched isoforms, dAPC and hAPC2, suggesting independent gene duplications. All APCs have six Arm repeats; a putative seventh Arm repeat is much more divergent and is not identifiable in dAPC2. The NH 2 -terminal conserved region (61% identity to dAPC vs. 44% identity to hAPC) distantly resembles the Arm repeat consensus and may form one or two degenerate Arm repeats. APC family members also share similarity COOH-terminal to the Arm repeats. hAPC has two sets of repeated βcat binding sites, the 15 and 20 amino acid repeats . dAPC2 shares two of the three 15 amino acid repeats of dAPC. dAPC and dAPC2 have five 20 amino acid repeats, among which are interspersed SAMP repeats . dAPC has four SAMP repeats, whereas dAPC2 has two. dAPC2 ends 40 amino acids after the last SAMP repeat. We generated antisera to a dAPC2 fusion protein ; antisera from two independent rats immunized with this antigen both recognize a single set of protein isoforms of ∼155–170 kD in embryonic extracts (they occasionally weakly cross-react with proteins of ∼120 and > 200 kD). In contrast, the preimmune sera do not recognize any proteins on immunoblots of embryo extract, supporting the specificity of the antisera. Further, as we show below, the migration on SDS-PAGE of the putative dAPC2 protein is altered in a dAPC2 mutant, consistent with these protein isoforms representing the genuine dAPC2 protein. The predicted molecular mass of dAPC2, 117 kD, is smaller than the observed molecular mass. However, an epitope-tagged version of the dAPC2 open reading frame expressed in human SW480 colon carcinoma cells also migrated at much higher apparent molecular mass than predicted from the sum of the predicted molecular mass of the dAPC2 coding sequence plus that of the epitope . This suggests that the large apparent molecular mass of dAPC2 is a property of its migration on SDS-PAGE. We examined the developmental profile of dAPC2 expression during embryogenesis . dAPC2 is present in the preblastoderm embryo (presumably maternally contributed), and levels remain relatively constant through the first half of embryogenesis, then drop sharply. As hAPC is phosphorylated , we suspected that the dAPC2 isoforms might be phosphorylation variants. To test this, we immunoprecipitated (IPed) dAPC2 from embryos and treated the IPs with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase. PP2A treatment reduced the apparent molecular mass of dAPC2; this effect was abolished if the PP2A inhibitor okadaic acid was included during incubation . Further, if embryonic cells were dissociated and incubated in tissue culture medium, the apparent molecular mass of dAPC2 decreased ; this effect was also abolished by okadaic acid, suggesting that it is mediated by endogenous phosphatases. Parallel alterations in Arm phosphorylation support this hypothesis . Taken together, these data suggest that the dAPC2 isoforms reflect, at least in part, differential phosphorylation. hAPC and dAPC bind to βcat and Arm, respectively. We tested whether dAPC2 also interacts with Arm in vivo. We immunoprecipitated Arm from embryonic extracts, and, in parallel, IPed proteins with a control mAb, anti-myc. dAPC2 specifically co-IPed with Arm from both early and older embryos , but did not co-IP with the control anti-myc antibody. We were unable to detect Arm in anti-dAPC2 IPs (data not shown); because the antigen for the dAPC2 antisera includes the Arm binding region, these sera might not recognize a dAPC2–Arm complex. We also found that a dAPC2 fragment containing the putative βcat binding sites co-IPed with βcat when expressed in the human colorectal cancer cell line SW480 (data not shown). The hAPC–βcat interaction is direct, and is mediated by the 15 and 20 amino acid repeats of hAPC and the Arm repeats of βcat ; the analogous region of dAPC binds Arm . To test whether dAPC2 directly interacts with Arm, we used the yeast two-hybrid system , examining whether dAPC2's 15 and 20 amino acid repeats interact with the full set of Arm repeats of Arm (R1–13), or with the centralmost Arm repeats (R3–8; the binding site for Drosophila E-cadherin and dTCF). For comparison, we tested the 15 and 20 amino acid repeats of dAPC . The full 15 and 20 amino acid repeat regions of both dAPC and dAPC2 strongly interact with the entire Arm repeat region and with R3–8. We also tested 31–34 amino acid fragments carrying individual 15 or 20 amino acid repeats of dAPC and dAPC2 (selected as good matches to the consensus). Individual 15 amino acid repeats of either dAPC or dAPC2 interacted with both the entire Arm repeat region of Arm and with R3–8. An individual 20 amino acid repeat of dAPC also interacted with both Arm fragments. A single 20 amino acid repeat of dAPC2 interacted strongly with Arm repeats 1–13; its interaction with R3–8 was much weaker. To demonstrate that our anti-dAPC2 antisera are specific in situ, we determined that preimmune sera do not specifically stain any structures in Drosophila embryos, even at concentrations 10-fold higher than those we used below (data not shown). Our anti-dAPC2 sera also specifically stain mammalian cells engineered to express dAPC2 but not nontransfected cells (data not shown). The specificity of staining in situ is further supported by the change in intracellular localization seen in a dAPC2 mutant (see below), and by the fact that antisera from a second rat immunized with this antigen recognize a similar set of cellular structures (at least during midembryogenesis, the stage we examined). Thus, we used our anti-dAPC2 antisera to characterize its expression and subcellular localization. During nuclear division cycles 10–13, which take place without cytokinesis in the peripheral cytoplasm of the embryo, dAPC2 shows dynamic changes in subcellular localization, coincident with those of actin . Sequential changes in MT organization as nuclei proceed through mitosis direct reorganization of the cortical actin cytoskeleton . Before nuclei migrate to the periphery, actin is found at the cortex in a random reticulum. When nuclei reach the periphery, actin condensations appear in interphase and prophase above each nucleus, forming an actin bud which overlays a cytoplasmic bud. This separates the mitotic apparatus of one nucleus from that of its neighbor. As division proceeds to metaphase, actin redistributes from the crown of the bud to its lateral cortex, forming an oblong ring around each spindle. During anaphase, actin redistributes into discs above each newly formed nucleus. Centrosomes and their associated MTs direct the changes in actin distribution, although the mechanism responsible for this interaction is not known. In cycle 10–13 embryos, dAPC2 colocalizes with actin at all stages of mitosis (we could not test for colocalization with Arm, as its levels at these stages are too low to detect its localization). The dAPC2/actin colocalization is most prominent in the microvillar projections at the surface of the bud in interphase and prophase . At metaphase and anaphase, dAPC2 and actin condensations are observed at the lateral cortex of the bud ; dAPC2 staining is somewhat less intense here relative to actin. Toward the base of the bud, condensations of actin and dAPC2 are also found in the region of the centrosome and asters . These dAPC2 condensations occur within 0.3–0.5 μm of the surface of the embryo (data not shown), and thus are most prominent above the spindle apparatus; kinetochore MTs are not in uniform focus until ∼1.25 μm from the surface of the embryo. The location of these dAPC2/actin condensations above the plane of the spindle places them in a position to interact with the astral MTs as they reach toward the cortex. During later nuclear cycles when pseudocleavage furrows are present, more defined dots of actin and dAPC2 staining are sometimes observed in the region of the centrosomes. In one of our wild-type stocks, which was infected with the bacterial endosymbiont Wolbachia (visible as small propidium iodide–positive bodies), we saw an additional APC2 localization. Wolbachia associate with astral MTs in Drosophila and thereby disperse into newly formed cells . In infected embryos, dAPC2 localizes with the actin cytoskeleton as in uninfected stocks, and also associates with bacteria at the asters . Another astral MT-associated protein, the kinesin-like protein KLP67A, is also reported to associate with bacteria . EM studies have shown that the bacteria are encapsulated within a cytoplasmic vacuole attached to astral MTs via an electron-dense bridge, possibly composed of cellular MT-associated proteins . dAPC2's localization to the aster region of noninfected embryos and its association with bacteria suggest that dAPC2 may contribute to the binding of the vacuole to the asters. After cellularization, dAPC2 is still enriched in the region of MTs. Increased levels of cytoplasmic dAPC2 are observed in mitotic domains (groups of cells undergoing synchronous mitosis) . Here, cytoplasmic condensations of dAPC2 are observed in the region of the spindle in metaphase and anaphase , but are absent in prophase or telophase ; serial sections revealed that these cytoplasmic condensations are most prominent within 2–4 μm of the cell apex. In mitotic domains of a Wolbachia -infected strain, we observed punctate condensations of dAPC2 near the spindle poles, presumably astrally associated bacteria , consistent with dAPC2 localization to bacteria associated with preblastoderm asters. dAPC2 is also expressed in dividing cells of the larval brain . The optic lobes contain two proliferative regions, the inner and outer proliferative zones. dAPC2 is highly expressed in dividing cells of the proliferative zones and in their immediate progeny, but not in differentiated neurons . In contrast, Arm is not enriched in the proliferative zones but is enriched in axons. In the ventral nerve cord, Arm is found in axons, whereas dAPC2 is found in midline glial cells . In contrast, dAPC localizes to axons, at least in embryos . However, in larval neuroblasts (neural stem cells) dAPC2 and Arm share a striking asymmetric distribution. Neuroblasts divide asymmetrically to produce a large neuroblast and a smaller ganglion mother cell, which will divide symmetrically to produce two neurons . The asymmetric division requires specific orientation of the mitotic spindle. Inscuteable (Insc), localized in a crescent opposite the future daughter cell during prophase and metaphase, is required for both spindle orientation and localization of the neural determinants Prospero and Numb . In larval neuroblasts, both dAPC2 and Arm colocalize to a cortical crescent next to the future daughter cell; this crescent also includes the neural determinant Prospero . In contrast to other asymmetric neuroblast components , the dAPC2 and Arm crescents are present even at interphase . In some neuroblasts, cortical actin also accumulates in a crescent with dAPC2 , whereas in others this association is less apparent . To examine the relationship between dAPC2 and the spindle, we triple-labeled neuroblasts with antibodies against phosphohistone, β-tubulin, and dAPC2 . One pole of the spindle apparatus colocalizes with the dAPC2 crescent; dAPC2 is enriched at this point relative to the rest of the crescent . We also observed low levels of dAPC2 at the opposite cortex at this stage of the cell cycle, the position of which often coincided with the other spindle pole . Whereas cortical dAPC2 associated with spindle poles, neuroblasts did not have cytoplasmic condensations of dAPC2 around the central spindle as were observed in epidermal cells. dAPC2 is also asymmetrically localized in embryonic neuroblasts . In nondividing cells, dAPC2 also associates with the cell cortex, and colocalizes with actin. In the embryo, dAPC2 is most strongly expressed in the epidermis and other epithelial cells. In the epidermis, dAPC2 is enriched at the cell cortex and is also found throughout the cytoplasm in a punctate distribution . At the cortex, dAPC2 appears as numerous punctate condensations of protein which are most prevalent at the apical end of the lateral cell surface but are also found more basally. The most intense staining of dAPC2 appears at points of contact between multiple epidermal cells . dAPC2 condensations often colocalize with condensations of actin and phosphotyrosine (data not shown), although actin and phosphotyrosine associate with the cortex more continuously. In fully polarized epithelial cells like the embryonic hindgut or the larval imaginal discs , dAPC2 is enriched in adherens junctions, where it colocalizes with Arm; dAPC2 also accumulates on the apical plasma membrane . The intracellular distribution of dAPC2 , in contrast to that of Arm , is not modulated in a segmental fashion. A strikingly different localization of dAPC2 occurs in the epidermis after stage 15. dAPC2 becomes organized into very large apical structures in segmentally repeated subsets of ventral epidermal cells , just before the stage at which these cells initiate denticle formation. The dAPC2 structures occur specifically in anterior epidermal cells of each segment and colocalize with similar actin structures , which likely represent larval denticle precursors. Although dAPC2 colocalizes with actin in many tissues, it does not colocalize with actin in all contexts. For example, during cellularization, actin is prominent at the cellularization front, whereas dAPC2 is enriched at the apical cortex (data not shown). In addition, as we noted previously, at the cortex of epidermal cells actin is present at the membrane in a continuous fashion, whereas dAPC2 is restricted to regions of most intense actin staining. Finally, dAPC2 is not found with actin in cytokinesis furrows . Thus, although dAPC2 associates with the actin cytoskeleton, the context-dependent nature of this association suggests that it is regulated. Biochemical analyses also suggest that dAPC2 associates with the cell cortex. When we fractionated 0–6-h-old embryos into soluble (S100) and membrane-associated (P100) fractions, dAPC2 partitioned almost equally into these two fractions . In contrast, Arm was almost exclusively in the membrane fraction at this stage. The isoforms of dAPC2 in the membrane fraction migrated more rapidly on SDS-PAGE than those in either the soluble fraction or the total cell lysate ; because these isoforms are not detectable in total lysate, we suspect that they may arise during fractionation by dephosphorylation. To examine whether dAPC2 might associate with the membrane via a glycoprotein, we used Con A–Sepharose, which can be used to isolate membrane glycoproteins as well as proteins associated with them (e.g., Arm) . A subset of dAPC2 specifically bound to Con A in extracts from 0–6-h embryos . Thus, dAPC2 may be anchored to the cortex via a transmembrane glycoprotein. We mapped dAPC2 to polytene region 95F1–2 on the third chromosome by in situ hybridization to wild-type and deficiency chromosomes. dAPC2 is removed by Df(3R)crb89-4 and Df(3R)crb87-4 but not by Df(3R) crb87-5 (data not shown). All three deficiencies remove crumbs and thus have a null crumbs phenotype ; thus, the severe epidermal fragmentation made examination of cuticular pattern impossible. During a genetic screen for suppressors of wg , we isolated a temperature-sensitive mutation which mapped to this genomic interval by complementation with the same deficiencies, and had a phenotype consistent with that of a negative regulator of Wg signaling (see below). Thus, we evaluated it as a candidate dAPC2 mutation, sequencing dAPC2 from the mutant and comparing its sequence to that of dAPC2 in the parental stock from which the mutant was derived, and in several other wild-type stocks. The mutant and parental chromosomes share 33 polymorphisms relative to the wild-type Canton S; only 8 altered the protein, and most changes are conservative . There is only a single difference between the parental chromosome and the mutant: deletion of three nucleotides, leading to deletion of serine 241. This serine residue falls within an alpha-helix in the third Arm repeat (by analogy to the Arm repeats of β-catenin) . The length of this alpha-helix is invariant among APC family members, and this residue is either serine or alanine (a conservative change) in all APCs. Thus, we refer to this allele as dAPC2 Δ S . Whereas homozygous mutant embryos accumulate normal levels of dAPC2, mutant dAPC2 migrates more rapidly on SDS-PAGE than wild-type protein . A portion of dAPC2 in heterozygous mutants, which are wild-type in phenotype, also migrates abnormally (data not shown), suggesting that this is an intrinsic property of mutant dAPC2 rather than a consequence of the mutant phenotype. The subcellular localization of dAPC2 in dAPC2 Δ S mutants was dramatically altered at both the permissive (18°C) and restrictive (25°C) temperatures. At the restrictive temperature, dAPC2 association with the cell cortex is essentially abolished, rendering the protein almost completely cytoplasmic . At the permissive temperature, some cortical dAPC2 remains . In heterozygotes, dAPC2 protein localization is intermediate between mutant and wild-type, as if mutant protein localizes incorrectly despite the presence of wild-type protein . The loss of phosphorylated dAPC2 isoforms observed above may be a consequence of the loss of cortical association. We also examined the localization of dAPC ΔS mutant protein at the restrictive temperature in other tissues. Although dAPC ΔS is found in apical buds in the preblastoderm embryo , it no longer associates with actin structures as does the wild-type protein . Furthermore, dAPC2 ΔS does not associate with the apical plasma membrane in the wing imaginal epithelia, marked by the presence of cortical actin . In the larval neuroblasts, dAPC2 ΔS is largely cytoplasmic , although an association with the cortex is sometimes observed . dAPC2 Δ S is viable and fertile at the permissive temperature (18°C). At the restrictive temperature (25°C), dAPC2 Δ S homozygous mutants derived from heterozygous mothers are viable, indicating that maternal contribution of dAPC2 is sufficient for embryonic development. Heterozygous embryos derived from homozygous mutant mothers are wild-type and survive to adulthood, suggesting that zygotic function is also sufficient. Mutant embryos derived from mutant mothers (referred to below as dAPC2 Δ S maternal/zygotic mutants) have severe abnormalities in their embryonic body plan. On the ventral surface, wild-type embryos show segmentally repeated denticle belts interspersed with naked cuticle . In dAPC2 Δ S maternal/zygotic mutants, denticle belts are replaced with an almost uniform expanse of naked cuticle , as is observed when wg is ubiquitously expressed . The dorsal surface also has an array of pattern elements marking specific cell fates ; cells receiving Wg signal secrete fine hairs. On the dorsal surface of dAPC2 Δ S maternal/zygotic mutants, many more cells secrete fine hairs , as they do when wg is ubiquitously expressed (data not shown). Thus, maternal/zygotic loss of dAPC2 function activates Wg signal transduction both dorsally and ventrally, suggesting that wild-type dAPC2 helps negatively regulate this pathway. Perturbing dAPC2 function at defined developmental time points supports this hypothesis. At the permissive temperature, dAPC2 mutant embryos develop normally into adults and a homozygous mutant stock can be maintained. When we shifted homozygous mutant embryos up to the restrictive temperature at 4 h after egg laying (AEL), they secreted uniform naked cuticle, like animals at the restrictive temperature throughout development. Progressively later upshifts result in intermediate cuticle defects, with increasing numbers of denticles secreted, until by 10 h AEL the pattern is essentially wild-type (data not shown). Conversely, shifts from the restrictive temperature down to the permissive temperature at 4 h AEL fully rescue the pattern, whereas progressively later downshifts result in more and more naked cuticle replacing the ventral denticle belts. Thus, dAPC2 activity is required between 4–10 h AEL, the same time window during which wg acts . Somewhat surprisingly, dAPC2 function may be dispensable for adult patterning; mutant embryos shifted up to the restrictive temperature after 10 h and cultured continuously at this temperature develop into apparently normal adults. This could be the result of partial activity of the dAPC2 Δ S allele. However, we suspect that dAPC2 Δ S is at least a strong hypomorph, as placing this allele over a deficiency for the region both in the mother and the zygote, does not increase the severity of the embryonic mutant phenotype at restrictive temperature . We carried out epistasis analysis to position dAPC2 with respect to other components of the signal transduction pathway. wg; dAPC2 Δ S double mutant embryos (with dAPC2 Δ S mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle , suggesting that dAPC2 is downstream of wg . There are two possible explanations for the fact that the double mutant does not show the same phenotype as the dAPC2 single mutant: either dAPC2 Δ S is not null, or the negative regulatory machinery remains partially active in the absence of dAPC2. If dAPC2 Δ S is not null, we reasoned that repeating the epistasis test with dAPC2 Δ S in trans to a deficiency removing dAPC2 (Df(3R)crb87-4) might further reduce dAPC2 function, producing a double mutant phenotype more similar to that of dAPC2 Δ S alone. However, when we did this, there was no change in the double mutant phenotype , suggesting that dAPC2 Δ S may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of dAPC2 . Embryos maternally and zygotically mutant for both dishevelled ( dsh ) and dAPC2 show a phenotype indistinguishable from the dsh single mutant , as do embryos maternally mutant for both dsh and dAPC2 that are zygotically dsh/Y; dAPC2 Δ S/Df(3R)crb87-4 . Likewise, arm; dAPC2 and dAPC2; dTCF double mutants (derived from dAPC2 homozygous mothers) are indistinguishable from arm or dTCF single mutants . Thus, dsh , arm , and dTCF all act genetically downstream of dAPC2 ; this was expected for arm and dTCF , but was surprising for dsh . Loss of dAPC2 also leads to ectopic activation of Wg-responsive genes. One target is wg itself. If the Wg pathway is constitutively activated by removing zw3 function or by expressing constitutively active Arm , an ectopic stripe of wg RNA is induced in each segment. A similar ectopic stripe of wg RNA is seen in dAPC2 Δ S maternal/zygotic mutants . Similarly, the domain of expression of a second Wg target gene, engrailed ( en ), is expanded relative to wild-type , as it is in zw3 mutants or in the presence of activated Arm. In addition, a novel phenotype was observed. In dAPC2 Δ S maternal/zygotic mutants , the levels of Wg protein are higher and Wg extends more cell diameters away from wg -expressing cells than in wild-type . These effects on Wg protein do not appear to be accounted for solely by ectopic activation of wg RNA, as they are detected beginning at stage 9 before induction of ectopic wg , and they are not observed in embryos expressing activated Arm . Thus, the efficiency of Wg protein transport appears to be enhanced in dAPC2 mutants. dAPC2 mutant embryos still respond to Wg signaling, as segmental stripes of stabilized Arm remain . In dAPC2 Δ S maternal/zygotic mutants, levels of cytoplasmic Arm in all cells are elevated, but cells receiving Wg signal continue to accumulate more Arm than their neighbors . In contrast, zw3 loss of function results in uniform accumulation of cytoplasmic Arm in all cells, eliminating the Arm stripes . Immunoblot analysis of Arm protein from dAPC2 Δ S maternal/zygotic mutants revealed an accumulation of hypophosphorylated Arm . This effect was not as dramatic as that seen in a zw3 mutant , but was similar to that seen upon ubiquitous expression of Wg using the e22c-GAL4 driver (data not shown). Thus, the effect of dAPC2 Δ S on Arm levels is intermediate between that of wild-type and that of zw3 loss of function, suggesting that negative regulation of Arm is reduced but not completely abolished in dAPC2 Δ S . As dAPC2 Δ S activates Wg signaling, we examined whether the change in its localization was simply a consequence of pathway activation. When we activated Wg signaling by ubiquitous Wg expression (via the e22c-GAL4 driver) or by removing zw3 function, the localization of dAPC2 was essentially unchanged, suggesting that pathway activation is not sufficient to eliminate cortical dAPC2 . There was also no apparent change in dAPC2 protein levels or isoforms in zw3 mutants relative to wild-type ; this was somewhat surprising as GSK phosphorylates hAPC , and suggests that dAPC2 can be phosphorylated by another kinase. The current model for hAPC function suggests that it is part of the destruction machinery for βcat and thus, negatively regulates Wnt signaling. dAPC negatively regulates the Wg pathway in the Drosophila eye , although surprisingly not in other tissues. We examined the function of dAPC2, which shows a broader pattern of expression. dAPC2 interacts directly with Arm and negatively regulates Wg signaling in the embryonic epidermis, helping trigger Arm destruction. dAPC2 mutant embryos resemble zw3 mutants in cuticle phenotype and in ectopic activation of Wg target genes. One novel phenotype of dAPC2 Δ S is a broadening of the stripes of Wg protein, suggesting an effect on Wg transport . This is not observed when Wg signaling was activated by other means, suggesting that dAPC2 may have novel roles in Wg signaling. In zw3 mutants, cytoplasmic Arm levels rise sharply . The effect of the dAPC2 Δ S mutation on Arm was similar but less severe. The current model for the destruction machinery is that Zw3, Axin, and APC function as a complex, facilitating Arm phosphorylation by Zw3 and thus targeting it for destruction . Axin can bind GSK/Zw3 and βcat/Arm independently of APC. Perhaps in the absence of dAPC2, Zw3 may still phosphorylate Arm, but not as effectively, explaining why loss of dAPC2 affects Arm stability less severely than does loss of Zw3. However, this conclusion is tempered by the fact that dAPC2 Δ S is not a protein-null, and in addition, other APC family members may play redundant roles. Our epistasis tests between dAPC2 and other components of the Wg pathway generally conform to earlier models of APC function, but also suggest further complexity. As expected, dAPC2 acts downstream of wg and upstream of arm and dTCF . However, the suppression of wg by dAPC2 Δ S is incomplete. As above, this may be because dAPC2 Δ S is not null, because dAPC2 is not completely essential for Arm downregulation, or because of redundancy. In contrast to zw3 , dAPC2 Δ S is genetically upstream of dsh . However, the relative positioning of dAPC2 and Dsh will not be definitive until a protein-null allele of dAPC2 is available. Most current models place Dsh upstream of the destruction machinery, but the recent discovery that Dsh, along with Axin, APC, and Zw3/GSK, is a component of the destruction complex reveals that these proteins may function as a network rather than as a linear series, making the results of epistasis tests more difficult to interpret. For example, the epistasis relationships might be explained if dAPC2 regulated assembly of Dsh into the destruction complex. In the absence of dAPC2, Dsh might constitutively turn off the destruction complex, activating signaling; thus, loss of dAPC2 would have no effect if Dsh is also absent. The localization of dAPC2 to large membrane-associated structures is intriguing. Axin and Dsh also accumulate in large punctate, often cortical structures when overexpressed in vertebrate cells, and like dAPC2, a fraction of Axin associates with a glycoprotein . Colocalization experiments will reveal whether cortical dAPC2 puncta contain other components of the destruction machinery. In light of these data, the inability of dAPC2 Δ S to associate with the plasma membrane may be informative. Loss of serine 241 likely affects the secondary structure of the Arm repeats, which may affect dAPC2 binding to a protein partner at the membrane. A membrane-bound localization of the destruction complex, perhaps via dAPC2, could be essential for optimal function of the Wg pathway. Both mislocalization of mutant dAPC2 ΔS protein and the slight residual activity of the destruction complex in these mutants could be explained if Arm destruction continues, albeit at greatly reduced levels, in the cytosol. These speculative ideas can be tested in the future by examining colocalization of dAPC2 and other components of the destruction complex in wild-type embryos and in the various mutant backgrounds. Although dAPC and dAPC2 clearly negatively regulate the Wg pathway, misexpression of APC in Xenopus suggested an apparent positive role in Wnt signaling . APR-1, the closest C. elegans APC relative, also appears to be a positive effector of Wnt signaling . However, APR-1 is very distantly related to the APC family. APR-1's Arm repeats are only slightly more similar to those of APC than to the Arm repeats of Arm . Whereas APR-1 has two highly divergent SAMP repeats , it does not contain the conserved NH 2 -terminal region or recognizable 15 or 20 amino acid repeats. Perhaps APR-1 is not an APC homologue, but instead plays a distinct role in the pathway. dAPC2 Δ S adults are viable and morphologically normal, suggesting that dAPC2 may not be required for critical functions such as patterning imaginal discs. Since the phenotypic severity of dAPC2 Δ S homozygotes is similar to that of dAPC2 Δ S/Deficiency , this allele is likely to be at least a strong hypomorph for Wg signaling in the embryonic epidermis. Although it is possible dAPC2 only functions there, its widespread expression at other stages suggests otherwise. Whether or not dAPC2 Δ S is a null, dAPC2 may still serve other functions. The specific effects of dAPC2 (these data) and dAPC mutations suggest that in some contexts they may be redundant. The possible other functions of dAPC2 remain to be tested by examining the effect of dAPC2 mutations on processes such as neuroblast divisions, and by characterizing dAPC dAPC2 double mutants. Previous studies of APC in vertebrate cultured cells revealed that APC localizes to the membrane and cytoplasm , where it can associate with MTs . Our biochemical and localization studies of dAPC2 reveal a complex relationship between dAPC2 and the actin and MT cytoskeletons, suggesting potential functions for dAPC2 in regulation of the cytoskeleton. dAPC2 colocalizes with actin in many but not all cell types, suggesting a regulated interaction. The association between the actin cytoskeleton and dAPC2 may occur via Arm and α-catenin, although in some places where dAPC2 and actin colocalize, there is little or no detectable Arm. The colocalization of dAPC2 and actin is intriguing given the effects of Wnt/Fz signaling on planar polarity in Drosophila . In the wing, the best studied example, Fz signaling triggers asymmetric polymerization of actin, leading to development of an actin-based wing hair in the distal vertex of each hexagonal wing cell . The colocalization of actin and dAPC2 during the onset of denticle formation was particularly striking in this context, because the process of denticle formation is very similar to that of wing hair formation in the nature of the structure, its strict orientation in the plane of the tissue, and in its cell biological and genetic bases. This raises the possibility that Wg/Wnt signaling directly affects the actin cytoskeleton and thus tissue polarity, using dAPC2 as an effector. Although dAPC2 does not contain the basic region thought to mediate MT association of hAPC, our data are consistent with the possibility that dAPC2, like hAPC , may associate with MTs under certain circumstances. The data for a microtubule association of dAPC2 are less robust than those suggesting association with actin. Whereas dAPC2 does not prominently localize to most microtubule-based structures (nor does hAPC, unless overexpressed), dAPC2 localized to several places consistent with a role in anchoring microtubules. In preblastoderm embryos, when actin is essential for tethering the spindle to the membrane , dAPC2 colocalizes with cortical actin and subcortical actin puncta. Subcortical dAPC2 is concentrated just above the spindle, placing it in a position to interact with astral MTs as they reach toward the cortex. Both dAPC2 and actin also localize to a dot-like structure which may be the centrosome. In postblastoderm embryos, dAPC2 is subtly enriched in the vicinity of the spindle. The asymmetric localization of dAPC2 in dividing neuroblasts is also consistent with a possible role for dAPC2 in linking the spindle to the cortex. During neuroblast mitosis, the spindle is specifically oriented . Insc, which localizes to a crescent opposite the future daughter cell from late interphase through metaphase, coordinates the neuroblast asymmetric cell division . Other proteins are likely to act in this process; e.g., Bazooka acts upstream of Insc . In C. elegans and yeast , actin or actin-associated proteins localize asymmetrically in cells in which spindle orientations are specified, suggesting a role for actin in this process. The position of actin in a crescent next to the future daughter cell in a subset of the neuroblasts suggests that actin may affect spindle orientation in Drosophila neuroblasts as well. dAPC2 may also play a role in this process. Within the crescent, dAPC2 localization was strongest in the region of the spindle pole. During later stages of mitosis, although dAPC2 remains enriched in a crescent next to the future daughter, dAPC2 also localizes to the cortex on the opposite side of the cell, often in the region of the other spindle pole. In contrast to other asymmetrically localized components of the neuroblast, dAPC2 localizes to a crescent during all stages of the cell cycle; in fact, the dAPC2 crescent was most apparent during interphase and prophase. dAPC2 and actin could also act as polarity markers for other proteins; actin is required for the asymmetric localization of Insc, Prospero, and Staufen . We emphasize that the dAPC2/MT connection remains speculative. In the future we must directly test whether dAPC2 associates with MTs, whether it can affect spindle orientation, and whether dAPC2 and Arm function in the neuroblast asymmetric cell division. We raise this issue in light of the influence of Wnt signaling on mitotic spindle orientation in both C. elegans embryos and in Drosophila sensory cells . In C. elegans , Wnt signaling controls spindle orientation independent of transcription , suggesting that the Wnt pathway directly targets the cytoskeleton. Since dAPC2 regulates Wg/Wnt signal transduction and appears to have connections to the cytoskeleton, it is a candidate for a direct effector of this process. RNA interference studies of C. elegans relatives of Arm (WRM-1) and APC (APR-1) did not reveal defects in spindle orientation , suggesting the existence of a branch to the cytoskeleton upstream of APC and Arm. However, because RNA interference may not completely remove gene function, and because of the divergence between APR-1 and the APC family noted above, the involvement of Arm and dAPC2 in the pathway remains plausible. These data are also intriguing in light of studies of the hAPC binding protein EB1 , which colocalizes with the spindle, centrosome, and asters . Budding and fission yeast EB1 homologues are required for spindle assembly and stability . However, it is worth noting that dAPC2 appears to lack the binding domain for EB1 identified in hAPC. In summary, our results define a role for Drosophila APC2 in the negative regulation of Wg signaling in the embryonic epidermis, and raise the possibility that it may act upstream of Dsh. This supports the idea that different APC family members operate in different tissues. The localization of dAPC2 in vivo, together with previous studies of hAPC in cultured cells, raises the possibility that dAPC2 acts as an effector molecule through which Wnt signaling influences the cytoskeleton. Finally, because dAPC2 associates with the actin cytoskeleton in contexts where no Wg signaling is thought to occur, such as in pre-blastoderm embryos, dAPC2 may play more fundamental roles in cytoskeletal regulation. Such functions may be revealed by further genetic analyses of dAPC and dAPC2 . Note Added in Proof: While this manuscript was in review, a related work was published by Yu, X., L. Waltzer, and M. Bienz. 1999. Nature Cell Biol. 1:144–151. | Study | biomedical | en | 0.999998 |
10491394 | E. coli BUG 968 strain and BUG 896 strain expressing the membrane form of IcsA were grown at 37°C in 2YT in the presence of 100 μg/ml ampicillin until midexponential phase was reached. Listeria strain Lut12 (pActA3) overexpressing ActA was grown at 37°C in brain/heart infusion medium in the presence of 7 μg/ml chloramphenicol and 5 μg/ml erythromycin until midexponential phase was reached. Bacteria were kept frozen in 30% glycerol at −80°C. Polyclonal rabbit antibodies were raised against human N-WASP protein residues 473 -EVMQKRSKAIHSSDED- 488 (C412 antibodies) and against recombinant human N-WASP protein obtained from baculovirus (NW011 antibodies). Anti-Arp3 polyclonal antibodies (AR3) were raised in rabbits against Arp3 residues YEEIGPSIVRHNPVFGVMS. Affinity-purified C421, NW011, and AR3 antibodies were used for Western blot and immunofluorescence, respectively, at 10 μg/ml concentration. Antibovine profilin polyclonal antibodies were raised in rabbits and used in Western blots at a 1:5,000 dilution. Human platelet extracts were prepared from outdated unstimulated preparations as previously described . Bovine brain extracts were prepared as follows. Brain was homogenized in 1 vol of 0.1 M MES, pH 6.8, 1 mM EGTA, 0.5 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, and clarified by centrifugation at 10,000 g for 30 min at 4°C. Tubulin and neurofilaments were removed by centrifugation for 1 h at 150,000 g after one cycle of microtubule assembly at 37°C for 30 min in the presence 0.5 mM GTP, 4 M glycerol, and 0.25 mM MgCl 2 . The supernatant (S 2 ) was stored at −80°C. Histidine-tagged human N-WASP was expressed in High Five insect cells. Human cDNA was cloned in pFast Bac Htb (Life Technologies, Inc.) and recombinant baculovirus constructed using standard protocol of transfection–recombination in insect cell lines. High Five insect cells were infected at a multiplicity of infection of 2. Cell culture was performed in suspension, in High Five serum-free medium, under vigorous shaking at 27°C for 40 h. Protein purification was performed by DEAE cellulose at pH 7.8 and cobalt affinity chromatography, as described by Laurent et al. 1999 . Recombinant histidine-tagged human VASP was expressed and purified as described previously . The NH 2 -terminal (Nt) and COOH-terminal domains (VCA) of human N-WASP were expressed and purified as GST fusion proteins in E . coli . Human cDNA regions encoding N-WASP Nt (residues 1-276) and VCA (residues 392-505) were amplified by PCR from human brain cDNA using oligonucleotides phNW1 (5′-ccggaattcATGAGCTCCGTCCAGCAGCAG-3′), phNW276 (5′-ccgctcgagTCACTTGCCTCCGCAGTTCATTTTTAAC-3′), and oligonucleotides phNW392 (5′-ccggaattcCCTTCTGATGGGGACCATCAG-3′), phNW505 (5′-ccgctcgagTCAGTC-TTCCCACTCATCATC-3′), respectively. N-WASP Nt and VCA PCR fragments were cloned in EcoRI and XhoI sites in pGEX4T-1 plasmid, respectively, to generate pCENHN and pCECHN plasmids. GST-Nt and GST-VCA protein expressions were obtained using BL21 E . coli strain according to standard induction and purification procedures. GST-Nt and GST-VCA glutathione Sepharose beads were kept at 4°C until use. GST-Nt protein was eluted from beads and VCA protein was obtained by thrombin cleavage. Both proteins were dialyzed against 10 mM Tris-Cl − , pH 7.5, 1 mM DTT, 50 mM KCl, and 0.01% NaN 3 , frozen on liquid nitrogen and stored at −80°C. The concentration of VCA was determined spectrophotometrically using an extinction coefficient ∈ 0.1% of 0.467 cm −1 at 278 nm calculated from the amino acid sequence, and a mol wt of 12,900 D. Due to the acidic character of VCA, the Bradford protein assay gave underestimated values of the concentration. Human Cdc42 was also expressed as a GST fusion protein in E . coli . Human cDNA was cloned in pGEX2T expression plasmid and was a kind gift from Dr. Alan Hall (Medical Research Council, University College, London, UK). Protein was expressed in E . coli JM109, purified on glutathione Sepharose as described, and kept on beads at 4°C in 10 mM Tris-Cl − , pH 7.6, 150 mM NaCl, 2 mM MgCl 2 , 0.1 mM DTT, and 0.1 mM GTP. When required, GST-Cdc42 was loaded with GDP or GTPγS on beads by a 1-h incubation at room temperature on a wheel in 50 mM Tris-Cl − , pH 7.6, 150 mM NaCl, 2 mM MgCl 2 , 1 mM DTT, 10 mM EDTA, and 0.1 mM GDP or GTPγS. The exchange reaction was stopped by addition of 10 mM MgCl 2 . IcsA 53-508 protein was purified as a GST fusion protein as described in Suzuki et al. 1996 . Bovine Arp2/3 complex was purified from bovine brain extracts by a novel two step method as follows. 100 ml of S 2 was dialyzed against 50 mM MES, pH 6.8, containing 1 mM DTT, 1 mM EDTA, and 1 mM MgCl 2 (buffer A), and loaded on an SP-Trisacryl column (2.5 × 12 cm) equilibrated in buffer A. After a wash step with 30 mM KCl, the Arp2/3 complex was eluted with 80 mM KCl in buffer A. This fraction was equilibrated in 20 mM Tris-Cl − , pH 7.5, containing 25 mM KCl, 1 mM DTT, 1 mM MgCl 2 , 0.5 mM EDTA, and 0.1 mM ATP-Tris, pH 7.5 (buffer B), and loaded on a GST-VCA glutathione Sepharose column (0.5 × 1 cm) in buffer B. Most of the proteins were washed off the column by buffer B, the remaining non-Arp2/3 proteins were eluted by 0.2 M KCl in buffer B, pending a small loss of Arp2/3 activity, and the pure Arp2/3 complex was eluted with 0.2 M MgCl 2 in buffer B . After dialysis against buffer B, the protein was concentrated using a Centriprep 30 cartridge (Amicon). The Arp2/3 complex was supplemented with 0.2 M sucrose and stored at −80°C. The activity of Arp2/3 along the purification steps was monitored spectrofluorimetrically, using the ability of Arp2/3 to activate the branched polymerization of actin filaments in the presence of VCA. The assay (80 μl) contained 2.5 μM Mg-G-actin (10% pyrenyl-labeled), 0.5 μM VCA, and 10 μl of the tested fraction in 5 mM Tris-Cl − , pH 7.8 buffer, 1 mM DTT, 0.2 mM ATP, 0.1 M KCl, and 1 mM MgCl 2 . The time course of increase in pyrenyl-actin fluorescence was followed. The activity of Arp2/3 was detected by the steep sigmoidal increase in fluorescence, indicative of branched polymerization. The activity of Arp2/3 was easily observed, even in the S 2 fraction. Alternatively, the activity of Arp2/3 was detected by the video-microscopy bacterial motility assay, using the property of Arp2/3 to initiate the formation of actin clouds at the surface of Listeria incubated in 4 μM rhodamine-labeled actin. The results of the two tests were in good agreement with each other, but the spectrofluorometric assay was more convenient and easily amenable to a quantitative estimate of the amount of Arp2/3 in the tested fractions by comparison with a calibration series of time courses performed with known concentrations of pure Arp2/3. The concentration of Arp2/3 complex was determined spectrophotometrically using an extinction coefficient of 224,480 M −1 · cm −1 at 278 nm, derived from the amino acid sequence of the seven polypeptides composing the 223,949-D complex. A good agreement was obtained between the spectrophotometric measurement and the bicinchoninic acid assay, with BSA as a standard. Typically, 1.2 nmol (0.26 mg) of pure Arp2/3 complex was obtained from 100 ml brain S 2 supernatant. Arp2/3 complex was depleted from platelet extracts using a variation of the coprecipitation method described by Machesky and Insall 1998 . Various amounts of glutathione Sepharose-coupled GST-VCA beads (20, 40, and 80 μg) were incubated with 25 μl of platelet extracts for 30 min at 4°C on a rotating wheel. Beads were pelleted at 5,000 g for 15 s. Depletion of the Arp2/3 complex was monitored by Western blotting of aliquots of protein extracts and beads. Add-back was done using the Arp2/3 complex purified from bovine brain. VASP immunodepletion was carried out as described previously . Add-back was done using the recombinant VASP purified from insect cells. Profilin depletion was carried out by two consecutive poly- l -proline chromatography steps, as described by Laurent et al. 1999 . Profilin depletion was monitored by Western blotting. Add-back was done using the purified wild-type profilin or the H133S mutant profilin, described as unable to bind poly- l -proline . Actin was purified from rabbit muscle and isolated as Ca-ATP-G-actin by gel filtration on Sephadex G-200 in G buffer (5 mM Tris-Cl − , pH 7.8, containing 0.1 mM CaCl 2 , 0.2 mM ATP, and 1 mM DTT). Actin was pyrenyl- or rhodamine-labeled, and converted into Mg-G-actin before polymerization, as described by Carlier et al. 1997 . Mg-actin was polymerized by addition of 1 mM MgCl 2 and 0.1 M KCl (physiological ionic conditions). When actin was polymerized in the presence of gelsolin, no EGTA was added to the solution, and Ca-ATP-G-actin supplemented with the desired amount of gelsolin was polymerized by addition of 2 mM MgCl 2 and 0.1 M KCl. Thymosin β4 was purified from bovine spleen as described by Pantaloni and Carlier 1993 . Gelsolin purified from human plasma was a kind gift from Dr. Yukio Doi (University of Kyoto, Kyoto, Japan). Spectrin-actin seeds were isolated from human erythrocytes as described by Casella et al. 1986 . Critical concentration plots were performed by serial dilution of pyrenyl-labeled F-actin solutions in F buffer (5 mM Tris-Cl − , pH 7.8, containing 1 mM DTT, 0.2 mM ATP, 0.1 mM CaCl 2 , 1 mM MgCl 2 , and 0.1 M KCl) and incubated overnight at room temperature. Polymerization assays were performed using the change in pyrenyl-actin fluorescence. Measurements were carried out in a Spex Fluorolog 2 instrument thermostated at 20°C, with excitation and emission wavelengths of 366 and 407 nm, respectively, and appropriate filters placed on the excitation and emisssion beams to eliminate stray light and bleaching, which interfere in polymerization in the presence of Arp2/3 (Pantaloni, D., manuscript in preparation). The G-actin sequestering activity was measured by the shift in critical concentration plots with gelsolin-capped filament barbed ends. The equilibrium dissociation constant, K d , for the actin-sequestering protein complex was derived from the measurements of the concentration, [A 0 ], of unassembled actin at steady state (abscissa intercept of the critical concentration plots) and of the critical concentration, [A C ], determined in the absence of sequestering agent. The following equation was used: K d = [A C ] · ([S 0 ] − [A 0 ] + [A C ]) / ([A 0 ] − [A C ]), in which [S 0 ] represents the total concentration of sequestering protein. Rates of filament growth from barbed and pointed ends were measured using spectrin–actin seeds or gelsolin-capped filaments, added at time zero to a solution of 1 μM MgATP–G-actin (10% pyrenyl-labeled) in polymerization buffer. The gelsolin-capped filament solution contained 5 μM F-actin polymerized in the presence of 16.7 nM gelsolin for 2 h, and diluted 20-fold into the G-actin solution. Frozen bacteria were thawed and spun down at 4,000 g for 5 min at 22°C, resuspended at 6 × 10 9 bacteria/ml in XB buffer (10 mM Hepes, pH 7.7, 0.1 M KCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , and 50 mM sucrose). Motility assays were performed by mixing 4 μl of thawed extracts supplemented with 12 μl of a mixture containing 5 μM F-actin, 0.38% methyl cellulose, 6 mM DTT, 2 mM ATP/MgCl 2 , 1.5 × 10 −3 % DABCO, 1 μM rhodamine-actin, and 2 × 10 8 bacteria/ml. An aliquot of 2.5 μl was squashed between a 22 × 22 mm coverslip and a slide, sealed with VALAP, and incubated for 5 min at room temperature before motility measurements. Fluorescence microscopy observations and data acquisition were carried out as previously described . N-WASP-coated E . coli (IcsA) were prepared as follows: 20 μl of a suspension of 6 × 10 9 bacteria/ml were incubated with 5 μl of 1 μM N-WASP for 5 min at room temperature. Bacteria were pelleted, washed, and resuspended in 20 μl of XB buffer and kept on ice. The N-WASP–IcsA complex remains stable for several hours on ice. HeLa cells were infected as follows: overnight culture of Shigella M90T strain was diluted in tryptic soy broth, grown to midexponential phase, centrifuged at 5,000 g for 5 min, washed, and resuspended at 10 8 bacteria/ml (M90T) in MEM, 50 mM Hepes, pH 7.5. HeLa cells, seeded on glass coverslips, were washed three times with MEM, overlaid with the bacterial suspension, and centrifuged for 10 min at 800 g . Bacterial entry was allowed by 30 min incubation at 37°C. Infected cells were washed with MEM five times and incubated with MEM, 50 mM Hepes, pH 7.5, with 50 μg/ml of gentamicin for a further 1 h at 37°C, to allow intracellular spread. For immunofluorescence staining, infected cells were permeabilized with 0.2% Triton X-100 in 0.1 M MES, pH 7.4, 1 mM MgCl 2 , 1 mM EGTA, 4% PEG 6000, and then fixed for 30 min in 3.7% paraformaldehyde in PBS. After quenching with NH 4 Cl 50 mM in PBS for 10 min and saturation for 30 min in PBS-BSA 2%, infected cells were incubated with specific mono- or polyclonal antibodies. Affinity-purified antihuman N-WASP polyclonal NW011 antibodies and polyclonal Arp3 antibodies were used at 10 μg/ml concentration. Texas red-conjugated secondary antibodies (Nycomed Amersham Inc.) were used. F-actin was visualized by FITC-phalloidin (0.1 mg/ml; Sigma Chemical Co.). Preparations were examined with a confocal laser scanning microscope (Zeiss Axiophot). Glutathione Sepharose coupled to GST–IcsA or to GST was equilibrated in buffer P (20 mM Tris-Cl − , pH 7.5, 100 mM KCl, 1 mM MgCl 2 , and 0.1% BSA). 100 pmol of protein-bound beads and 80 pmol of purified N-WASP or VCA were mixed in 100 μl (total vol) and incubated for 1 h at 4°C on a rotating wheel. Supernatants were collected. Beads were rinsed twice with buffer P. Free and bound N-WASP and VCA were detected by Western blotting after resolving the proteins by SDS-PAGE. Actin-based motility of E . coli (IcsA) has been observed in Xenopus egg extracts . Motility was observed as well in HeLa cell extracts and murine or bovine brain extracts, but not at all in human platelet extracts, which, in contrast, supports actin-based motility of Listeria . This puzzling observation suggested that an essential factor for Shigella motility is missing in platelets. The requirement of N-WASP for Shigella motility in Xenopus egg extracts has recently been demonstrated . Immunoblotting using polyclonal NW 011 antibodies that recognize both WASP and N-WASP , showed that N-WASP was present in brain extracts, but absent in platelets, which, in contrast, contain the lower molecular weight WASP homologous protein mainly expressed in hematopoietic cells , whose mutations result in the Wiskott-Aldrich syndrome . The endogenous WASP protein, homologue of N-WASP in platelets, apparently cannot bind IcsA. To check whether the absence of N-WASP was responsible for the lack of E . coli (IcsA) motility in platelet extracts, recombinant human N-WASP was expressed using the baculovirus system and purified . The interaction of recombinant N-WASP with IcsA was verified by successful binding of N-WASP on glutathione Sepharose beads to which the GST–IcsA fusion protein was attached . The isolated VCA of N-WASP, in contrast, did not bind to IcsA . Actin-based motility of E . coli (IcsA) was restored by addition of N-WASP to platelet extracts. Specifically, upon addition of 10 nM N-WASP, actin clouds were observed. Comets were observed upon addition of 50 nM N-WASP, optimum movement was seen at 100 nM N-WASP, and inhibition occurred at 1 μM N-WASP. Neither bacterial motility nor actin polymerization were observed under the same conditions with control BUG 968 E . coli bacteria that did not express IcsA (see Materials and Methods). Consistent with the apparent high affinity of N-WASP for IcsA, when E . coli (IcsA) bacteria were incubated with N-WASP at concentrations as low as 0.1 μM, washed, and added to platelet extracts, bacterial actin-based motility was readily observed . Bacteria induced actin polymerization and moved at rates similar to Listeria (4 μm/min, SD = 1, n = 20). In contrast, when control BUG 968 E . coli bacteria that did not express IcsA were preincubated with N-WASP, washed, and added to platelet extracts, they failed to induce actin assembly and to move. Consistent with these results, beads coated with GST–IcsA 53-508 induced actin assembly at their surface in platelet extracts only when they were first preincubated with N-WASP . Control GST beads, preincubated in the same N-WASP solution, and washed, failed to induce actin assembly in platelet extracts. Neither actin assembly nor movement was observed when bacteria or GST–IcsA beads were preincubated with VCA instead of N-WASP. Accordingly, bacterial movement and actin assembly were not inhibited by the presence of a 20-fold molar excess of VCA over N-WASP in the preincubation step. Hence, VCA does not displace IcsA-bound N-WASP. These results demonstrate that interaction of IcsA with N-WASP is crucial for IcsA-mediated actin polymerization and bacterial motility, and that VCA does not interact with IcsA. The N-WASP protein was proposed to initiate actin assembly at the surface of bacteria by interacting with actin via its two verprolin homology regions and/or its cofilin homology sequence . However, when either the E . coli (IcsA) bacteria or the GST–IcsA beads precoated with N-WASP were incubated in a solution of rhodamine-labeled F-actin, no initiation of actin assembly was observed at their surface (data not shown), indicating that, in itself, the actin-binding property of N-WASP is not sufficient for inducing the actin assembly seen in platelet extracts. Recently, the association of the Arp2/3 complex with Scar1, a member of the WASP family, was demonstrated to enhance the actin nucleation activity of Arp2/3 complex in vitro . To investigate the role of Arp2/3 complex in Shigella motility, platelet extracts were at least 80% depleted in Arp2/3 complex by GST-VCA beads . Actin polymerization and motility of E . coli (IcsA) coated with N-WASP and of Listeria were completely abolished in those Arp2/3-depleted extracts. Add-back of 0.15 μM pure Arp2/3 from bovine brain to the depleted platelet extracts restored bacterial movement at a rate of 1 ± 0.2 μm/min, i.e., 30% of the speed observed in the mock-depleted extracts. The N-WASP–coated E . coli (IcsA) bacteria, N-WASP–bound GST–IcsA beads, or Listeria bacteria were then incubated in a solution of rhodamine-labeled F-actin containing pure Arp2/3 from bovine brain . Formation of actin clouds around the bacteria and the beads was immediately observed . These results demonstrate that IcsA-bound N-WASP interacts with Arp2/3 complex, and that the IcsA–N-WASP–Arp2/3 complex is able to initiate local actin assembly in a solution of pure actin. Previously, we have shown, using phalloidin, that G-actin, not F-actin, was recruited by Arp2/3 at the Listeria surface . Even though actin was polymerized in the assay, it is the G-actin that coexists at steady state with the filaments that are shuttled onto barbed ends created by Arp2/3. To get more insight into the respective roles of N-WASP and Arp2/3 in Shigella movement, immunolocalization of the two proteins in infected cells was carried out. N-WASP colocalized with IcsA at the surface of Shigella , whereas the Arp2/3 complex was found in the entire actin tail . In conclusion, Arp2/3 interacts with N-WASP to initiate actin assembly at the surface of Shig ella , but the interaction of Arp2/3 with N-WASP is not permanent, and the Arp2/3 complex remains incorporated into the assembled filaments. In contrast, the interaction of N-WASP with IcsA is probably very strong and in slow equilibrium. To understand how the interaction between IcsA-bound N-WASP with Arp2/3 complex leads to the initiation of actin filament assembly at the surface of Shigella , the biochemical properties of the individual proteins of this complex machinery were examined in in vitro experiments. The Arp2/3 complex purified from bovine brain was able to induce the branched polymerization of actin filaments , like the Acanthamoeba complex . The activity of the Arp2/3 complex appeared enhanced by either the VCA domain of N-WASP or by the entire N-WASP protein in the same concentration-dependent manner. At 2.5 μM actin, the optimum activation was observed at 0.5 μM VCA or N-WASP. The activation decreased at higher concentrations of VCA or N-WASP . The maximum extent of activation was fourfold higher with VCA than with N-WASP. The effect of N-WASP, but not of VCA, was further stimulated (about threefold) by Cdc42-GTPγS, not by Cdc42-GDP , in agreement with Rohatgi et al. 1999 . In conclusion, in the whole N-WASP protein, the VCA domain is accessible to Arp2/3 complex and capable of activating Arp2/3 function in actin assembly. The recombinant IcsA protein greatly stimulated the N-WASP–Arp2/3-induced polymerization of actin in a concentration-dependent, saturable fashion . In the presence of 20 nM Arp2/3 complex and 130 nM N-WASP, 80% of the maximal polymerization rate was reached upon addition of 30 nM IcsA, indicating that maximal activity is associated with the formation of a ternary Arp2/3–N-WASP–IcsA complex in which the equilibrium K d of IcsA is in the nanomolar range. The activation reached with IcsA was greater than with Cdc42. Interestingly, IcsA by itself did not affect the polymerization of actin alone, but at concentrations as low as 0.1 μM, it activated the Arp2/3 complex to some extent in the absence of N-WASP , which indicates that IcsA interacts with Arp2/3 complex. In the presence of N-WASP, IcsA, and Arp2/3, a true triangular complex is formed between the three proteins at the surface of Shigella , enhancing the stability of the edifice. The VCA of N-wasp was described as a filament depolymerizing filament severing protein . These conclusions do not appear confirmed in the present work. The interaction of isolated VCA with actin was examined in steady state and kinetic experiments, on the dynamics of pointed and barbed ends separately. When filament barbed ends are capped by gelsolin, VCA induced a shift in the critical concentration plots . Addition of increasing amounts of VCA to F-actin at a given concentration caused a linear decrease in F-actin at steady state, leading to eventual complete depolymerization at high concentration of VCA. The slope of the plot was independent of the actin concentration. Hence, VCA behaves like a G-actin sequestering protein when barbed ends are capped. A value of 0.8 ± 0.1 μM was derived for the equilibrium K d of the VCA–G-actin complex, from analysis of the data in Fig. 5 , a and b. When barbed ends were free, the behavior of VCA was different. No efficient sequestration of actin was observed. In the presence of 5 μM VCA, a modest shift of the critical concentration plots from 0.12 to 0.21 μM unassembled actin was observed, while a 3.5-fold larger shift (up to 0.7 μM unassembled actin) was expected, using the K d value of 0.8 μM. These results are reminiscent of the effect of profilin in actin assembly and they demonstrate that when barbed ends are free, the concentration of ATP–G-actin at steady state is lowered by VCA, suggesting that VCA–actin complex, like profilin–actin, participates in barbed end assembly. Specifically, in the experiment shown in Fig. 5 c, the amount of unassembled actin, [A] 0 , at steady state is 0.21 μM, which distributes into free G-actin, [A], and VCA–actin complex, [VA]. The concentrations of free G-actin and VCA-actin are linked by the law of mass action: ([A] · ([V] total − [VA]) / [VA] = K d ); and by the sum: [A] 0 = [A] + [VA]; resulting in a quadratic equation that has a single solution: [A] = 0.03 μM, [VA] = 0.18 μM. The concentration of free actin, thus, appears fourfold lower than 0.12 μM, the value that is measured in the absence of VCA. Evidence for the participation of VCA–actin complex in barbed end assembly was directly provided by measurements of the rate of filament growth. The rate of elongation from G-actin at pointed ends, initiated by gelsolin–actin complex, was totally inhibited by VCA , confirming that the VCA–actin complex does not polymerize onto pointed ends. In contrast, growth of barbed ends initiated by spectrin–actin seeds, was inhibited only by 30% at saturation by VCA, indicating that the VCA–actin complex associates to barbed ends with a rate constant 30% lower than G-actin . Again, this result is strikingly similar to the one obtained with profilin . Like profilin, VCA delivers G-actin subunits to growing barbed ends and dissociates from the barbed end after incorporation of actin, in a manner coupled to ATP hydrolysis. The coupling to ATP hydrolysis was demonstrated to be thermodynamically required to account for the decrease in partial critical concentration of actin . Spontaneous polymerization of actin was inhibited by VCA, exactly as observed with profilin (data not shown). Hence, VCA–actin complex, like profilin–actin complex, is able to productively associate to barbed ends, but not to nucleate F-actin. This result accounts for the decrease observed in the activation of Arp2/3 complex at high VCA or N-WASP concentration , which correlates with the saturation of G-actin by VCA. At low concentrations, VCA-actin interacts with Arp2/3 complex to enhance nucleation, but when all G-actin is bound to VCA, actual inhibition of nucleation occurs. Full-length N-WASP did not display the G-actin sequestering activity of VCA, when barbed ends were capped by gelsolin. No depolymerization of gelsolin-capped F-actin was measured at steady state, and only weak inhibition in the rate of growth at the pointed ends of gelsolin-capped filaments was detected by addition of N-WASP. Instead, N-WASP bound to F-actin and stabilized the filaments, as shown by the observed decrease in critical concentration . A slight decrease in the slope of the pyrenyl fluorescence critical concentration plots indicated that the fluorescence of F-actin was partially quenched upon binding N-WASP. The binding to F-actin and the decrease in critical concentration were also observed in a sedimentation assay . The NH 2 -terminal fragment of N-WASP protein (Nt, residues 1–276) stabilized the filaments and affected the slope of the critical concentration plots of gelsolin-capped filaments exactly like the full-length protein , and bound to F-actin in sedimentation assays, which indicates that the Nt contains the F-actin binding site. The affinity of N-WASP for F-actin was roughly estimated to 2 to 5 10 5 M −1 from these data. The binding to F-actin presumably counteracted and prevented observation of the sequestering activity of the COOH-terminal part of N-WASP in solutions of F-actin. When barbed ends are free, the critical concentration is low (0.1 μM), hence, the stabilizing effect of N-WASP (decrease in critical concentration) was best observed in the presence of a sequestering protein like thymosin β4. The amount of unassembled actin at steady state then is amplified by the sequestering effect of thymosin β4, making any change in critical concentration easily measurable. The sequestering effect of thymosin β4 was drastically reduced by N-WASP, as the result of the decrease in critical concentration . Addition of 30 μM thymosin β4 to 2 μM F-actin caused depolymerization of 1.28 μM actin into thymosin β4–actin complex, consistent with a value of 0.1 · (30 − 1.28) / 1.28 = 2.2 μM for the K d of the thymosin β4–actin complex. Upon addition of N-WASP to this solution, the concentration of F-actin at steady state increased. At saturation by N-WASP, only 0.5 μM actin was unassembled, which, by solving the quadratic equation expressing the combination of the laws of mass conservation and mass action, corresponded to 0.47 μM thymosin β4–actin complex and 0.03 μM G-actin. Hence, the barbed end critical concentration was decreased three- to fourfold by N-WASP. Sedimentation assays confirmed the conclusions derived from fluorescence measurements . In the presence of IcsA, the binding of N-WASP to F-actin was strengthened , and N-WASP–bound IcsA then cosedimented with F-actin. In conclusion, binding of IcsA to N-WASP enhances the affinity of both its COOH-terminal domain for Arp2/3 complex and its NH 2 -terminal domain for F-actin. To test the postulate that the binding of vinculin to IcsA would serve to recruit VASP , which would have the same function in Shigella and Listeria movement, immunodepletion of VASP in platelet extracts was performed as described . Total depletion of VASP was checked by immunoblotting. The movement of E . coli (IcsA) precoated with N-WASP was not affected by removal of VASP ( Table ). Control samples were prepared in parallel to verify that Listeria , in contrast, did not move at all in VASP-depleted extracts, in agreement with previous observations . In conclusion, the putative vinculin–VASP interaction is unlikely to play a role in Shigella movement. Depletion of profilin from platelet extracts by extensive poly- l -proline chromatography led to a 40% decrease in the rate of propulsion of both Listeria and E . coli (IcsA). Add-back of pure bovine spleen profilin (1 μM) restored 80% of the rate measured in mock-depleted extracts. It has been postulated that binding of profilin to the proline-rich regions of N-WASP could play a functional role in filopodium extension, which is, like Shigella movement, mediated by N-WASP-induced actin polymerization. To test this possibility, the add-back of profilin was performed using H133S-mutated profilin that fails to bind to poly- l -proline . We first checked that the mutated profilin bound actin in a functional fashion strictly identical to either the bovine profilin or the recombinant human wild-type profilin. In brief, H133S profilin sequestered actin when barbed ends were capped, participated in barbed end assembly when they were uncapped, and the H133S profilin–actin complex associated to barbed ends in a growth assay at the same rate as the wild-type profilin–actin complex (data not shown). H133S profilin enhanced the rate of bacteria movement in profilin-depleted extracts to the exact same extent as bovine profilin in three reproducible experiments ( Table ), demonstrating that, by interacting directly with actin and not with a proline-rich protein, profilin enhances actin-based motility. Accordingly, we recently showed that the function of profilin in Listeria movement is independent of VASP, another proline-rich protein also initially thought to act by recruiting profilin. The stimulation of local actin assembly by Arp2/3 complex in response to different stimuli is thought to be pivotal in the generation of a large diversity of cellular extensions (lamellipodia, filopodia, neural growth cone, etc.), suggesting that a variety of connectors must link the different members of the Rho family to the Arp2/3 complex and activate actin polymerization by a common mechanism, with different partners. Our work shows that lessons can be learned from Shigella to understand the molecular mechanism of Cdc42-induced filopodium extension mediated by actin polymerization. Recently, Rohatgi et al. 1999 showed that N-WASP relays Cdc42 signals and directly activates the Arp2/3 complex in Xenopus egg extracts and in vitro with purified N-WASP and Arp2/3 complex. We extend this work to show that the Shigella IcsA protein mimics Cdc42, to greatly enhance the affinity of N-WASP for Arp2/3 complex, thus assembling a tight IcsA–N-WASP–Arp2/3 ternary complex at the bacterial surface, in a conformation that has maximal activity in actin assembly. N-WASP activation by IcsA explains why Shigella actin-based motility is independent of the Rho GTPases . Our findings, combined with results from other laboratories , can be summarized using a thermodynamic scheme expressing that: both Arp2/3 complex and N-WASP exist in solution in two conformations, inactive (X) and active . The active form of Arp2/3 complex (A*) stimulates actin polymerization. The active form of N-WASP (W*) exposes two functionally active domains involved in actin-based motility, which may be masked in the inactive state by intramolecular interaction . The VCA binds Arp2/3 complex and G-actin, and the NH 2 -terminal domain binds F-actin. Our data indicate that the F-actin binding site is most likely located in the WH1 domain, which also interacts with the verprolin homologue WIP. This formulation accounts for the fact that the Arp2/3 complex by itself has a weak nucleating activity and that N-WASP by itself is able to bind F-actin and G-actin weakly (this work). Binding of IcsA (I) to N-WASP shifts the N-WASP equilibrium toward the W* form, exposing the VCA domain and the F-actin binding domain, thus enhancing the affinity of N-WASP for active Arp2/3 complex and F-actin . Binding of N-WASP to Arp2/3 complex shifts the Arp2/3 equilibrium toward the A* form, increasing the amount of nucleating complex. It is implicit here that to nucleate actin polymerization, Arp2/3 complex binds G-actin. In the presence of IcsA and N-WASP, the Arp2/3 complex equilibrium is maximally shifted toward the A* form, in a triangular A*–W*–I complex. In this complex, protein–protein bonds are formed between the three partners (A-W, W-I, and A-I). Hence, each of the three components increases the stability of the complex. It is very likely that in vivo the actin-polymerization machinery is highly integrated and works on an all-or-none basis, i.e., the Arp2/3 complex switches from an inactive to a fully active form upon interacting with the COOH-terminal region of N-WASP, which itself is exposed only when Cdc42 has switched to its active conformation by binding GTP. Separating the components of this machinery by purification may cause structural alterations that partially unmask the protein interfaces normally buried in the inactive state. Detailed studies of the effect of solution variables on the activity of pure Arp2/3 complex will certainly help to understand the physical–chemical basis of N-WASP and Arp2/3 activation. VCA has a high affinity for Arp2/3 complex (a rough estimate of 10 7 M −1 can be derived from the polymerization curves), allowing easy depletion of Arp2/3 from extracts and a straightforward purification procedure by affinity chromatography. At variance with a previous report , VCA does not appear to bind to IcsA and elicit actin polymerization at the surface of Shigella . The reason for the discrepancy with Suzuki et al. 1998 remains obscure, but may relate to the different methods used to probe the interaction. We conclude that VCA is not sufficient for IcsA binding and that this interaction probably also requires residues present outside of the VCA domain. Since WASP and N-WASP are very similar in sequence, it is surprising that WASP, which is abundant in platelets, does not appear able to bind IcsA. A possible explanation is that WASP in platelets may be in strong interaction with other ligands, competing with IcsA binding. It may be noted, in this respect, that while the CRIB domains of WASP and N-WASP are very similar in sequence, differences exist in the binding of Cdc42-Y40C . Whether Cdc42 and IcsA directly compete for the same site to activate N-WASP is not known, but raises an interesting issue concerning the possible multiple ways of activation of N-WASP. The interface of the CRIB domain of N-WASP with GMPPCP-Cdc42 is known from NMR studies . It is also known that the glycine-rich regions of IcsA are involved in the association with N-WASP . The structural aspects of N-WASP activation await more detailed investigation. Like Shigella , other pathogen model systems may help discover the multiple effectors linking Arp2/3 complex to different signaling pathways. Analysis of the interaction of the COOH-terminal and NH 2 -terminal domains of N-WASP with actin provides insight in the mechanism by which actin polymerizes at the surface of Shigella . The two domains of N-WASP that become exposed upon binding to IcsA fulfill different functions. The COOH-terminal domain binds G-actin and Arp2/3 complex. The nucleation and filament branching activities of Arp2/3 imply that G-actin binds to Arp2/3 complex, perhaps by interacting with Arp2 or Arp3 (Pantaloni, D., manuscript in preparation). We propose, in agreement with Machesky and Insall 1998 and Rohatgi et al. 1999 , that the binding of G-actin in the nucleation complex is facilitated by association of the VCA domain to Arp2/3 complex, because in the VCA–actin complex, actin is positioned in the right orientation to interact with Arp2/3, in the structure of a barbed end nucleus, hence the free energy of G-actin binding to Arp2/3 is lowered. Second, in the filament elongation step, while the VCA domain functions like profilin in barbed end assembly, shuttling actin subunits onto growing barbed ends, the NH 2 -terminal domain of N-WASP maintains the filament in the vicinity of Shigella surface. We propose that the two domains of N-WASP work together with actin as expected for a motor of insertional polymerization at the surface of Shigella . Note that it is the treadmilling of actin filaments (which is a dissipative process) that supports unidirectional filament growth and promotes steady movement. N-WASP acts as a molecular ratchet that transduces actin polymerization into force. N-WASP is the first protein described thus far that possesses an integrated profilin-like function and locally lowers the critical concentration of G-actin. Whether other proteins share the same property in different cellular contexts is an exciting possibility. The observed localization of Arp2/3 complex in the actin tail of Listeria and of Shigella correlates with its localization in the lamellipodium . In the present work, the combination of motility assays with the biochemical analysis of the effects of Arp2/3 complex and N-WASP on actin polymerization brings a functional insight into the immunolocalization studies. While N-WASP remains bound to IcsA as the bacterium is moving , the Arp2/3 complex is activated by interacting with N-WASP transiently to nucleate and branch the filaments, then Arp2/3 complex remains incorporated in the actin meshwork as it is formed. We conclude that the Arp2/3 complex present in the actin tail (and in the lamellipodium) is no longer activated by N-WASP. This view is quite different from the currently expressed one . The Shigella bacterial model, therefore, proves to be a good tool to understand the mechanics of lamellipodium extension. Comparison of the Listeria and Shigella actin-based motility mechanisms provides interesting information on the different strategies available to induce actin assembly. Listeria does not harness a cellular activator of Arp2/3 complex. Instead, the ActA protein itself elicits the two functions of activated N-WASP. The central proline-rich domain of ActA binds VASP, which in turn binds F-actin, thus mediating the attachment of the filaments to the bacterium, as does the NH 2 -terminal domain of N-WASP in Shigella . Whether the NH 2 -terminal domain of ActA, which activates nucleation by Arp2/3 complex, acts like VCA by interacting with the p21-Arc subunit of Arp2/3 complex and with G-actin, possibly in a profilin-like fashion, is an open issue that may be of general relevance in the stimulation of actin assembly and which deserves investigation. Remarkably, the NH 2 -terminal domain of ActA contains the acidic sequence DEWEE homologue of the DEWED in the extreme COOH-terminal region A of VCA, and a sequence KKRRKAIASS similar to the QKRSKAIHSS sequence in the cofilin homology region of VCA, but does not contain the verprolin homology regions present in VCA. The possibility is raised that Arp2/3 complex is activated in a similar way by different effectors. Removal of VASP from platelet extracts does not affect the movement of Shigella , while VASP is essential in the propulsion of Listeria . This result rules out the postulated mechanism according to which VASP would work, in both Shigella and Listeria systems, as a profilin recruiter, to enhance actin assembly in an actin-based motility (ABM) complex . Incidentally, the physical relevance of that mechanism was questionable, since diffusion of profilin-actin is very fast and dissociation of profilin from a proline-rich target would introduce a kinetically limiting step in filament assembly. Overall, our results are in general agreement with the conclusion derived by Goldberg 1997 that vinculin, hence the vinculin–VASP interaction, is not required in Shigella motility. Our data further show that the function of profilin in actin-based Shigella movement is mediated by binding actin directly, without interacting with the proline-rich region of N-WASP. Similarly, we demonstrated that the function of profilin in Listeria movement is independent of VASP . VASP in Listeria and N-WASP in Shigella mediate attachment of the actin tail to the bacterial surface. Hence, the mechanisms of movement of these two pathogens are similar in their physical principles, and some of the operators are the same (Arp2/3 complex and actin), whereas some functions are elicited by different players. Although the interaction of IcsA with N-WASP is essential in generating Shigella motility, N-WASP-coated bacteria fail to actively move in a solution of F-actin at steady state in the presence of Arp2/3 complex. As pointed out earlier , regulatory factors that maintain G-actin at a high steady state concentration, by regulating the dynamics of actin assembly, are important missing ingredients in the reconstitution of movement . | Study | biomedical | en | 0.999996 |
10491395 | Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. All photographic images were compiled using Adobe Photoshop, Adobe Illustrator, and/or Canvas (Deneba Systems) software. The printed images are representative of the original data. Human nonmuscle β-actin was cloned by PCR from a human cDNA library and was fused in-frame with the enhanced GFP gene into the BamHI-XhoI sites of the vector pEGFP-C1 (Clontech Inc.) to generate a fusion between the COOH terminus of EGFP and the NH 2 terminus of beta-actin in the expressed protein. PCR-derived sequences were confirmed by dideoxy sequencing. MDCK clone II/G cells were obtained from the laboratory of Gerrit van Meer (Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, The Netherlands) and have been described previously . MDCK cells were transfected with pEGFP-actin using the lipofectamine protocol from GIBCO BRL. Clones were selected in 400 μg/ml G418 (Geneticin; GIBCO BRL). Low passage aliquots of drug-resistant MDCK clones were frozen and stored in liquid nitrogen: clones were passaged in DME containing 10% FCS (Gemini) and 400 μg/ml G418 for a maximum of 4–6 wk. For experiments, all cells were grown in DME containing 10% FBS and antibiotic-antimycotic (GIBCO BRL) at 37°C in a humidified incubator containing 5% CO 2 . Antibiotic-antimycotic was removed from cultures used for bacterial infections. For the preparation of lysates, 5 × 10 5 MDCK cells were plated on 35-mm tissue-culture dishes and extracted after 24 h in culture. For the preparation of SDS lysates, MDCK cells were extracted in hot SDS buffer containing 1% SDS, 10 mM Tris-HCl, pH 7.5, and 2 mM EDTA, and then scraped from the petri dish with a rubber policeman. Samples were boiled for 15 min and insoluble material was removed by centrifugation at 12,000 g for 15 min. For preparation of Triton X-100 lysates, MDCK cells were extracted for 15 min at 4°C with CSK buffer (0.5% Triton X-100, 10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, and 3 mM MgCl 2 ) containing a protease inhibitor mix (1 mM Pefabloc, 1 mM benzamidine, and 10 μg/ml each of aprotinin, pestatin, and leupeptin). Pefabloc, pepstatin, and leupeptin were purchased from Boehringer Mannheim Biochemicals. After extraction, cells were scraped from the petri dish with a rubber policeman and insoluble material was pelleted by centrifugation at 12,000 g for 30 min. Pellets were washed twice with CSK buffer, resuspended in SDS buffer, boiled 10 min, and insoluble material was removed by centrifugation at 12,000 g for 15 min. Protein concentrations were determined using the BCA protein assay reagent kit (Pierce Chemical Co.). Portions of the lysates were boiled for 5 min after adding one-third volume of 4× SDS reducing sample buffer and separated by SDS-PAGE in 7.5% polyacrylamide gels . Lysates were immunoblotted with anti-actin monoclonal antibody (clone C4; Boehringer Mannheim; 1:500) and HRP secondary antibody as described previously . 1 × 10 5 MDCK cells were plated on collagen-coated coverslips in 35-mm tissue-culture dishes and fixed after 24 h in culture. Cells were washed once in Dulbecco's PBS and fixed for 20 min at RT with 2% formaldehyde in Dulbecco's PBS, or cells were washed once, extracted 5 min at 4°C with CSK buffer, washed again, and fixed with 2% formaldehyde. Cells were washed with blocking buffer, incubated 10 min with rhodamine-phalloidin (Molecular Probes; 1 unit per coverslip) in blocking buffer, washed, and processed for microscopy as described previously . Photomicrographs were taken with an Axiophot inverted fluorescence microscope (Carl Zeiss Inc.). L . monocytogenes strain 10403S was grown 12–15 h in 2 ml BHI (Difco) at room temperature without agitation. Cells were pelleted and resuspended in PBS (0.9 mM CaCl 2 , 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 0.5 mM MgCl 2 , 137 mM NaCl, and 8.1 mM Na 2 HPO 4 ) or DMEM containing 10% FBS, and then 50–100 μl was added to cells growing on glass coverslips in 2 ml DMEM with 10% FBS. After 60–90 min, cells were rinsed three times with PBS and fresh DMEM/FBS added. 30 min later, in most cases, gentamicin sulfate was added to a concentration of 30–50 μg/ml. Infected cells grown on glass coverslips were observed 5–20 h after infection after mounting on a stage whose temperature was maintained at 37°C. Cells were overlaid with phenol red-free DMEM with 10% FBS and buffered with 20 mM Hepes (pH 7.3), and covered with a thin layer of silicone DC-200 fluid (Serva) to prevent evaporation. Observations were performed on a Nikon Diaphot-300 inverted microscope equipped with phase contrast and epifluorescence optics. Time–lapse video microscopy was achieved with an intensified charge-coupled device (CCD) camera (Dage-MTI; GenIISys/CCD-c72) or a cooled CCD camera (NDE/CCD; Princeton Instruments) and Metamorph (Universal Imaging) software. Phase contrast/fluorescence image pairs were recorded every 10–30 s, with eight video frames averaged per image (intensified CCD) or 50-ms exposures (cooled CCD). Cells grown on glass coverslips were fixed in 3% formaldehyde for 30 min at room temperature in PBS after 7–10 h of infection. Cells were then washed twice in PBS at room temperature and extracted with CSK buffer at room temperature for 10 min. Cells were washed twice in PBS at room temperature for 10 min. Cells were blocked in PBS containing 1% BSA, 1% calf serum, and 50 mM NH 4 Cl for 1 h at room temperature, then in primary antibody solution containing mouse anti-E-cadherin antibody (Transduction Laboratories) for 2 h at room temperature or overnight at 4°C. Cells were then washed three times in PBS with 0.2% BSA and incubated in Texas red–conjugated mouse secondary antibody solution for 1 h at room temperature. Cells were washed three times in PBS and 0.2% BSA and mounted in 90% glycerol in 20 mM TRIS. Three-dimensional images were recorded with a cooled CCD camera (Photometrics Ltd.). Optical sections (1,023 × 1,023 pixels) of thickness 0.10–0.25 μm were recorded with a 100× oil immersion objective (Olympus Corp.). All aspects of image collection were controlled by a Silicon Graphics workstation (Silicon Graphics Corp.), on a deconvolution microscopy system (Applied Precision). Infected cells were observed on the microscopy workstation described for video microscopy. DMEM + 10% FBS with 0.5% (wt/vol) methyl cellulose was added to the heated chamber and protrusions imaged. Cellular ATP synthesis was inhibited by addition of 50 μM 2,4-dinitrophenol and 10 mM 2-deoxy- d -glucose. Inhibitors were removed by rinsing in fresh DMEM + 10% FBS with 0.5% methyl cellulose. Cells were incubated 1–3 h in 1–10 μM TRITC in DMEM containing 10% FBS and antibiotic-antimycotic (GIBCO BRL). Cells were rinsed 1–2 times over the next 1–5 h with fresh media, then trypsinized and replated with unlabeled cells, and grown at least 12 h before infection. Cells were suspended in 2 ml LISS buffer (239 mM sucrose and 5 mM Hepes, pH 7.3), to which was added 4–10 μl of a 0.25% (wt/vol) stock solution of 3, 3′-dioctadecyloxacarbocyanine perchlorate (DiOC 18 ; Molecular Probes) in dimethylformamide (DMF). Stock solution was prepared by mixing DiO and stearylamine octadecylamine in a 5:1 (wt/wt) ratio and dissolved in chloroform, heated to 50°C, precipitated on ice with two volumes of methanol, and pelleted by centrifugation. The pellet was dried then dissolved in 1,000 μl DMF, heated to 50°C, and centrifuged. Cells were incubated for 20 min with gentle agitation, rinsed twice with LISS, and plated in DMEM + 10% FBS. TRITC-labeled and unlabeled cells were plated together and incubated 12–40 h. After infection, DMEM + 10% FBS containing 1 μM bafilomycin A in DMSO was added. This concentration inhibits acidification of endocytic vacuoles in mammalian cells . Cells were incubated at least 20 min before observation of protrusion dynamics by video microscopy. Infection of J774 and metabolic labeling were performed as previously described with some modifications . J774 cells were infected with the wild-type strain 10403S, isogenic mutants DP-L2296 ( Δmpl ) , or DP-L1942 ( ΔactA ) . The cells were washed with PBS at 30 min post-infection (p.i.), and gentamicin (10 μg/ml) was added at 1 h p.i. Cells were starved for methionine at 3.5 h p.i., and host protein synthesis was blocked with anisomycin (30 μg/ml) and cycloheximide (22.5 μg/ml) 15 min before labeling. At 4 h p.i., cells were pulse-labeled with 35 S-methionine (90 μCi/35-mm dish) for 10 min, and chased with cold methionine (2 mM) and chloramphenicol (20 μg/ml) for 15 min. At that point, the medium was replaced with a potassium buffer, pH 6.5 (133 mM KCl, 1 mM MgCl, 15 mM Hepes, 15 mM MOPS), cold methionine, and chloramphenicol, with or without nigericin (10 μM) . Treatment with nigericin allows the pH to equilibrate across all membranes. After 15 min, samples were washed with cold PBS, lysed in 2× sample buffer, boiled, and separated by 10% SDS-PAGE. Photobleaching of cells was performed and analyzed on the Nikon microscopy workstation described above. Infected cells were prepared as described in that section. Bleaching was performed by narrowing the field diaphragm on a 100-W mercury arc lamp, exposing the region to be bleached for 3–5 min through a Chroma FITC filter. For protrusions, bleached regions were chosen so as to exclude the entire cell body except for the protrusion. Clouds were chosen for bleaching based on proximity to cell edges, to minimize photodamage to the cell body and the GFP it contained. Immediately after bleaching, lamp intensity was attenuated to 10% by a neutral density filter and 8-frame averaged images were collected every 7 s to follow recovery. For analysis, the integrated pixel intensity of each cloud or protrusion was measured using the Measure Brightness function of Metamorph. Additionally, the intensity of a nearby unbleached region of cytoplasm was measured for each frame and used to normalize intensities of regions of analysis, to compensate for fluctuations in lamp intensity and photobleaching due to image capture. The standard intensity never fell below 90% of its original value. To characterize the exponential recovery of fluorescence, normalized intensities were fit to the following perturbation-relaxation equation: 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*}F_{ \left \left(t\right) \right }=F_{0}+ \left \left(F_{{\mathrm{{\infty}}}}-F_{0}\right) \right \left \left(1-e^{-kt}\right) \right {\mathrm{,}}\end{equation*}\end{document} after Salmon et al. 1984 . F (t) is the normalized fluorescence ratio at time t after photobleaching, and k is the first-order rate constant, which describes the rate of recovery. F 0 is the fluorescence intensity measured immediately after bleaching, and F ∞ is the asymptotic value to which fluorescence intensity recovers after bleaching. Data was fitted to and the half-time ( t 1/2 ) to recovery from F 0 to F ∞ was calculated using the equation: 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*}t_{{1}/{2}}={ \left \left({\mathrm{ln\;2}}\right) \right }/{k}{\mathrm{.}}\end{equation*}\end{document} Infected cells grown on glass coverslips were fixed in 3% formaldehyde for 30 min at room temperature and then incubated in FITC-phalloidin in PBS for 30 min at room temperature. They were then rinsed with PBS for 5 min and mounted on slides in 90% glycerol in 20 mM Tris-HCl. Cells were viewed with the Nikon microscopy workstation described above, using a 100× oil immersion objective. Primarily infected cells were defined as (in descending order of importance): (a) cells with the most bacteria, (b) cells in the center of an infectious focus, and (c) cells with the greatest fraction of bacteria possessing tails. Secondarily infected cells were cells with fewer bacteria surrounding the perimeter of the primarily infected cell, and tertiarily infected cells were at least one cell away from the primarily infected cell and contained fewer bacteria than other cells in the focus. After 8 h, primary, secondary, and tertiary infections could no longer be unambiguously defined. Video 1. Supplement to Fig. 1 c. MDCK canine kidney cells, which constitutively express green fluorescent protein fused with actin, are infected with Listeria monocytogenes . Available at http://www.jcb.org/cgi/content/full/146/6/1333/F1/DC1 Video 2. Supplement to Fig. 3 . Ricochets and protrusions can be seen in this monolayer, which was confluent for ∼72 h preinfection. Ricochets are marked by yellow R's and protrusions by red P's. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F3/DC1 Video 3. Supplement to Fig. 4 . The majority of fitful movement occurs along the axis of the protrusion. Speeded up 120×. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F4/DC1 Video 4. Supplement to text. In the presence of methyl cellulose, addition of ATP synthesis inhibitors quickly stops all protrusion movement. One protrusion still drifts; however, the chamber was not free of flow, and on average protrusions moved less than 1 μm in 10 min. Washing with inhibitor-free media restores fitful movement. Available at http://www.jcb.org/cgi/content/full/146/6/1333/DC2 Video 5. Supplement to Fig. 6 . Collapse of the protrusion from an ovoid to spherical geometry. A long protrusion moving into a non-GFP-expressing cell collapses. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F6/DC1 Video 6. Supplement to Fig. 8 (a–c). Vacuole lysis. Three protrusions/vacuoles can be seen spreading from one cell to another. Shortly after collapse of the protrusion (especially apparent in the lowest protrusion), the membrane signal disappears but the actin signal remains. Phase contrast: lower right corner. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F8/DC1 Video 7. Supplement to Fig. 8 d. Actin flash. Here, a protrusion is formed, undergoes fitful movement, stillness and collapse, followed shortly by a bright flash of actin intensity temporally coincident with average times for vacuole lysis. The membrane was not labeled in this case. Available at http://www.jcb.org/cgi/content/full/146/6/1333/F8/DC1 Since any pathogenic relationship requires contributions from both the pathogen and the host, selection of a model host system is important. We chose to study intercellular spread of L . monocytogenes in MDCK cells, whose columnar epithelial morphology makes them ideal subjects both practically (given their extensive cell-cell contacts) and physiologically (since L . monocytogenes initially invades its human host across the columnar epithelium of the intestine). However, bacteria inside these cells are difficult to observe by phase-contrast microscopy, since the cells are tall and contain many intracellular phase-dense structures. To solve this problem, an MDCK cell line expressing GFP-actin was selected based on its normal cytoskeletal organization and gross morphology . Examination by Western blot revealed that the amount of GFP-actin expressed was <2% of total monomeric actin , although expression levels varied from cell to cell. Upon cell extraction in buffer containing Triton X-100 , GFP-actin was more abundant in the Triton X-100 soluble fraction than the pellet compared with endogenous actin. Nevertheless, we detected GFP-actin incorporation into actin filaments that stained with phalloidin. The level of GFP-actin incorporated into filaments was low enough that it did not appear to disrupt the structure of the filament, but was sufficient to observe actin filament structure and dynamics in living cells . The GFP-actin enabled simultaneous phase-contrast and fluorescence video microscopic observation of L . monocytogenes –induced actin comet tail formation and movement , and actin-dependent bacterial motility in these cells was similar to that previously described in other cells not expressing GFP-actin with movement speed averaging 0.137 μm/s (SD = 0.065, n = 48). Using this system, we were able to observe many incidences of intercellular spread, and thereby analyze, for the first time, stages in transfer of bacteria and membrane protrusions between neighboring cells. The first hypothesized step in intercellular spread is formation of a membrane-bound protrusion into a neighboring cell. We frequently observed this step in infected MDCK cells. Here, L . monocytogenes –containing membrane protrusions are defined as extracellular if formed from a host surface free of cell-cell contacts, and intercellular if formed from one cell and protruding into an adjacent cell. Intercellular protrusions must therefore distend two membranes from the point of their inception, one from the recipient and the other from the donor cell. Only these protrusions permitted the ultimate release of a bacterium into another cell. Intercellular protrusions ranged in length from 3 to 18 μm (average length: 8.5 μm, SD = 4.3, n = 16). Since the host cells were many focal planes deep, intercellular protrusions into neighboring cells were easy to distinguish from those protruding apically into the extracellular medium. In making a protrusion, the bacterium did not simply distend the lipid bilayer: it pulled the associated cellular structure along with it . Adherens junctions, mediated by the transmembrane protein, E-cadherin, line the lateral plasma membrane in the form of puncta in MDCK cells partially polarized by growth on coverslips . We noted that intercellular protrusions induced by bacteria were lined by E-cadherin puncta with a distribution similar to that on the lateral membranes of cell-cell contacts. This suggests that adherens junctions maintain their geometry in the plasma membrane with respect to one another despite the distension of the membrane during protrusive activity. Some extracellular protrusions displayed similar E-cadherin staining ; however, extracellular protrusions that did not contain detectable levels of E-cadherin were morphologically indistinguishable from those that did , suggesting that E-cadherin on noncontacting membranes does not play an appreciable role in creating the protrusion structure. Surprisingly, a protrusion did not form on every occasion that a bacterium was propelled by its actin tail into the plasma membrane . Instead, the bacterium occasionally ricocheted off the membrane and continued to race around the cell. A single bacterium might ricochet off one face of the plasma membrane and immediately form a membrane-bound protrusion from another, demonstrating that the ricochet events were dependent on properties of the host cell membrane, rather than a bacterial deficiency. We examined cells to learn more about the basis for ricochet versus membrane protrusion events. There did not appear to be particular regions of the cell membrane that exhibited a predilection for a ricochet or protrusion event, and neither the instantaneous velocity nor angle with which the bacterium struck the membrane was predictive of which fate would result. Bacteria moving at rates >0.02 μm/s and striking the membrane at an angle ≥30° were equally likely to have been members of the protrusion or ricochet subsets. However, the probability of a ricochet was highly dependent upon the age of the MDCK monolayer . The percentage of membrane contacts resulting in ricochets increased with monolayer age. A possible cause of this change could be structural alterations commensurate with development of cell polarity. MDCK cells are known to undergo structural changes during maturation of the monolayer on solid or permeable substrates . These include extensive cell-cell contact formation as cells increase their height, and formation of a microtubule lattice oriented vertically with respect to the substrate along basolateral membranes . It is possible that the change in susceptibility of the plasma membrane to bacteria-induced protrusions is due to changes in the extent of cell-cell adhesion and/or the development of an organized submembranous cytoskeleton. We observed that membrane-bound intercellular protrusions frequently underwent a period of erratic motility (which we termed fitful movement) followed by a period of stillness before their uptake (see next section). 62% ( n = 29) of intercellular protrusions exhibited fitful movement in the recipient cell's cytoplasm . The remaining 38% exhibited little or no movement throughout their duration. Of the protrusions that underwent fitful movement, 78% moved erratically for 7–15 min (average = 10.3 min, SD = 2.3, n = 15) followed by an additional 10–25 min of stillness. 60% of that population was stalled for 2–3 min before beginning fitful movement . The remaining protrusions which moved ( n = 4) exhibited fitful movement throughout the duration of their time in the protrusion. This fitful movement is consistent with continued polymerization of actin at the bacterial surface, as most of the motion occurs in a vector along the protrusion's axis of formation . We plotted the bacterial path as a function of time with respect to axes defined by the three time points which constituted the initial direction of the intrusive pathway as it crossed the cell border (x axis) . We found that 73% (SD = 9, n = 17) of the motility was along the protrusion axis , strongly suggesting that the bacterium was largely responsible for fitful movement of the protrusion. Extracellular membrane-bound protrusions into free space exhibit rapid erratic movement . When we inhibited the Brownian component of that movement by increasing extracellular fluid viscosity, we observed fitful movement of the extracellular protrusions similar to that of intercellular protrusions . Under these experimental conditions, bacteria moved more slowly and in a directed fashion, occasionally taking steps backward, as intercellular protrusions do, but did not stop moving completely. We monitored these protrusions for motility and found that they displaced an average of 11.1 μm over 10 min (SD = 8.1, n = 10) with an average instantaneous velocity of 0.021 μm/s (SD = 0.018). When host cell ATP production (and therefore actin polymerization) was inhibited by addition of 50 μM 2,4-dinitrophenol and 10 mM 1-deoxyglucose, cytoplasmic bacteria stopped moving in <90 s. Likewise, extracellular protrusions stopped virtually all movement, displacing an average of 1.1 μm over 10 min (SD = 1.5) with an average instantaneous velocity of 0.0018 μm/s . 5 min after washing out these inhibitors, the average displacement of the protrusions returned to 10.0 μm over 10 min (SD = 9.6), with average instantaneous velocity 0.016 μm/s (SD = 0.015), which is similar to movements of protrusions in untreated cells (see above). Taken together, these experiments demonstrate that the fitful movement of membrane-bound protrusions is most likely due to bacterially directed actin polymerization, though it is possible that the recipient cell may make a minor contribution by tugging on the protrusion. Since polymerization-based movement of protrusions requires ATP, it is probable that cessation of that movement following entry of the protrusion into a recipient cell corresponds to the pinching off and sealing of the donor cell's protrusion membrane. This would prevent further generation of actin-based force as it denies bacterial access to the cellular pool of ATP. Extracellular protrusions were not taken up by nearby cells, even when constrained by them. We captured occasional ( n = 4) video sequences of membrane-bound protrusions that lay confined in the intercellular space between two neighboring cells. After >1.5 h observation, they were never taken up by these cells, though they occasionally twitched back and forth along the protrusion axis. Furthermore, we never observed a protrusion that was initially extracellular taken up by a neighboring cell, indicating that classical, actin-based phagocytosis of membrane-bound protrusions does not occur frequently, if at all, in these cells. Since we never observed bacterial escape from extracellular protrusions ( n > 300), we concluded that the receiving cell must actively pinch off a protrusion that has intruded into its cytoplasmic space. In other words, the bacterium initially plays the active role, pushing the protrusion directly into the cytoplasm of the neighboring cell, which then takes over the active role to engulf the protrusion. Approximately 30–40 min (average = 34 min, SD = 7, n = 14) after the start of membrane-bound protrusion into the recipient cell (corresponding to 15–25 min after the cessation of fitful movement), a distinct morphological change in protrusion geometry was apparent in all but one case . Initially, membrane protrusions in recipient cells were an elongated ovoid in shape. Subsequently the protrusion appeared to shrink, and then snap to a roughly spherical morphology over a period of 30–150 s. Visually, the protrusion appeared to have been released from tension, as if it had been pinched off. Of the four protrusions that exhibited constant fitful movement, two underwent this geometric collapse after >1 h of observation. The other two failed to collapse even after 90 min. We did not observe any correlation between entry speed and protrusion length, or entry speed and time before the change in geometry, suggesting that these variables did not depend on the activity of the bacterium . These results, combined with the properties of fitful movement (see above) lead us to conclude that protrusion uptake occurs in two steps, the timing of which are primarily determined by the recipient host cell. In the first, the supply of ATP to the bacterium is cut off, as the bacterium becomes sealed in a single membrane structure. In the second, which occurs ∼20 min later, tension is released as the second membrane closes off and the protrusion snaps to a spherical shape. Shortly after this second step, the vacuole lyses. Interactions between the membranes supplied by the donor and recipient cells and bacterial lysis of the double membrane vacuole are important steps in intercellular spread, but difficult to observe using the heretofore described system of GFP-actin expressing MDCK cells. Therefore, we added a plasma membrane label to the donor cell. Cells were incubated with TRITC, which covalently and nonspecifically labels membrane proteins on lysine residues, and were then coplated with unlabeled cells. Protrusions in the act of spreading from labeled into unlabeled cells were selected for observation. Simultaneous time–lapse recording of phase contrast and the two fluorescent channels, GFP-actin and TRITC, was performed. In every case examined ( n = 9), the membrane TRITC signal vanished 1–5 min (average = 2.4, SD = 1.4) after the change in geometry, always fading over a time interval <50 s . In the presence of bafilomycin A 1 , which inhibits vacuolar acidification , the geometric collapse of the protrusion occurred in 9 out of 10 cases examined, but the membrane TRITC signal remained detectable for at least 1 h in 8 of those 9 cases, at which time we stopped recording. In the 9th case, the signal disappeared after 45 min of observation. These manipulations and observations lead us to conclude that: (a) The internalized membrane protrusion becomes a sealed vacuole; (b) Subsequent acidification of the vacuole is not required for the abrupt change in the geometric shape of the protrusion; and (c) Vacuolar acidification dramatically increases the efficiency of bacterial escape from the double membrane. Throughout the period after cessation of fitful movement, but preceding the abrupt loss of the membrane TRITC signal, we noticed that the strength of the GFP-actin signal declined . This could represent photobleaching, but could also in part be due to vacuolar acidification, since GFP intensity declines with decreasing pH . Coincident with or one frame (20–30 s) before vacuolar lysis, a sudden increase in GFP-actin fluorescence intensity (flash), followed by a significant decrease in intensity, was observed in 37% of cases examined . This flash might be expected if the GFP-actin surrounding the bacterium was freed from the acidified vacuole and returned to a neutral pH, at which time a fraction of it is free to diffuse away into the recipient cell's cytoplasm. Since we detected a GFP signal for up to 30 min after intercellular spread around the bacterium in recipient cells that did not express detectable levels of GFP-actin, we conclude that a fraction of the GFP-actin from the donor cell depolymerized very slowly after escape from the secondary vacuole. This was unexpected, as actin turnover in L . monocytogenes tails is rapid, with an average half-life of 33 s in PtK2 cells . Furthermore, even in highly organized actin structures such as stress fibers in PtK2 cells, individual actin filaments have a half-life of <4 min . We hypothesized that the stabilization of actin around the bacterium is the legacy of the lysed protrusion. Therefore, we examined actin turnover in extracellular and intercellular membrane protrusions. The rate of actin turnover in intercellular and extracellular protrusions was examined by FRAP. For these measurements, cells were grown at medium density in the presence of 0.5% methyl cellulose, which permitted formation of extracellular membrane-bound protrusions and actin polymerization-dependent movement, but inhibited Brownian motion (see above). Neither extracellular ( n = 5) nor intercellular protrusions ( n = 10) exhibited any measurable recovery of fluorescent actin along the length of the protrusion >10 min of observation after complete photobleaching (27% of photobleached protrusions exhibited some very gradual recovery within 1 μm around the bacterial surface, particularly the tail-associated end, where new polymerization is expected to occur). These results indicate that actin filaments in protrusions, like those in clouds surrounding bacteria that have just escaped from a secondary vacuole, are highly stabilized, with a half-life significantly >10 min. This is consistent with our unpublished observations that bacteria-containing extracellular membrane-bound protrusions persist for >30 min and continue to react with phalloidin along their length, demonstrating the presence of persistent actin filaments, even when the protrusion is extending very slowly. Bacteria associated with uniform actin clouds in the cytoplasm were also photobleached and their fluorescence recovery measured. Unbleached clouds were observed to be at steady state (their fluorescence intensity and area neither increased nor decreased), demonstrating that the rate of actin polymerization must equal that of depolymerization. Hence FRAP, which measures polymerization rates, allowed us to calculate filament half-life. Unlike clouds surrounding bacteria which have just escaped from the secondary vacuole, filaments in ordinary cytoplasmic clouds turn over at rates close to that in actin tails (average = 45 s, SD = 15, n = 8). Bacteria that had escaped from a secondary vacuole did not recover motility over a period of >30 min of observation ( n = 13). After initial invasion, bacteria require 1–3 h to acquire tails and begin movement . This is due in part to the fact that actA expression is not induced until after the bacteria escape from the primary vacuole . Therefore, we hypothesized that the delay in the recovery of bacterial movement might be due to inhibition or destruction of existing ActA protein by acidification of the secondary vacuole. To observe effects of pH on ActA stability, we turned to the well-characterized model of J774 cells . In these cells (unlike MDCK cells), L . monocytogenes infects with very high efficiency, so newly synthesized ActA protein can be readily detected. Using pulse–chase experiments we found that the ActA protein is cleaved in a pH-dependent manner reminiscent of the acid-dependent cleavage of proPC-PLC (see introduction), which is proteolytically activated in the secondary vacuole by the metalloprotease Mpl . Cells were pulse-labeled with 35 S-methionine for 10 min, followed by a 15-min chase. After the chase, the medium was replaced with a buffer at pH 6.5 (see Materials and Methods). In lanes 3, 6, and 9, 10 μM nigericin was added to allow pH to equilibrate across all membranes. Three bands corresponding to phosphorylated forms of ActA were observed. The ActA was cleaved in the wild-type strain in a pH-dependent manner (lanes 2 and 3), but not in the mpl mutant (lanes 5 and 6). In an Mpl-deficient strain, no cleavage of ActA was observed (lanes 8 and 9). This strongly suggests that Mpl, which is activated upon acidification, mediates ActA proteolysis in the secondary vacuole. This destruction of ActA explains why newly escaped bacteria do not immediately recruit actin from the recipient cell and reinitiate movement in the cytoplasm. To further examine the kinetics of the recovery of motility in epithelial cells, we examined individual infectious foci in MDCK cells fixed at different time points after infection . Most intercellular spread into secondary cells occurred between 4 and 5 h after infection, with tertiary infections following 2.5 h later at a similar rate . The initial baseline percentage of foci with secondary infections (2–3 h) is probably due to primary infections occurring simultaneously in neighboring cells, as insufficient numbers of bacteria have acquired tails at this time for intercellular spread to have occurred . Interestingly, as only 6.9% of cells are infected at t = 2 h, it is highly improbable ( P < 0.01 by chi-squared test) that primary infections of neighboring cells (apparent in 12% of foci) are independent events. This may be due to a sensitization effect. It has been observed that transient calcium spikes occur during L . monocytogenes invasion and also during invasion by at least one other invasive bacterial species . This ion flux could be translated to adjacent cells via gap junctions, thereby sensitizing the cells and increasing the likelihood of bacterial invasion in neighbors. The increase in secondary infections was not followed by immediate recovery of bacterial motility . The fraction of moving bacteria in secondary cells followed a curve almost identical to that obtained from primarily infected cells, but with a shorter lag time and slightly steeper slope. To determine how many bacterial divisions were required before initiation of motility, we counted the total number of bacteria in cells that contained 1–2 bacteria with tails. To exclude the possibility that other, motile bacteria had already left the cell, primary cells with infected neighbors were not counted. For secondarily infected cells, spreading to a tertiary cell or back to the primary cell are equally probable; thus, we excluded secondary cells after 7 h infection, when tertiary infections were first observed. Primary cells contained an average of 6.3 bacteria per cell when 1–2 tails were observed (SD = 2.6, n = 18), suggesting approximately three divisions were required for acquisition of motility. Long-term video observation in PtK2 cells also indicates that an average of three divisions are required before bacterial motility is initiated (Theriot, J.A., unpublished results). Secondarily infected cells, however, required only two bacterial divisions, with an average bacterial population of 4.0 per cell hosting 1–2 bacterial tails (SD = 1.5, n = 28). This difference may reflect the time required to upregulate transcription of ActA after initial infection versus the shorter time required to merely translate and polarize ActA after its proteolytic destruction in the secondary vacuole. Out of 711 cells observed, only one was inhabited by a single, moving bacterium, and it was unclear whether this bacterium's sister could have recently spread to a neighboring cell. All other cells with tail-associated bacteria possessed at least one other bacterium, strongly suggesting that motility cannot initiate except after at least one bacterial division. The near-absolute dependence of motility on bacterial division is consistent with the model that division is responsible for the establishment of ActA polarity and therefore actin polarity . We were surprised to observe that when TRITC-labeled cells were incubated with unlabeled cells in the absence of L . monocytogenes , 5–18% of the unlabeled cells adjacent to labeled cells contained 1–2 TRITC-labeled vacuoles . Percentages varied greatly according to plating density and seed ratio, and did not appear to increase over time (20–72 h). Vacuoles ranged in diameter up to ∼0.5 μm, and exhibited some saltatory movement inside cells. To confirm that these vacuoles did not merely contain labeled extracellular matrix proteins or products of trypsinization, we colabeled TRITC-dyed cells with the dialkylcarbocyanine DiO, a lipophilic tracer. Multiple DiO-containing vesicles were observed inside unlabeled cells, and a subset of these, usually those largest in size, were also TRITC-labeled. To our knowledge, this is the first demonstration that MDCK cells engulf portions of their neighbors' membranes. L . monocytogenes intercellular spread may capitalize on this natural paracytophagic behavior. Since the columnar epithelium of the intestine is the initial target of L . monocytogenes infection, monolayers of MDCK columnar epithelial cells (albeit derived from kidney) provide a useful model system in which to study pathogenicity at the cellular level. The extensive characterization of this cell line as a polarized epithelium increases its utility. It is clear from our work that the host cell is not merely a passive victim of L . monocytogenes infection, but extensively cooperates in perpetuating the infection as an accomplice in intercellular spread. Intercellular spread by L. monocytogenes can be described by a stereotyped sequence of contingent events that are highly reproducible. Fig. 12 a shows the timeline for spread of a typical bacterium, using average times for each event. First, the bacterium is propelled by actin-based motility into a membrane-bound protrusion, which undergoes bacterially directed fitful movement for ∼15 min. Cessation of this movement represents cutoff of the bacterium from the donor cell's pool of ATP or possibly other factors. The protrusion's morphology is maintained for some time, then is suddenly released from tension. Within minutes, the vacuole is lysed in an acidification-dependent manner, leaving the bacterium free in the cytoplasm of the recipient cell. Recovery of motility occurs much later, after at least one bacterial division. The process requires approximately one bacterial generation time ; however, bacteria were rarely observed to divide during intercellular spread (in the membrane-bound protrusion or vacuole), so the process may have an inhibitory effect on division. This is consistent with the observation that bacteria deficient in PC-PLC have slower growth unless intercellular spread is blocked by addition of cytochalasin B . Uptake of membrane-bound protrusions has been described as being phagocytic in nature by analogy to phagocytic uptake of bacteria by macrophages . However, intercellular spread is fundamentally different, requiring that the cell not only physically enclose the bacterium but also pinch off the donor cell's membrane. Our data suggest that protrusion uptake requires two steps: first, the donor membrane is closed off and second (15–25 min later) the recipient cell's membrane seals . Since bacteria are never observed to escape from or lyse extracellular protrusions despite many hours of observation, the recipient cell must play an active role in pinching off that protrusion and permitting vacuolar lysis. In this model, the first host-dependent step is the closure of the donor membrane, which corresponds to the cessation of fitful movement . This is analogous to the inhibition of fitful movement in extracellular protrusions by host cell ATP depletion. After the donor membrane is sealed, the protrusion's morphology is maintained under tension by existing cell-cell adherens junctions . The host proteasomes, which are known to degrade LLO , will no longer have access to bacterially secreted proteins. We speculate that the recipient cell does not recognize the open-ended distension of its membrane as an endosome, the ovoid protrusion will not be acidified, and the acid-dependent proteins LLO and PC-PLC will not be activated. Collapse of the protrusion to a roughly spherical geometry marks the closure of the recipient cell membrane . Now the cell recognizes the endocytosed vacuole as a target for acidification. How this recognition occurs remains an open question. The sudden drop in pH activates LLO and Mpl; the latter cleaves proPC-PLC to its active form and also cleaves ActA. Vacuolar lysis occurs rapidly . The temporal coupling between protrusion collapse and vacuolar lysis is striking in that they are consistently separated by <5 min. This timing is consistent with biochemical observations that proteolytic activation of PC-PLC occurs very rapidly, with maximal amounts accumulated within 5 min of acidification (Marquis, H., unpublished observations). The observation that inhibition of acidification decreases the efficiency of vacuolar lysis is consistent with the phenotype of bacteria deficient for the acid-activated PC-PLC which exhibit decreased intercellular spread . It has been shown that cadherin-cadherin interactions are required for intercellular spread of Shigella flexneri , a Gram-negative pathogen that also undergoes actin-based motility and may spread from cell to cell by similar mechanisms. Our observations suggest that this is not a molecular requirement, but rather a need for membrane adhesion that creates a cellular architecture that permits intrusion of a bacterial membrane-bound protrusion into the cytoplasmic space of a neighboring recipient cell. The two-step model presented above suggests a plausible resolution to the biochemical problem of regulating two separate membrane fusion events. The model is also consistent with the observation that protrusions which exhibit fitful movement throughout their duration are taken up only after extremely long periods of time or not at all, as it posits their donor membrane was not sealed. Further studies will elucidate the mechanism by which this occurs. After escape into the cytoplasm of the recipient cell, actin from the donor cell remains associated with the bacterium for up to 30 min or more, indicating that is not turned over. This is in contrast to cytoplasmic bacteria surrounded by uniform actin clouds in which the actin is rapidly turned over at a rate similar to that in comet tails (half-lives of ∼30–45 s). However, slow actin turnover around secondarily spread bacteria is consistent with the actin turnover rate in protrusions, which is much slower than that in comet tails. We suggest that intimate association of the bacterial comet tail with the host's membrane in these protrusions forces interactions between actin and other membrane-associated host proteins that stabilize the actin filaments associated with the bacterium. Characterization of such interactions is beyond the scope of this report, but this hypothesis is supported by previous data. Ultrastructural examination of cytoplasmic comet tails reveals short, randomly oriented actin filaments that are 0.2–0.3 μm long . However, tails in protrusions possess two filament populations; one is comprised of short, random filaments, the other is comprised of long (≥1 μm) filaments that are axially oriented along the long axis of the protrusion . This second population, which is not found in cytoplasmic tails, may be stabilized by its proximity to membrane-localized proteins responsible for forming cytoskeleton-membrane associations. Ezrin and radixin, members of a family of proteins responsible for stabilizing actin-membrane associations, have been localized to a subset of tails that may correspond to bacteria-containing protrusions . This may also explain the tendency of L . monocytogenes to occasionally slide back out of short extracellular protrusions in PtK2 cells which do not posses microvillus-like structures. We have never observed this behavior in MDCK cells, which exhibit many microvilli on their apical surface. It is possible that the presence of microvillus-stabilizing proteins in MDCK cells cause them to recognize the bacterial actin tail in close association with the membrane as a structure worthy of stabilization. PtK2 cells are less likely to express or positively regulate the proteins required for such stabilization. The composition and behavior of L . monocytogenes -containing protrusions could prove a useful probe of host actin-membrane interactions. Membrane-dependent stabilization of actin filaments along the length of the protrusion does not prevent further polymerization at the tip, which is apparently responsible for the fitful movement of extracellular and intercellular protrusions. Similar actin-dependent fitful movement has been described for dendritic spines in neurons . Recovery of motility after intercellular spread is not immediate for two reasons. First, the actin turnover rate is very low in clouds surrounding bacteria that have just spread into a neighboring cell (see above). More importantly, new actin polymerization is inhibited by the acid-dependent cleavage of ActA. Not only does the new bacterium need to replace its ActA, but it must polarize it. Other studies have reported that ActA is asymmetrically distributed in a gradient along the bacterial surface of bacteria . The simplest explanation is that this gradient is generated by bacterial elongation during division combined with a slow turnover rate of ActA of ∼3 h (Moors, M.A., and D.A. Portnoy, manuscript in preparation) that leaves the zone of septation relatively free of ActA. This explanation is consistent with our observations that in cells bacteria only begin moving immediately after division and that stopped bacteria will not start moving again unless they divide ( n > 100). Our data also suggest an explanation for the difference in tail acquisition time between primarily and secondarily infecting bacteria. Transcription of the actA gene followed by translation requires more time than just translation to recover sufficient surface ActA density for motility. However, this alone cannot account for the lag in tail recovery, since bacteria begin to nucleate actin at least one generation before they acquire tails . Thus we conclude that bacterial division acts to reorganize the symmetric actin cloud into an asymmetric structure capable of generating unidirectional force. Our work shows that epithelial cells do not actively internalize extracellular protruding membranes containing bacteria. Instead, cell to cell spread requires that a bacterium actively drive a membrane protrusion to intrude into the cytoplasm of the adjacent cell in order to trigger uptake. After that, the recipient cell takes over, with uptake occurring in two distinct phases, each considerably longer than classical mechanisms of phagocytosis, which normally requires only a few minutes. It is surprising that an epithelial cell should ingest a membrane-bound protrusion, considering that the only thing that the recipient cells should recognize is the surface of its neighbor. We cannot exclude the possibility of a bacterially produced presentation molecule that lines the external surface of the protrusion and directs the recipient cell to engulf it; however, there is no evidence for such a signal. If such a molecule existed, it would either require posttranslational modification and plasma membrane targeting by the host cell or a multicomponent bacterial secretory system which could insert the signal directly into the host cell membrane during bacterium-membrane contact. The former is unlikely, since it requires secretion in the protrusion but modification and targeting in the cell body, though it is possible the signal is produced in the cytoplasm and then targeted ubiquitously to the host membrane. The latter is unlikely since genetic screens have failed to find a signal, upstream regulatory components, or components of a secretory system with the required properties in L . monocytogenes . It is also possible that the bacterium modifies the interactions of host membrane proteins at the protrusion tip, however, this is unlikely since extracellular membrane-bound protrusions which are confined between cells are not taken up by their neighbors. Finally, unrelated bacterial pathogens, such as S . flexneri , also undergo actin-based motility to spread from cell to cell and no signal molecule has been found. It is possible that both bacteria evolved independent mechanisms for intercellular spread; however, the simpler explanation is that they are both taking advantage of the same natural host process. The engulfment and uptake of membrane from one cell into another, as described here for the lateral spread of bacteria, also occurs in several natural processes. One little-understood but well-known example is the formation of dendrites by melanophores to deliver melanin-containing vesicles to numerous nearby keratinocytes . Dendrite formation is actin-dependent , and the required molecules for melanin transfer are unknown. It is possible that this protrusive activity triggers uptake of proffered vesicles by keratinocytes much as L . monocytogenes protrusion triggers uptake by recipient cells. Another example of plasma membrane exchange includes a set of several cell signaling events in which a transmembrane ligand on the surface of one cell binds to a cognate receptor on an adjacent cell and is internalized. The Drosophila proteins boss and sevenless are internalized in this manner. The uptake of boss–sevenless complexes can be inhibited by mutations in the hook gene, a factor required by the recipient R7 cell of Drosophila ommatidium for such endocytosis , whose mutation also results in sharply bent bristles , a potential cytoskeletal defect. That gene also inhibits endocytosis of the transmembrane ligand delta , which binds notch . Multivesicular bodies in R7 cells costain for delta and boss , suggesting that this endocytic mechanism may depend only on either the boss/sevenless or delta/notch interaction, or neither. Similarly, the C . elegans Lag-2 transmembrane ligand, a homologue of delta , can be internalized into adjacent cells via interaction with its receptor Glp-1 . Although none of these studies has demonstrated uptake of membrane phospholipids across cell borders, we have shown that both membrane proteins labeled on the surface with TRITC and the lipophilic dye DiO are found inside adjacent, unlabeled cells in membrane-bounded compartments indicating constitutive, albeit rare, engulfment of membranes by adjacent cells. We also note that epithelial cells frequently take on the role of semi-professional phagocytes to digest their apoptosing neighbors, thereby eliminating the need for macrophages to perform this function . Very little is known about semi-professional phagocytosis by epithelial cells . At least six genes critical for this process have been identified in C . elegans ; three of these mutants also manifest defects in cell migration . One of these genes, ced-5 , has been demonstrated to function in extending the membrane of the engulfing cell over the apoptotic cell , and it is a homologue of DOCK180, a Drosophila melanogaster protein that has been implicated in cell motility and integrin-mediated signaling. When recruited to the membrane, DOCK180 induces cell spreading and, in the presence of EGF, causes cells to take on a highly protrusive, dendritic morphology . This link between protrusive cytoskeletal activity and the natural process of neighbor consumption of apoptotic cells underscores the possibility that L . monocytogenes intercellular spread could co-opt a natural host process, inducing uptake simply by initiating an actin-stabilized protrusion that intrudes into the cytoplasmic space of a neighboring cell. The type of membrane engulfment observed in these genetically characterized pigment delivery, signaling, and apoptotic systems is a normal feature of epithelial cell biology, and we suggest the descriptive term paracytophagy (eating of nearby cells) for this widespread behavior. L . monocytogenes , an excellent bacterial cell biologist, appears to have exploited the tendency of epithelial cells to engulf bits of their neighbors to enable its efficient intercellular spread. Video microscopy has enabled us to observe the dynamic physical and temporal constraints under which this host-pathogen relationship has evolved. | Other | biomedical | en | 0.999996 |
10491396 | Wild-type Psn full-length cDNAs ( Psn+14 and Psn − 14 variants) were cloned into the pUAST vector as EcoRI–XbaI fragments. Four missense mutation constructs were generated by standard PCR-based site-directed mutagenesis. The mutated Psn cDNAs were cloned into pBS-SK and confirmed by sequencing, then inserted into the pUAST vector downstream of the UAS regulatory region. The D-ALG3 and loop truncation constructs were generated by the same strategy after PCR with the following primer pairs: D-ALG3, 5′-CAAGAGTGGTCAGAATTCAAAATGGAACGTGTGGC-3′, 5′-TACTGTAAGACTCTAGATGTGTCCTTG-3′; Loop, 5′-TCTATTTGGGAATTCAAAATGGTCCTTTCGCC-3′, 5′-GGCCACAAAGCTCTAGATTTAGGTCGTCCAGTC-3′. pHS-GV was made from N + -GV3 by PCR with the primer pair: 5′-GAAAGCGGTCGCGGCCGCCAAAATGAAGCTTCTGTC-3′, 5′-AACCCCCGATATCTCACCCACCAAAGTCGTC-3′ and inserted into the NotI/StuI sites of pCaSpeR-hs. To produce transgenic flies, each construct was injected into w 1118 embryos as described in Spradling and Rubin 1982 . Fly culture and crosses were carried out according to standard procedures. To generate N ts1 ; vg (quadrant enhancer) -lacZ or N ts1 ; ac-lacZ larvae, homozygous N ts1 females collected at the permissive temperature (18°C) were crossed to homozygous vg (quadrant enhancer) -lacZ flies or ac-lacZ flies . Hemizygous N ts1 male progeny from these crosses were mated to homozygous N ts1 females to generate N ts1 mutant larvae with appropriate lacZ markers. These crosses were kept at the nonpermissive temperature (29°C) for different time periods before larvae were analyzed by acridine orange staining and β-galactosidase staining. For scanning electron microscopy (SEM), adult flies were dehydrated sequentially in 25, 50, 75, and 100% ethanol for >12 h each, and two 100% ethanol steps before critical point drying with hexamethyldisilazane (Electron Microscopy Sciences, Inc.). The flies were then mounted on stubs, sputter coated with gold, and imaged using a JEOL 6400 scanning electron microscope. Plastic sections were prepared as described in Tomlinson and Ready 1987 . Cobalt sulfide staining of pupal retinae was done as described in Wolff and Ready 1991 . Acridine orange stainings were done by dissecting eye imaginal discs in Ringer's solution, placing the tissue in 0.2 mg/ml acridine orange in Ringer's solution for 4 min, and mounting the discs in Ringer's solution for immediate fluorescence photomicroscopy . β-galactosidase detection was performed as described in Ye et al. 1999 . Immunostainings were done as described in Ye and Fortini 1998 , using the following primary antibodies: rat anti-ELAV mAb 7E8A10, 1:200 dilution ; mouse anti-Psn Ab L7, 1:100 dilution . Adult wings were removed and mounted in DPX mountant (Electron Microscopy Sciences, Inc.) for photomicroscopy. TUNEL assays were performed using the Oncor S7110 kit. In brief, imaginal discs were fixed with 2% paraformaldehyde for 20 min and washed in PBS-DT (1× PBS, 0.3% Triton X-100, 0.3% deoxycholate) for 20 min on ice. Discs were then washed with PBS four times for 5 min each, and then transferred to equilibration buffer for 5 min, followed by working-strength reaction buffer containing terminal deoxynucleotide transferase. The discs were incubated with the enzyme in a 96-well plate in a humidified chamber at 37°C for 1.5 h. The reaction was stopped by transferring the discs into working-strength stop/wash buffer for 10 min, and discs were then rinsed in three changes of PBS, followed by staining with either working-strength antidigoxigenin–fluorescein solution or antidigoxigenin–AP solution (Boehringer Mannheim Corp.) for 45 min and two more PBS washes for 10 min each. Discs were then immunoreacted with mouse anti-Psn Ab L7, 1:100 dilution, as described above or developed following the S7110 kit instructions. Drosophila S2 cell transfections, Western immunoblot analysis, and antibody stainings were performed as described in Fehon et al. 1990 and Ye et al. 1999 , using Notch construct pMTNMg and Notch mAbs C17.9C6 and C458.2H , Myc mAb 1-9E10.2, and β-tubulin mAb E7 (University of Iowa Developmental Studies Hybridoma Bank). In view of the extensive neuronal cell death seen in Alzheimer's disease, and the increased susceptibility of cultured cells to apoptotic stimuli caused by overexpression of normal and mutant variants of mammalian PS proteins, we sought to determine whether presenilin is associated with apoptosis in a genetically tractable experimental organism. We therefore produced loss-of-function genetic lesions in the Drosophila Presenilin ( Psn ) gene and analyzed patterns of cell death in developing tissues of the mutant animals. Imaginal discs from the Psn mutant larvae are considerably smaller than age-matched wild-type control discs, and Psn mutant eye discs lack almost all differentiating neurons posterior to the morphogenetic furrow . To determine whether this neuronal cell loss occurs by apoptosis, we examined third-instar Psn mutant imaginal discs using the TUNEL assay for apoptotic nuclear fragments . A dramatic increase in the number of apoptotic cell bodies is readily detected in the Psn mutant eye discs . Similarly high levels of apoptosis are observed in other imaginal tissues, including the wing and leg discs, when presenilin function is eliminated (data not shown). Our data are consistent with a role for presenilin in either the regulation of apoptosis itself, or in developmental patterning events that lead to high levels of apoptosis when they are not executed properly. To investigate further the ability of presenilin proteins to influence apoptotic events in the context of a multicellular organism, we overexpressed Drosophila Psn in transgenic fly tissues and compared the results to analogous experiments that have already been performed exclusively in mammalian cultured cells. The Drosophila Psn gene undergoes alternative splicing to generate two protein isoforms, one of which contains an additional 14 amino acids in the hydrophilic loop domain . We ectopically expressed both the short wild-type isoform (termed Psn−14) and the long wild-type isoform (termed Psn+14), as well as four different Alzheimer's disease-linked missense mutations and three truncated forms of fly presenilin in third-instar larval eye discs using the GAL4-UAS system . As driver constructs, we used sev-GAL4 , which expresses GAL4 under the control of the sevenless promoter, and GMR-GAL4 , in which GAL4 is downstream of a multimerized copy of the binding site for the Glass transcription factor. These regulatory sequences are active in different neuronal subsets of the immature retina and they have been used extensively in studies of cell death in the eye . The sevenless gene is strongly expressed in the R3, R4, and R7 photoreceptor cell precursors and cone cell precursors, while GMR is more generally expressed in all cells posterior to the morphogenetic furrow . The expression of Psn constructs in eye discs was confirmed by antibody staining, excluding D-ALG3 , which does not encode epitopes recognized by available Psn antibodies . Flies bearing one copy of UAS-Psn + 14 or UAS-Psn − 14 , together with either sev-GAL4 or GMR-GAL4 , are morphologically indistinguishable from wild-type, as are flies bearing the GAL4 driver constructs alone . At 25°C, two copies of UAS-Psn+14 (termed 2X UAS-Psn+14 ) with one copy of sev-GAL4 produce a rough eye phenotype, consisting of irregular ommatidial packing, occasional ommatidial fusions, and missing bristles . Tangential sections of these eyes revealed that pigment cells are missing while the photoreceptor cell array is largely normal . In general, similar results were obtained with UAS-Psn+14 and UAS-Psn − 14 , so UAS-Psn+14 was used for subsequent experiments, unless noted otherwise. Two copies of UAS-Psn+14 driven by one copy of GMR-GAL4 result in a stronger rough eye phenotype characterized by fusion of the lens material of adjacent ommatidia and loss of interommatidial bristles, producing a glossy external eye surface . A similar glossy eye phenotype is caused by facet alleles of the Notch gene, and is due to a specific defect in primary pigment cell development . Most pigment cells are missing in GMR-GAL4, 2X UAS-Psn+14 retinae, resulting in a nearly complete absence of the pigment cell lattice between photoreceptor cell arrays of different ommatidia . Cobalt staining of retinae from pupae shows that almost all ommatidia have the normal number of cone cells, but display missing primary, secondary, and tertiary pigment cells, consistent with the adult eye phenotype . To test whether the two alternatively spliced Psn variants are functionally equivalent or antagonistic, we generated flies bearing two copies of UAS-Psn+14 and one copy of UAS-Psn − 14 in addition to GMR-GAL4 . The rough eye phenotype caused by two copies of UAS-Psn+14 alone is strongly enhanced by one copy of the short form of Psn. The lens surfaces are completely fused together into a smooth glossy sheet that is almost devoid of interommatidial bristles . These data suggest that the long and short Psn protein isoforms function in a similar manner in this assay. Although the sevenless promoter is not active in pigment cells and bristle precursor cells, these two types of cells are the major ones that are affected in our transgenic lines expressing Psn under sev-GAL4 control. One possible explanation for this discrepancy is that Psn overexpression may decrease the number of cells available for eye patterning by affecting cell proliferation or cell death. In this case, cells recruited during the later stages of ommatidial assembly, such as pigment cells and bristle cell groups, would be disproportionately affected because all cell types of the adult eye are recruited from a common pool of uncommitted progenitor cells in the third-instar larval eye disc . Given the apoptotic effects of PS1 and PS2 in cultured mammalian cells, we examined programmed cell death in third-instar larval eye discs using acridine orange staining, which specifically labels apoptotic cells, but not necrotic cells, in Drosophila . Although a low level of cell death is normally seen posterior to the morphogenetic furrow of the eye disc, a significant increase in apoptosis is consistently observed in discs carrying two copies of UAS-Psn+14 and either the sev-GAL4 or GMR-GAL4 transgene . Since increased levels of apoptosis are also a feature of the Drosophila Psn gene loss-of-function mutant phenotype, our data raise the possibility that the proapoptotic effects of mammalian PS proteins may actually represent a partial loss of Psn function due to protein oligomerization and aggregation when ectopic Psn is expressed at a very high level . To determine if Psn-induced apoptosis is cell autonomous, we analyzed third instar eye imaginal discs of genotype sev-GAL4, UAS-Psn+14 with TUNEL labeling in conjunction with Psn antibody staining. The sev promoter is strongly active in only a subset of cells at this stage of eye development, namely the R3, R4, and R7 photoreceptor cell precursors, the cone cell precursors, and one or two so-called mystery cells that are transiently associated with the five-cell ommatidial precluster. The ability of these precursor cells to be identified solely on the basis of their positions in nascent ommatidia enabled us to directly correlate TUNEL-positive apoptotic cell nuclei with the corresponding cell bodies expressing high levels of Psn . Psn is distributed throughout the cytoplasm, but is excluded from the nucleus . The TUNEL method, however, specifically stains fragmented DNA within nuclei of apoptotic cells and thus labels a different subcellular compartment than the Psn antibody staining. Nevertheless, careful analysis of nearby optical sections using confocal microscopy clearly reveals that both TUNEL and Psn antibodies label numerous identical cells in all the discs analyzed , confirming that the Psn-induced cell death is a cell-autonomous process and that only a minority of cells overexpressing Psn undergo apoptosis. We also noticed that the assembly of the photoreceptor cell clusters is largely unperturbed, despite the increase in cell death, as the normal mirror symmetric patterning of R3 and R4 photoreceptor cell precursors along the eye equator can still be visualized by Psn antibody staining . It is apparent that the apoptotic cells are rapidly removed from photoreceptor cell precursor clusters and replaced with neighboring cells that generally are not eliminated by apoptosis. Although the Alzheimer's disease-linked PS mutations are not complete loss-of-function alleles , it is not clear if they are partial loss-of-function or gain-of-function mutations. All Alzheimer's disease-linked mutations occur at amino acid residues that are conserved in the Drosophila Psn protein, allowing us to introduce these mutations into the fly protein and assess their apoptotic effects in transgenic animals. Four missense mutations, N141I, M146V, L235P, and E280A were tested in this manner. The N141I mutant of PS2 has been shown to induce more apoptosis in certain cell types, compared with wild-type PS2, and may thus represent a gain-of-function mutation . Both the M146V and E280A mutants of PS1 increase the ratio of Aβ42/Aβ40 by increasing the level of the more neurotoxic and amyloidogenic Aβ42 cleavage product of amyloid precursor protein . The L235P mutation was identified in a family with an onset of Alzheimer's disease as early as age 29 , and may therefore represent a particularly severe mutant form of presenilin. In addition, a truncated Psn protein named D-ALG3, consisting of the most COOH-terminal 100 amino acids, two loop constructs consisting of either the long or short variable hydrophilic loop, and transmembrane domain 7 (TM7) following the loop were also included in our analysis. These constructs were chosen because D-ALG3 resembles truncated mammalian PS proteins that confer resistance to cell death in PC12 cells , and the loop may be a cytoplasmic domain required for protein–protein interactions. The apoptotic activities of mutant presenilins were assessed by the following criteria: their ability to generate a rough eye phenotype in flies bearing two copies of each mutant transgene and GMR-GAL4 ; their ability to modify the eye phenotype of GMR-GAL4, 2X UAS-Psn+14 flies; and their ability to modify the eye phenotype of flies expressing the Drosophila death-domain protein Reaper under GMR promoter control ( Table ). At least five independent transgenic lines were analyzed for each construct. Only the M146V substitution produces a rough eye phenotype in two independent transgenic lines out of five analyzed. The remaining mutations and Psn fragments fail to produce rough eye phenotypes when expressed under GMR-GAL4 control, but all four Alzheimer's disease-associated mutants enhance the rough eye phenotype of GMR-GAL4, 2X UAS-Psn+14 flies, and M146V and N141I also enhance the phenotype of flies bearing GMR-reaper . Although the ALG-3 fragment of PS2 inhibits programmed cell death in PC12 cells, the equivalent segment of Drosophila Psn, D-ALG3, instead weakly enhances the GMR-GAL4, 2X UAS-Psn+14 phenotype, indicating that it may possess weak apoptotic activity. The two loop variant fragments possess no modifying activity in these transgenic assays. To gain further insight into the mechanism of Psn-induced cell death, we tried to suppress this apoptosis by coexpression of Drosophila cell death inhibitors (DIAP1 or DIAP2) or the baculoviral survival factor p35. DIAP1 and DIAP2 are Drosophila homologues of baculoviral inhibitor of apoptosis proteins (IAPs), and can block programmed cell death induced by proapoptotic factors or mutations . Baculoviral p35 protein inhibits caspases and thus blocks apoptosis in many species . In the presence of any of these antiapoptotic proteins, the Psn-induced rough eye phenotype is largely suppressed, as revealed by the more regular external eye surface, the more normal trapezoidal pattern and orientation of R1-6 photoreceptor cells, and a restoration of the pigment cell lattice between ommatidia . The observation that Psn-induced rough eye phenotypes are suppressed by coexpressing either DIAP1, DIAP2, or p35 confirms that increased cell death is the main cause of the rough eye phenotype. Psn-induced programmed cell death may thus be mediated by the conserved caspase pathway of apoptosis or, alternatively, it may be circumvented by inhibition of this pathway. The ability of wild-type and, to a lesser extent, mutant forms of Drosophila Psn to induce low levels of apoptosis similar to that seen in developing imaginal tissues of Psn loss-of-function mutants suggests that the apoptotic effects of Psn may be a secondary consequence of reduced or dominant-negative Psn activity during developmental patterning. In C . elegans and mice, presenilin proteins have been shown to facilitate Notch signaling, and worms or mice lacking presenilin activity display typical Notch or lin-12/glp-1 loss-of-function phenotypes . Similarly, flies lacking functional Psn gene activity exhibit embryonic neurogenic phenotypes and imaginal disc phenotypes that are characteristic of impaired Notch signaling . Moreover, elevated levels of apoptosis have been noted previously in wing imaginal discs of flies having the partial loss-of-function heteroallelic Notch genotype N ts /N 55e11 ; neur A101 / + . These observations raise the possibility that the apoptotic effects of PS overexpression may be due to a primary interference with Notch signaling, followed by elimination of cells that have not adopted their proper cell fate by a normal corrective mechanism of developmentally controlled apoptosis . Our in vivo genetic model for Psn-mediated apoptosis allowed us to examine the potential involvement of Notch signaling in the apoptotic response, an important issue that has not been possible to assess in the widely used mammalian cell culture assays for Psn-induced apoptosis. First, we wished to determine if the UAS-Psn constructs that cause apoptosis in the Drosophila eye when driven by GMR-GAL4 are able to produce Notch pathway phenotypes in other tissues when expressed using suitable GAL4 driver constructs. We found that several GAL4 driver lines that are active in the wing and cuticle anlagen are indeed capable of producing adult Notch -like phenotypes in the wing blade and thorax, including wing margin notching, vein thickening, ectopic wing margin bristles, ectopic wing vein campaniform sensilla, ectopic thoracic macrochaetae, and missing thoracic microchaetae . These phenotypes are consistent with the notion that Psn overexpression leads to dominant-negative effects, since Psn loss-of-function mutants exhibit similar Notch -like phenotypes . To determine if Psn-induced apoptosis might be an indirect effect of reduced Notch activity, we first analyzed apoptosis in imaginal wing discs of the conditional temperature-sensitive Notch mutant N ts1 . Increased levels of programmed cell death are spatiotemporally correlated with progressive loss of Notch activity as visualized by reduced wing-pouch–specific expression of the Notch target gene reporter vg (quadrant enhancer)- lacZ and expansion of proneural cell clusters positive for ac-lacZ expression in the presumptive notum region , suggesting that developmental patterning defects caused by reducing Notch activity directly lead to elimination of affected cells by apoptosis . Second, we tested whether reduction in the dosage of the wild-type Notch gene or coexpression of constitutively activated Notch is able to suppress or enhance Psn-related apoptotic phenotypes. We found that the rough eye phenotype of GMR-GAL4, 2X UAS-Psn+14 is strongly enhanced in an N 54l9 mutant background bearing only one functional copy of the Notch gene . Apoptosis caused by either Psn overexpression or removal of Psn gene function is also dramatically suppressed by coexpression of constitutively activated Notch in the retina . These studies show that genetic removal or overexpression of Psn in developing Drosophila tissues is able to induce Notch -like phenotypes, as well as apoptosis, and that when genetic methods are used to compensate for effects on Notch signaling, the levels of apoptosis are dramatically reduced. Our results, together with the observed correlation between impaired Notch signaling and high levels of developmental apoptosis, offer a potential explanation of Psn-mediated apoptosis as a developmental response to a primary failure in cellular patterning events requiring Psn activity for proper Notch synthesis or signaling. To further elucidate the molecular mechanism underlying the Psn overexpression phenotypes, we analyzed Notch processing and trafficking in S2 cells cotransfected with Psn and Notch. When expressed before Notch induction, wild-type Psn and the various Psn mutants lead to reduced Notch protein levels, affecting both the full-length and the processed COOH-terminal fragments of Notch . In agreement with our genetic results, the biochemical effect appears to be more pronounced for wild-type Psn than for the mutant forms. The expression of a nonmembrane-bound control protein, Suppressor of Hairless, is not affected by either the wild-type or the mutant Psn proteins . The effect on Notch synthesis is unlikely to be due to increased protein degradation, because ectopic expression of the same Psn construct in S2 cells after Notch induction has no detectable effect on Notch protein levels (data not shown). In addition, live cell surface immunostainings reveal that Notch protein is trafficked and inserted into the cell membrane normally in spite of the reduced protein levels caused by Psn overexpression . In mice, removal of PS1 results in a severe loss of neural progenitor cells in specific regions of the brain, suggesting that PS1 may normally play a neuroprotective role during neurogenesis . However, other studies have shown that overexpression of PS1 and PS2 in mammalian cell lines sensitizes the cells to apoptosis induced by trophic factor withdrawal or Aβ peptide, indicating that presenilin may promote cell death triggered by certain stimuli . In addition, COOH-terminal fragments of PS1 and PS2 are reported to have antiapoptotic activity in similar assays , and contradictory data exist concerning the proapoptotic effects of wild-type and Alzheimer's disease-associated mutant variants of PS1 . We therefore sought to analyze the role of presenilin in promoting or preventing apoptosis in a multicellular experimental organism using both classical genetics and a transgenic approach that closely parallels the overexpression strategies used in the mammalian cell culture studies. Using several loss-of-function lesions in the endogenous Drosophila Presenilin gene, we showed that mutant tissues lacking Psn activity display elevated levels of developmental apoptosis. Imaginal tissues are considerably smaller than age-matched wild-type control tissues, suggesting that apoptosis plays a significant role in reducing cell numbers in the Psn mutants. High levels of apoptosis are correlated with selective loss of photoreceptor neurons in imaginal eye discs of the mutants, consistent with suggestions that mammalian PS proteins may normally function as antiapoptotic factors required to protect neurons from degeneration. However, it is worth noting that apoptotic bodies are also readily detected in other cell types of the Psn mutants, indicating that the antiapoptotic effects are not limited to neuronal tissues in Drosophila . Our loss-of-function genetic analysis is in agreement with a recent study showing that antisense inhibition of PS1 in mammalian cell culture blocks neuronal differentiation and induces apoptosis . In our transgenic overexpression model, we find that wild-type Psn is most efficient at inducing apoptosis. Since Psn overexpression causes phenotypes that resemble Psn loss-of-function mutant apoptotic phenotypes, the proapoptotic activity of overexpressed Psn may actually be due to dominant-negative effects. Perhaps cells overexpressing Psn undergo apoptosis not because of a specific regulatory role of Psn in apoptosis, but rather due to a blockage of essential intracellular protein transport pathways by overaccumulation of Psn in the ER and Golgi compartments. Alternatively, overexpression of Psn may interfere with endogenous Psn function, resulting in phenotypes resembling those of Psn loss-of-function mutants. It has been shown previously that both NH 2 - and COOH-terminal PS1 fragments form oligomers in vitro . It is therefore possible that overexpression of wild-type Psn in vivo may promote excess or premature Psn oligomerization, resulting in the formation of nonfunctional complexes that deplete endogenous functional Psn within the cell or disrupt ER/Golgi compartment function. Our results suggest that the apoptotic effects of presenilin proteins may involve their well-documented role in Notch signaling . In C . elegans , the presenilin homologue Sel-12 is needed for normal cell-surface accumulation of the Lin-12 protein, a member of the Notch receptor family , and in Drosophila and mammals, presenilin is required for normal proteolytic processing of Notch during receptor assembly or ligand-induced signaling . We show here that the apoptotic effects of fly Psn are strongly dependent upon Notch gene dosage, and that the effects can be partially suppressed by supplying flies with constitutively activated Notch protein. Moreover, when Psn overexpression is restricted to certain developing structures, we observe typical Notch -like wing and neurogenic phenotypes. Based on these results, we suggest that the apoptotic effects of Psn overexpression may be a secondary consequence of impaired Notch signaling, perhaps due to a dominant-negative effect that interferes with Notch activity, resulting in cells that are developmentally misprogrammed and thus selectively eliminated by a normal corrective mechanism of apoptosis. This interpretation is consistent with the observation that increased amounts of cell death are correlated with reduced Notch signaling . As discussed above, previous studies have shown that Psn protein self-aggregates under some conditions ; similar aggregation might interfere with ER/Golgi compartment function in vivo. Our finding that overexpression of Psn in S2 cells leads to reduced Notch protein levels is consistent with this notion and provides a plausible molecular explanation for the Psn overexpression apoptotic and neurogenic phenotypes. This dominant-negative effect on Notch synthesis is distinct from the effect of Psn loss-of-function mutations, which cause overaccumulation of specific ∼120-kD Notch COOH-terminal fragments at the expense of other fragments . We have assessed the proapoptotic activity of different Alzheimer's disease-linked mutant variants and found that they possess less apoptotic activity than wild-type presenilin, consistent with the idea that this class of presenilin mutations represents partial loss-of-function mutations in nematodes and transgenic mice . In contrast to previous studies showing that Alzheimer's disease-associated missense mutations enhance the ability of PS1 and PS2 to induce cell death and that the COOH-terminal ALG-3 fragment blocks the proapoptotic activity of PS2 , the corresponding missense mutants of fly Psn induce less cell death than wild-type Psn, and the Drosophila Psn COOH-terminal D-ALG3 fragment appears to induce apoptosis only weakly in vivo. In view of the dominant-negative role that we suggest for overexpressed Psn, the reduced apoptotic activity of these mutant variants may reflect a decreased ability to interfere with endogenous presenilin function or a more rapid clearance of misfolded mutant proteins from the ER/Golgi compartment. Analogous partial loss-of-function effects on Aβ secretion of human mutant presenilins may be relatively weak under physiological conditions, but may contribute to gradual deterioration of brain tissue during the aging process and eventually trigger disease onset when neuronal loss reaches a certain threshold. Using genetic tools developed by others for the analysis of apoptosis in the Drosophila eye , we have shown that the rough eye phenotypes associated with Psn overexpression are partially suppressed when DIAPs or baculoviral p35 is coexpressed using the same promoter. This result confirms that apoptosis induced by overexpression of Psn is a major cause of the final adult eye phenotype, regardless of its developmental origins. In addition, the Psn effects are most likely to occur at a step upstream of the conserved caspase effector pathway of apoptosis or via a parallel pathway, since Psn-induced cell death is inhibited by p35, which antagonizes caspase activity, and DIAPs, which may block the ability of the Drosophila Reaper protein to activate the caspase effector cascade . It has been previously demonstrated that cells rescued from apoptosis can be physiologically functional, since blocking cell death by inhibiting caspase activity prevents blindness in Drosophila neurodegeneration mutants . In this regard, our results support the notion that preventing neuronal apoptosis may be a useful therapeutic strategy for counteracting the neurodegeneration associated with Alzheimer's disease. In summary, we demonstrate that both loss of Drosophila Psn activity and overexpression of Psn in the developing fly retina induce programmed cell death in a cell autonomous manner. The apoptotic effects of Psn may result from dominant-negative effects of the overexpressed presenilin, and are likely to be a consequence of interfering with Notch-mediated developmental signaling by impeding Notch synthesis. Apoptotic effects of fly Psn, and perhaps also those caused by mammalian PS proteins, may thus reflect the normal elimination of aberrant cells generated by a primary failure in intercellular communication and cell-fate specification. In addition, we demonstrate that Alzheimer's disease-linked Presenilin mutations may be partial loss-of-function mutations using different genetic criteria. Furthermore, Psn-induced rough eye phenotypes are highly sensitive to transgene dosages and modifying effects of other genes, including evolutionarily conserved components of the caspase-mediated apoptosis pathway. Psn transgenic flies may therefore be a useful genetic tool to dissect Psn function by performing genetic modifier screens for additional factors that interact with presenilin. | Study | biomedical | en | 0.999995 |
10491397 | Newborn (P0) and 4-d-old (P4) Sprague Dawley rat pups were anesthetized with methoxyflurane, the left eye was gently raised with forceps, and the optic nerve was transected with microscissors between the retinal excavation and lamina cribrosa. Alternatively, the left cornea of P0 rats was cut, the vitreous body and the lens removed, and the retina was mechanically ablated. The right optic nerves served as nonoperated controls. Rat pups (P0–P7) were anesthetized as described above and perfused through the heart with 4% paraformaldehyde in 0.08 M Sorensen's phosphate buffer (pH 7.6). The optic nerves were removed and immersed in the fixative (3 d, 4°C). Longitudinal, free-floating sections (20-μm thick) were cut on a freezing, sliding microtome. Nerves used for identification of oligodendrocytes were microwaved in citrate buffer (pH 6.0, three times for 3 min) before sectioning. Sections were placed in PBS containing 10% Triton X-100 and 1% H 2 O 2 (30 min), and incubated in PBS containing 3% normal goat serum (30 min). Sections were exposed to primary antibodies at 4°C (NG2 antibodies overnight, myelin basic protein [MBP] or activated caspase-3 antibodies for 5 d). The sections were then placed in biotinylated goat anti–rabbit Ig or goat anti–mouse Ig (60 min), and peroxidase-conjugated avidin (60 min). Immunoperoxidase staining was developed by metal enhanced diaminobenzidine (Pierce). The sections were treated with 0.04% osmium tetroxide (20 s), mounted in glycerol, and examined with a Zeiss Axiophot microscope. Progenitor cell density was quantified by counting NG2-labeled cell bodies in optic nerves from P0-transected and control optic nerves at P1, P2, P3, and P4 using a 40× objective lens, a rectile (10 × 10 grid), and Zeiss Axiophot microscope. From four to five control and transected optic nerves at each age, two to three longitudinal sections of the entire optic nerve were examined from the chiasm to the lamina cribrosa. The number of OPCs per grid area was determined and the total area analyzed in each section was calculated. The optic nerve sections were divided into three equally sized segments (chiasmal, mid, and retinal portions). For each portion, total areas and OPC numbers were calculated and expressed as OPCs/mm 2 tissue. Explants of isolated retinal, chiasmal, and intermediate regions of rat optic nerves were prepared from P0 and P4 rats as described previously . Explants were grown in collagen gels for at least 7 d and labeled with mAb O4 according to published protocols . The number of explants from each region of the nerve that contained oligodendrocytes at the end of the study was recorded, and the data from at least three separate experiments were pooled. To investigate the effect of nerve transection on progenitor cell process extension, the length of DAB-stained, bipolar NG2 cells was measured in P0-transected ( n = 26 cells) and control ( n = 44 cells) optic nerves at P1. Optic nerves were transected at P0 and the rat pups were killed at P2, 90 min after injection with bromodeoxyuridine (BrdU) (i.p.; 0.1 mg/g body weight) (Boehringer Mannheim). Sections were cut as described above and pretreated with 10% Triton X-100 in PBS (30 min), 2 N HCl (10 min), 4% sodium borate (10 min), and 3% goat serum in PBS (30 min). The sections were incubated with NG2 and BrdU antibodies overnight at 4°C, and were then exposed to biotinylated goat anti–rabbit Ig and Texas red–conjugated donkey anti–rat Ig (60 min), and FITC-conjugated avidin (60 min), and examined in a confocal microscope (Leica TCS-NT). To determine the effect of nerve transection on progenitor proliferation, the percentage of NG2-positive cells that were double-labeled by BrdU was compared in two to three sections from P0-transected ( n = 4) and control ( n = 4) optic nerves at P2. To investigate the effect of nerve transection on oligodendrocyte density, MBP-labeled cells were quantified in sections of P4-transected and control optic nerves at P5, P6, and P7, as well as in P0 retina-ablated and control optic nerves at P7. Two to three sections from four to five control and P4 retinal-ablated optic nerves were analyzed at each age. Total areas of optic nerve sections analyzed were determined as described above, and oligodendrocyte density was expressed as cells/mm 2 of tissue. Retinal regions of P4, P5, and P6 nerves that contained <10 oligodendrocytes per 40× field were excluded from the analysis. To examine the effect of axonal transection on oligodendrocyte process formation, the longest radial diameter of premyelinating oligodendrocyte cell bodies and associated processes was measured in sections from P4-transected ( n = 90 cells) and control ( n = 52 cells) optic nerves at P6. Sections were pretreated as described above for fluorescence immunostaining, incubated with proteolipid protein (5 d at 4°C), biotinylated goat anti–rat Ig (60 min), and FITC-conjugated avidin (60 min), and then examined in a confocal microscope. Analysis was restricted to cells that occupied clearly defined boundaries. Sections were pretreated as described for immunoperoxidase staining above and incubated with MBP and activated caspase-3 antibodies (5 d at 4°C). The sections were then treated with biotinylated goat anti–mouse Ig and FITC-conjugated donkey anti–rabbit Ig (1 h), and then with Texas red–conjugated avidin (1 h) and the nucleic acid binding dye propidium iodide. The preparations were examined in a confocal microscope as described above. To quantify the density of dying oligodendrocytes, the number of activated caspase-3–positive oligodendrocytes was counted in P4-transected and control optic nerves at P5, P6, and P7. Two to three sections from four to five control and P4-transected nerves were examined at each age. Only activated caspase-3–labeled cells with a clear oligodendrocyte-like shape or process morphology were counted. Optic nerve section area and dying oligodendrocyte density were determined as described above. Retinal areas of nerve sections not containing oligodendrocytes were excluded from the analysis. Primary antibodies are well-characterized and were used at the following concentrations: rabbit polyclonal NG2 antibody, 1:15,000 ; mouse monoclonal MBP antibody, 1:8,000 (SMI-99; Sternberger Monoclonals, Inc.); rat monoclonal PLP antibody, 1:250 (Agmed, Inc.); rat monoclonal BrdU antibody, 1:5,000 (Harlan Bio); and rabbit polyclonal activated caspase-3 (CM1) antibody, 1:20,000 (Idun Pharmaceuticals, Inc.). Secondary antibodies were purchased from Vector Laboratories. Unpaired t test was used for statistical analysis of progenitor proliferation indices, progenitor cell length, radial diameter of premyelinating oligodendrocytes, and oligodendrocyte density in P0 retina-ablated nerves. Analysis of variance was used for statistical comparison of progenitor cell density, oligodendrocyte density, and dying oligodendrocyte density in controls and transected optic nerves. The data were expressed as means ± SD. To verify that all axons degenerate after P0 retinal ablation and P4 transection, optic nerves were examined by electron microscopy. Retinal ablation and optic nerve transection were performed as described above, and the pups were anesthetized and perfused through the heart with 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.08 M Sorenson's buffer at 2, 14, 24, 48, and 96 h after treatment. Optic nerves were removed, immersed in the fixative (3 d at 4°C), postfixed with 1% osmium tetroxide (1 h), dehydrated, and embedded in Epon. Ultrathin sections were cut on an ultramicrotome (Ultracut E; Reichert), collected on Formvar-coated slot grids, counterstained with uranyl acetate and lead citrate, and examined in a Philips CM-100 electron microscope. Midportions of three optic nerves were examined at each time point. Previous in vitro studies suggest that OPCs enter the chiasmal end of the rat optic nerve before birth, and populate the entire nerve by P9 . However, direct demonstration of the distribution and morphology of OPCs in the intact and transected nerve has not been described. Therefore, sections of developing and transected optic nerve were labeled with antibodies specific for NG2, a sulfated proteoglycan that colocalizes with the PDGF receptor on OPCs in vitro and in vivo . Consistent with in vitro studies , NG2 antibodies progressively stained more optic nerve cells in a chiasmal-to-retinal gradient between P0 and P4. Most NG2-positive cells were elongated or stellate-shaped . Elongated cells were enriched at the migratory front of the NG2 cell population, always oriented parallel to developing axons, and their processes often terminated in growth cone–like extensions. The densities of elongated and stellate-shaped NG2 cells were quantified in chiasmal, mid, and retinal thirds of P0–P4 optic nerves . Collectively, there was a greater than sixfold increase in the density of NG2 cells between P0 and P4. In a chiasmal-to-retinal gradient along the developing optic nerve, elongated cells were detected before stellate cells, suggesting that the elongated cells were more immature and migratory. Elongated cells reached an optimal density (∼200–300 cells/mm 2 ) in all three regions of the optic nerve at P3. Between P2 and P4, the density of stellate-shaped cells progressively increased at all levels of the nerve, suggesting a transition from elongate to stellate morphology during maturation. To confirm that the distribution of NG2-positive cells accurately represented the location of oligodendrocyte precursors in the postnatal nerve, isolated explants of chiasmal, mid, and retinal thirds of P0 and P4 nerves were grown in vitro for 7 d and assayed for O4-positive cells. O4 recognizes sulphatide and other minor lipid components expressed by oligodendrocytes and some oligodendrocyte progenitors . Greater than 96% (34/35) of chiasmal-derived explant contained O4-positive cells at both ages. In contrast, 44% (13/29) of mid and 25% of retinal (14/56) explants contained O4-positive cells in P0 explants, whereas ∼80% of mid (10/12) and retinal (11/13) explants contained O4-positive cells in P4 explants. These data provide independent support for the migration of NG2-positive oligodendrocyte precursors along the postnatal optic nerve. To investigate whether the absence of axons influenced the developmental appearance, morphology, or density of OPCs, optic nerves were transected near the retina at P0 and analyzed at P1, P2, P3, and P4. The general distribution of NG2 cells was similar in sections from control and transected optic nerves. However, the morphology of NG2 cells was affected by nerve transection. Although NG2 cells could still be divided into two general morphologies, elongated and stellate , the processes of OPCs were generally shorter than those in control nerve. For example, processes of elongated cells in transected nerves at P1 were significantly shorter (66 ± 20 vs. 132 ± 52 μm) and more torturous than those in control nerves. Stellate cells in transected nerves extended fewer processes, and surface labeling of perikarya was more irregular . These data suggest that the loss of viable axons and/or pathological changes that follow axonal transection alters the elaboration of normal OPC morphology. To investigate whether axons influence OPC density, the number of NG2-positive cells per unit area of tissue was compared in sections from P1, P2, P3, and P4 control and P0-transected optic nerves. At all ages, the density of NG2 cells/mm 2 of tissue was similar in control and transected optic nerve . The progressive increase in NG2-positive cells after P0 optic nerve transection suggested that viable axons were not required for OPC proliferation. A BrdU incorporation assay was used to compare levels of NG2 cell proliferation in transected and normal nerves. In P2 control and P2 optic nerves transected at P0 , nuclei of both elongated and stellate NG2 were BrdU-positive after a 2-h pulse. Approximately 25% of all NG2-positive cells were BrdU-positive in both control and transected optic nerves , indicating that viable axons and neuronal electrical activity are not essential for OPC proliferation in vivo. To investigate the influence of axons on the development of differentiated oligodendrocytes, the distribution and morphology of MBP-positive cells were compared at P5, P6, and P7 in normal and optic nerves transected at P4 . In the normal optic nerve at the time of transection (P4), there was no detectable myelin, and MBP-positive oligodendrocytes were detected only in the chiasmal third of the nerve. These oligodendrocytes appeared in clusters of two to three cells, which extended multiple processes radially. 1 d later at P5, there was no detectable myelin, and MBP-positive oligodendrocytes were restricted to the chiasmal half of control nerves . These cells had a similar appearance to those at P4. In transected optic nerves at P5, there was no detectable myelin, and as in control nerves, the MBP-positive oligodendrocytes were restricted to the chiasmal half of the nerve . Compared with P5 control nerves, individual oligodendrocytes extended fewer and shorter processes (data not shown). In control nerves at P6, MBP immunoreactive oligodendrocytes were present throughout the chiasmal two thirds of the nerve. Although most MBP-positive cells had the appearance of premyelinating oligodendrocytes and occurred in clusters of two or three cells , some extended processes that ran parallel to axons. In P4-transected nerves analyzed at P6, MBP-positive cells were also detected throughout the chiasmal two thirds of the nerve . The morphology of these MBP-positive cells differed from those in control nerves in that their processes were shorter and more asymmetrically distributed . In control nerves at P7, MBP-positive oligodendrocytes were detected along the entire length of the nerves. Myelin was detected along the entire optic nerve, with highest concentrations at the retinal end. The MBP-positive cell bodies were less clustered at P7 than the premyelinating oligodendrocytes at P6, and were generally located in longitudinally oriented chains parallel to the axons . In P4 transected nerves analyzed at P7, myelin was not detected, although MBP-positive oligodendrocytes were distributed along the entire length of the optic nerve . These oligodendrocytes were frequently clustered without any particular orientation . To investigate whether axons influence oligodendrocyte density in the optic nerve, the number of MBP-positive cells per unit area of tissue was compared in sections of P5, P6, and P7 control and P4-transected optic nerves . In sections of P4 control nerves, MBP-positive cells were detected at a density of 380 cells/mm 2 of tissue . 1 d later at P5, the density increased to 700 cells/mm 2 of tissue. At P5, the density of oligodendrocytes was significantly reduced in P4-transected nerves (361 cells/mm 2 tissue). Although the overall distribution of oligodendrocytes increased in P6 control nerves, their density (622 cells/mm 2 tissue) was similar to that observed at P5. At P6, the density of MBP-positive oligodendrocytes was similar in control (621 cells/mm 2 tissue) and P4-transected (642 cells/mm 2 tissue) nerves. Between P6 and P7, the density of oligodendrocytes increased and was similar in both control (1,157 cells/mm 2 tissue) and transected (1,205 cells/mm 2 tissue) optic nerves. A consistent observation in transected optic nerves was an apparent reduction in the number and length of processes extending from premyelinating oligodendrocytes. Quantification of premyelinating oligodendrocyte process length in confocal microscopic images of control and P4-transected nerves at P6 detected a 50% reduction in the radial extensions of oligodendrocyte processes in the transected nerves . The number of processes was clearly reduced in transected nerves, although quantification was difficult due to the complexity of processes in normal nerves. Cell death of premyelinating oligodendrocytes is a characteristic feature of oligodendrocyte lineage development . Programmed cell death (PCD) requires the activation of a family of activator and effector cysteine protease enzymes referred to as caspases . Caspases are expressed as inactive proenzymes. Upon activation, caspases are cleaved into smaller catalytically active subunits. Activated caspase-3 is a downstream effector of PCD in a variety of cell types , including oligodendrocytes maintained in vitro . It is expressed as a 32-kD inactive proenzyme. The antibody used in this study selectively recognizes the enzymatically active p18 subunit of caspase-3 . To investigate whether P4 optic nerve transection influenced the extent of oligodendrocyte PCD, the number of dying oligodendrocytes was compared in sections from P5, P6, and P7 control and P4-transected nerves. Dying premyelinating oligodendrocytes were identified by fragmentation of myelin protein–positive processes, condensed nuclear chromatin, and the presence of activated caspase-3 . Double-labeling immunocytochemistry colocalized activated caspase-3 and MBP in sections of control and transected nerves . Double-labeled cells had the morphology of premyelinating oligodendrocytes. As described previously , most (97%) had fragmented MBP-stained processes and condensed nuclear chromatin as detected by propidium iodide staining (data not shown). The remaining 3% of activated caspase-3–positive oligodendrocytes were probably in early stages of PCD before process fragmentation and nuclear chromatin condensation. Occasional caspase-3–positive myelinating oligodendrocytes were detected in control nerves. Activated caspase-3–positive NG2 cells were not detected in either control or transected nerves (data not shown). Activated caspase-3–positive, MBP-negative cells were also detected. These cells did not extend activated caspase-3–positive processes and they were often located next to vessels. At P5, there was a small but significant increase in the proportion of dying oligodendrocytes in transected optic nerves . At P6 and P7, the percentage of activated caspase-3–positive oligodendrocytes was similar in control and transected optic nerves , indicating that the absence of viable axons and axonal electrical activity did not significantly increase the extent of oligodendrocyte cell death, 2 and 3 d after P4 nerve transection. The studies described above independently investigated the role of axons in (a) OPC appearance following P0 optic nerve transection; and (b) oligodendrocyte appearance after P4 optic nerve transection. To investigate if OPCs can colonize the optic nerve and produce oligodendrocytes in the absence of viable axons, retinas of newborn (P0) rats were mechanically ablated and the optic nerves analyzed for oligodendrocytes using immunocytochemistry and MBP antibodies 1 wk later at P7. Compared with control nerves , retina ablation at P0 drastically reduced the width of the optic nerve at P7 , but these nerves retained their normal length, and thus provided a substantial substrate for OPC migration. In electron micrographs from midregions of nerves 14 h after P0 retinal ablation, all axons were in advanced stages of degeneration (data not shown). Axonal fragments were detected 24 h after ablation, were rare at 48 h, and undetected at 4 d. The marked decrease in optic nerve volume also supports total loss of axons in this experimental paradigm. In both control and P0 retinal-ablated optic nerves, oligodendrocytes were present along the entire length of the nerve at P7. However, quantification of MBP-positive cells at P7 demonstrated a 50% reduction in the density of oligodendrocytes and a total absence of myelination following P0 retinal ablation. These studies establish that OPCs can colonize regions of the optic nerve and produce oligodendrocytes in the absence of viable axons. A prevailing notion suggests that retinal ganglion cell axons play a critical role in several aspects of the development of optic nerve oligodendrocytes . In this study, we demonstrate that axonal transection inhibited myelin formation, altered the three-dimensional organization of oligodendrocytes, and reduced the number and length of oligodendrocyte processes. However, the temporal and spatial appearance and density of optic nerve OPCs and oligodendrocytes were similar in control and transected optic nerves. These data demonstrated that oligodendrogenesis occurs in the absence of viable retinal ganglion cell axons and argues against direct axonal regulations of optic nerve oligodendrogenesis. Because axon-depleted nerves fail to grow radially, they do contain fewer OPCs and oligodendrocytes, indicating that axons indirectly influence oligodendrocyte lineage cell number by increasing optic nerve volume. This and previous studies that supported direct axonal regulation of oligodendrocyte lineage development both used optic nerve transection paradigms. However, they diverged in the methods used to identify and quantify OPCs and oligodendrocytes and in the postnatal age of optic nerve transection. Previous studies often used indirect immunocytochemical techniques (i.e., delivery of antibody in vivo by intraventricular transplantation of hybridoma cells) to identify and quantify OPCs and oligodendrocytes, and concluded that loss of viable axons caused decreased OPC proliferation and increased death of oligodendrocytes . This study used recently developed immunocytochemical methods to identify and quantify OPCs and oligodendrocytes in fixed sections of control and transected optic nerves. These methods label OPCs and premyelinating oligodendrocytes in a predictable temporal and spatial pattern during brain and optic nerve development, and they establish in this report that the percentage of BrdU-labeled OPCs and dying oligodendrocytes are similar in control and transected optic nerves. The OPC proliferation data may be consistent between the studies, and the differences reported may reflect the denominators used to express the data . In electron micrographs, all axons were fragmented and in advanced stages of degeneration by 14 h after transection and retinal ablation. Therefore, our studies established that oligodendrocyte production in vivo can occur in the absence of viable axons and neuronal electrical activity. Small amounts of axonal debris remained at 24 h after transection, and could have some effect on oligodendrocyte lineage. A similar time course of axonal degeneration was reported in optic nerves transected during the second and third postnatal week . Therefore, it is unlikely that axonal debris accounts for significant differences between this and previous studies. A major objective of this study was to investigate the role of viable axons on OPC colonization of optic nerve and their subsequent differentiation into oligodendrocytes. Past studies supporting axonal regulation of oligodendrocyte lineage analyzed optic nerves transected during the second and third postnatal week. It is possible that oligodendrocyte lineage is differentially regulated during different stages of optic nerve development. However, previous immunocytochemical studies detected normal numbers of oligodendrocytes 5, 10, 20, and 40 d after P21 optic nerve transection. In addition, myelin protein mRNA in these P21-transected optic nerves decreased gradually and depending on the mRNA, representing between 25 and 60% of mRNA levels in control nerves at 40 d after transection. Similarly, two additional studies failed to detect a decrease in oligodendrocytes after P18 or older optic nerve transection. These data indicate that oligodendrocytes do not die after P18 optic nerve transection and/or that new oligodendrocytes are generated. Both events are inconsistent with the hypothesis that viable axons directly regulate oligodendrocyte lineage. The distribution and morphology of OPCs in the intact developing optic nerve and the pattern of oligodendrogenesis in explants isolated from P0 and P4 optic nerve were consistent with previous analyses, suggesting that OPCs enter the chiasmal end of the rat optic nerve just before birth and reach confluency along the nerve by P9 . Elongated NG2-positive cells were enriched at the migratory front of the OPCs and had a morphology very similar to the migratory OPCs in the chick optic nerve . These cells were invariably oriented parallel with the optic axons and their process often terminated in growth cone–like structures. It seems likely that with maturation, some elongate cells develop into stellate cells, since the density of the stellate cells increased significantly in older nerves. The cellular substrates used by migrating OPCs are unknown. While axons are a candidate for such a substrate , our P0 retinal ablation study establishes that OPCs can migrate the length of the optic nerve in the absence of viable retinal axons, and migrate after P2 in the absence of detectable axonal fragments. Other potential substrates in the transected nerves include astrocytes and longitudinally oriented blood vessels. Indeed, NG2 cell bodies and the processes of NG2-positive cells frequently terminated near or associated with blood vessels. OPC migration on the extracellular matrix of astrocytes or endothelial cells would support the in vitro finding that specific integrin expression may regulate OPC migration . Previous studies suggested that OPC proliferation in the developing optic nerve was driven through release of the mitogen PDGF that was dependent on axonal electrical activity . By contrast, this study clearly establishes that viable axons and electrical activity are not essential for the mitosis of OPCs and for expansion of oligodendrocyte lineage cells in the optic nerve. Our data are consistent with the normal myelination in P9 optic nerves following intraocular injection of tetrodotoxin every 2 d starting at P0 . As suggested previously, our data support mitogenic stimulation of OPC proliferation by factors released from astrocytes and/or endothelial cells. Astrocytes are a major source of PDGF , and conditioned medium from purified cultured type I astrocytes is a strong mitogen for purified OPCs . In the normal rat optic nerve, in situ hybridization studies of myelin protein mRNA and the distribution of MBP describe a chiasmal-to-retinal gradient in the developmental appearance of oligodendrocytes. However, myelination proceeds in a retinal-to-chiasmal gradient , suggesting that oligodendrocyte maturation and the induction of myelination are independently regulated. Oligodendrocytes mature to the MBP-positive stage but fail to initiate myelination in the axon-free environment. Thus, the early stages of oligodendrocyte development can occur independently of direct axonal signals, but myelination is critically dependent on axons. Axonal control of myelination is further supported by the differences we observed in the apparent life span of premyelinating oligodendrocytes at the retinal and chiasmal ends of the normal optic nerve. Oligodendrocytes in the chiasmal end of the optic nerve do not myelinate for at least 2–3 d after their initial detection by MBP antibodies, whereas oligodendrocytes at the retinal end of the nerve can myelinate within 24 h or less. It has been proposed that survival of premyelinating oligodendrocytes is determined by a competition for axonally derived trophic signals, and cells that do not receive such signals within 2–3 d initiate a suicide program . The distribution of dying premyelinating oligodendrocytes along the length of the developing normal optic nerve suggests that there is not a defined interval between generation of premyelinating cells and the induction of PCD. Rather, it may be that oligodendrocyte PCD in normal optic nerves is induced by positive death signals or failed development. P4 axonal transection caused a reduction of oligodendrocyte density and a slight increase in the percentage of oligodendrocytes undergoing PCD at P5. However, 2 and 3 d after P4 optic nerve transection, the density of oligodendrocytes and percentage of oligodendrocytes undergoing PCD in control and transected nerves were similar. These data argue against the hypothesis that oligodendrocyte survival is primarily dictated by limiting quantities of axonally derived survival factors. The number of optic nerve myelin internodes formed is precisely matched to the number of axons requiring myelination. How such matching is achieved is unclear. One hypothesis is that the number of axons directly regulates the number of oligodendrocytes . Consistent with this notion, when the number of axons is increased in the optic nerve, oligodendrocyte numbers increased proportionally . Our data suggest that this simple correlation is incomplete and support an alternative mechanism. In axon-free transected nerves, OPC and oligodendrocyte densities are similar to control nerves, but their total numbers are decreased because the nerve fails to grow radially due to loss of axons and failed myelination. Since oligodendrocyte lineage cell density is regulated by a homotypic density-dependent inhibition of OPC proliferation in vitro , one possible mechanism operating in developing optic nerves is that the OPCs proliferate until they reach a critical density. Increasing or decreasing the size of the nerve through addition or loss of axons will change the potential area, and thus the number of OPCs and oligodendrocytes, but does not necessarily articulate direct axonal regulation of oligodendrocyte number. Retinal axons are required for the generation of the optic nerve during early development and they may directly regulate type I astrocyte colonization of the optic nerve, as astrocytes fail to develop in the optic stalk of mice that do not form retinas . This study indicates that endothelial cells or the astrocytes which bundle groups of developing axons provide sufficient substrates for colonization of optic nerves by OPCs, for proliferation of OPCs, and for production of oligodendrocytes. Endothelial cells have received little attention as a regulator of oligodendrocyte lineage. Closure of the blood–brain barrier mimics the in vitro conditions (no serum) that drives oligodendrocyte production from O2A cells . Astrocyte–endothelial interactions may regulate oligodendrocyte lineage by modulating astrocyte expression of mitotic, chemotactic, and survival factors and by restricting inhibitory serum factors following blood–brain barrier closure. If axons indirectly regulate oligodendrocyte numbers via type I astrocytes, our data demonstrate that this axonal influence occurs before P1, and viable axons need not be present at the time of OPC migration or oligodendrocyte production. The faithful match between myelin production and axonal length argues for axonal control of myelination. This study seriously questions direct axonal regulation of oligodendrocyte number during early stages of optic nerve development, and raises the alternative possibility that axons regulate the number of myelin internodes formed by individual oligodendrocytes. In support of this hypothesis, premyelinating oligodendrocytes in the corpus callosum and optic nerve extend many processes and form 30–40 thin, short myelin internodes . Premyelinating oligodendrocytes in the region of cerebral cortex and spinal cord extend fewer, longer processes and form few, thick long myelin internodes. Therefore, developing axons may directly regulate premyelinating oligodendrocyte process number, and in turn, myelin internode number, thereby assuring an appropriate match between oligodendrocyte number and myelination. This matching may reflect differences in the electrical activity of developing fiber tracks, or molecules specific to or present at varying concentrations on different populations of developing axons. | Study | biomedical | en | 0.999997 |
10491398 | Herbimycin B, sodium orthovanadate, genistein, cytochalasin B, and aprotinin were purchased from Sigma Chemical Co. LY294002, ML7, KT5720, bisindolylmaleimide I, and human recombinant tissue inhibitor of metalloproteinases (TIMP)-2 were from Calbiochem. Jasplakinolide was obtained from Cambridge Bioscience. A broad-range inhibitor of MMPs, BB3103, was provided by British Biotech Pharmaceuticals Ltd. Rat tail collagen, type I was purchased from Collaborative Biomedical Products. Antiintegrin mAbs used were: anti-α2, A2-IIE10 and A2-VIIC6 ; anti-α3, P1B5 and A3-IIF5 ; anti-α6, A6-BB ; anti-β1, clone P5D2 (provided by Dr. N. Hotchin, University of Birmingham, Birmingham, UK). Anti-TM4SF mAbs were: anti-CD9, C9-BB ; anti-CD63, 6H1 ; anti-CD81, M38 ; anti-CD82, M104 ; and anti-CD151, 5C11 . Other mAbs were: antiemmprin, 8G6 and 7E7 ; anti–MMP-9, 4H3; and anti–MMP-1, SE600 (British Biotech Pharmaceuticals, Ltd.). Anti–MMP-2 polyclonal antibody, AB808, was from Chemicon International. Antiphosphotyrosine (pTyr) mAb, clone 4G10, was purchased from Cambridge Bioscience. Anti–p85 PI3K mAb, clone U13, and anti-α6 mAb, clone GoH3, were purchased from Serotech. Various anti–c-Akt mAbs were purchased from New England Biolab. The MDA-MB-231 human breast cancer cell line was purchased from American Type Culture Collection and maintained in L-15 Leibovitz medium (Sigma Chemical Co.) supplemented with 15% FCS. HT1080/zeo and HT1080/CD9 cells were cultured in DMEM supplemented with 10% FCS. To study the cell behavior in the three-dimensional ECM environment, the cells were embedded into Matrigel (Becton Dickinson) according to the manufacturer's recommendations. In brief, MDA-MB-231 cells were detached from culture dishes and suspended in Matrigel solution at the final concentration of 5 × 10 4 cells/ml at 4°C. The cell suspension was aliquoted (250–300 μl) into a 24-well plate and left to solidify at 37°C for 30 min. The cells embedded into Matrigel were cultured in L-15 growth medium with or without FCS. For time-lapse video recording, Matrigel-embedded cells were cultured in the temperature controlled humidified box mounted on the stage of the inverted microscope (Axiovert 25; Carl Zeiss). Migration of MDA-MB-231 cells within Matrigel was recorded using the Hitachi CCD camera connected to the computer and the captured images were processed using the Image PC software package (Scion Co.). Cellular localization of integrins and tetraspanins on the surface of embedded MDA-MB-231 cells was analyzed by indirect immunofluorescence staining. The three-dimensional cultures were fixed with 1% paraformaldehyde/PBS for 20 min and washed three times with PBS for 1 h. The primary antibodies were applied to the cultures and incubated for 1 h at room temperature. After three 10-min washes in PBS, samples were incubated for 1 h at room temperature with FITC-conjugated goat anti–mouse antibody (Sigma Chemical Co.). After subsequent washes, the samples were analyzed using a Zeiss Axioscop. The immunofluorescence images were captured using the Coolview CCD camera (Photonic Sciences) and subsequently processed for analysis by using the Openlab software package (Improvision). A standard static adhesion assay (30–35 min) was carried out as described previously . When the effect of mAbs on adhesion was studied, cells were preincubated with mAbs at 4°C for 30 min and then aliquoted into 96-well plates precoated with various concentrations of Matrigel. For the invasion assay, an upper surface of 8-μm framed polycarbonate filters (NeuroProbe, Inc.), was coated at 17.5 μg/cm 2 of Matrigel for 30 min at 37°C. The lower compartments of the 96-well chemotactic Boyden chamber were filled with L-15 medium supplemented with 5% FCS. MDA-MB-231 cells (4 × 10 4 ) were suspended in serum-free L-15 media containing 30 μg/ml of BSA (no treatment) or mAb at a similar concentration. In the experiments using a mixture of anti-TM4SF antibodies, the mAbs C9-BB, 6H1, and 5C11 were combined at a final concentration of 10 μg/ml each. Untreated and mAb-treated cells were incubated for 1 h at 37°C before they were applied to the upper compartment of the chamber (chamber well). After incubation for 20 h at 37°C in 5% CO 2 and 95% air, the filters were fixed with methanol and stained with 1% crystal violet (Sigma Chemical Co.). The cells from the upper surface of the filter were wiped off, and the migrated cells were counted in five to eight randomly selected microscopic fields (×1,200) for each chamber well. All experiments were performed in triplicate. Production of MMPs by MDA-MB-231, HT1080/zeo, and HT1080/CD9 cells was analyzed by gelatin zymography as described previously . Cells suspended in the complete media were plated for 4 h on the 100-mm bacteriological dish precoated either with ECM ligands or immobilized antibodies. After three washes with serum-free L-15 media, cells were incubated in serum-free media for an additional 24 or 48 h before the conditioned media were collected for the analysis. The conditioned media were supplemented with Laemmli sample buffer, and the loading volumes of each sample were subsequently adjusted according to the cell number. The samples were resolved in 9% acrylamide SDS-PAGE containing 1 mg/ml gelatin. The gels were washed three times with 2.5% Triton X-100 for 1 h, and incubated for 24 h at 37°C in 50 mM Tris-HCl, pH 7.4, 10 mM CaCl 2 , 150 mM NaCl with 0.02% NaN 3 . The lytic bands were visualized by Coomasie brilliant blue R250 staining. After staining, the gels were scanned using the GI6000 Gel densitometer (Bio-Rad) and analyzed using the Molecular Analyst software package (Bio-Rad). Serum-starved MDA-MB-231 cells were plated on the immobilized mAbs or ECM ligands in serum-free DMEM for 1 h. Cells were lysed for 3 h at 4°C in 1% Brij 98 buffer containing 20 mM Tris HCl, pH 7.4, 140 mM NaCl, 10% glycerol, 1 mM Na 3 VO 4 , 1 mM NaF, 10 mM Na-pyrophosphate, 2 mM PMSF, 5 μg/ml aprotinin, and 10 μg/ml leupeptin. Insoluble material was pelleted at 13,000 rpm and the supernatants were precleared overnight at 4°C with goat anti–mouse Ig antibody immobilized on agarose beads (Sigma Chemical Co.). The precleared lysates were incubated for 3 h with the appropriate mAb captured on the goat anti–mouse Ig antibody immobilized on agarose beads. The immunoprecipitates were washed once with the lysis buffer, then twice with a solution containing 0.1 M Tris HCl, pH 8.0, and 0.5 M LiCl, then once with a solution containing 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.1 M NaCl, and finally, once with a kinase reaction buffer (40 mM Hepes, pH 7.4, 100 μM ATP, 5 mM MgCl 2 ). To initiate the kinase reaction, the beads were resuspended in 50 μl of the kinase buffer containing 30 μg of phosphatidylinositol (Sigma Chemical Co.) and 5 μCi [γ- 32 P]ATP. The reactions were terminated after 15 min with 60 μl 2 N HCl, and lipids were extracted with 160 μl of 1:1 (vol/vol) chloroform/methanol. The lipids were subsequently analyzed by TLC . Serum-free growth medium, conditioned by MDA-MB-231 cells for 24 h, was incubated with gelatin-conjugated Sepharose beads for 2 h at 4°C. The beads were washed three times with 50 mM Tris HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl 2 , and the captured material was eluted from the beads into Laemmli loading buffer at 95°C. Proteins were resolved in 10% SDS-PAGE, transferred to the nitrocellulose membrane, and developed with the specific anti-MMP antibody. Protein bands were visualized using HRP-conjugated secondary antibodies and Enhanced Chemiluminescence Reagent (Amersham Pharmacia Biotech). In this study we investigated whether integrin–tetraspanin protein complexes play a role in controlling invasive behavior of tumor cells. As a model system we used highly invasive mammary epithelial cells, MDA-MB-231. In preliminary experiments, we have established that MDA-MB-231 cells express five TM4SF proteins (e.g., CD9, CD63, CD81, CD82, and CD151) and each of them form complexes with α3β1 integrin (data not shown). Migration on planar surfaces coated with ECM proteins is a widely used paradigm for analyzing tumor cell invasion. Indeed, the data accumulated in these in vitro studies provided important insights into the molecular mechanisms that may control invasive behavior of tumor cells. However, it became increasingly obvious that cellular responses may change when cells are placed within a three-dimensional ECM environment . Thus, we rationalized that migratory behavior of tumor cells within a three-dimensional ECM could reflect an invasion process more accurately and would allow better understanding of the molecular mechanisms that control invasive migration. In the course of this study we examined the behavior of MDA-MB-231 cells within artificial EHS matrix (Matrigel). Time-lapse video microscopy has shown that within the first 2–4 h after embedding into Matrigel, characteristic short pseudopodial protrusions began to appear . These protruding structures were dynamic and typically retracted within 15–40 min. Although they remained round, most cells had undergone various types of motion, including wobbling, turning around, and slow directional movement . The average rate of cellular migration within Matrigel was 3.44 ± 0.8 μM/h (calculated observing 19 cells in 12 separate experiments). The random protrusive activity persisted over 48–72 h, with the protrusions becoming longer and more stable at later times, and then decreased in number when cells started to form round colonies. The cells exhibited similar behavior when they were plated on top of polymerized Matrigel (data not shown). These experiments have demonstrated that migration through three-dimensional ECM environment does not require cell spreading, but may rely on short invasive protrusions developed by tumor cells (see also below). We began investigating the role of α3β1–tetraspanin protein complexes in invasive migration by examining their distribution on the Matrigel-embedded MDA-MB-231 cells. In the initial experiments, we carried out whole-mount immunofluorescence staining using nonpermeabilized cells. When analyzing various focus planes, we found that α3β1 integrin and tetraspanins were concentrated within distinctive large clusters covering the cell body and at the tips of short thread-like protrusions . These microvilli-like protrusions extended from the main cell body and from the pseudopodia , thus producing characteristic hairy images. In contrast, clusters of α2β1 integrin were visibly smaller and not detected on the thread-like protrusions . Given their abundant presence at the tips of the microvilli-like cellular extensions, we addressed the question as to whether the α3β1–tetraspanin complexes have the potential to affect protrusive activity of MDA-MB-231 cells. Thus, we examined the effect of antitetraspanin mAbs on the morphology of the cells embedded in Matrigel. These experiments were performed under serum-free conditions to avoid growth factor interference. As in the presence of serum, during the first 24 h after embedding, the cells incubated without mAbs remained rounded, and only 25–30% of them extended one or two short (0.25–0.5 size of the cell body diameter) pseudopodial protrusions . In contrast, 45–70% of the cells incubated with mAbs to CD9, CD63, CD81, and CD151 developed long thin extensions that often exceeded the cell body diameter by 2–2.5-fold . When analyzed using time-lapse video microscopy, we found that in the presence of antitetraspanin mAbs, protrusive activity of the cells was not appreciably affected, whereas retraction of the invasive protrusions was attenuated, thus leading to their extended elongation . Notably, we observed that the mAb-treated cells could use these elongated protrusions to generate traction forces and facilitate directional movements within Matrigel . Importantly, the morphogenetic effect of the mAb to α3 integrin subunit was comparable to that of antitetraspanin mAbs . On the other hand, mAbs to α2 integrin subunit and emmprin, an abundant cell surface protein that also form complexes with α3β1 integrin , had no appreciable effect on cell morphology . Taken together, these results indicate that α3β1–tetraspanin complexes play an important role in controlling protrusive activity of the MDA-MB-231 cells. One explanation for the observed changes of cell morphology is that the mAbs could directly affect adhesive interactions between MDA-MB-231 cells and Matrigel. Thus, we tested the effect of the antitetraspanin and antiintegrin mAbs on attachment of the cells to various concentration of Matrigel. These experiments demonstrated that mAbs to the α3 integrin subunit or to tetraspanins (tested separately or in various combinations) neither diminished nor increased adhesion of MDA-MB-231 cells to Matrigel . Interestingly, we found that the function inhibitory mAb to the α2 or α6 integrin subunits also failed to interfere with the adhesion of the cells to Matrigel. On the other hand, an inhibitory mAb to the β1 integrin subunit almost completely blocked adhesion of MDA-MB-231 cells to the substrate. While these results clearly demonstrate that adhesive interactions of MDA-MB-231 cells with Matrigel matrix involve various β1 integrins, they also argue against a direct modulation of cell adhesiveness by the α3β1–tetraspanin protein complexes. We surmised that clustering with the antitetraspanin or anti-α3 mAbs may trigger signaling events that induced changes of morphology of the Matrigel-embedded cells. To begin investigating which signaling pathway(s) is involved in the mAb-mediated morphogenetic responses, MDA-MB-231 cells (untreated or treated with the anti-CD63 mAb) were cultured in Matrigel in the presence of various pharmacological inhibitors. The results of these experiments are shown in Fig. 6 . Orthovanadate and genistein inhibited protrusive activity of both untreated and mAb-treated cells, suggesting that the basal and antibody-induced protrusive activities involve functions of tyrosine phosphatase(s) and protein tyrosine kinase(s) . In agreement with this notion, herbimycin, another tyrosine kinase inhibitor, could mimic the morphogenetic effect of the mAb . Notably, a specific inhibitor of PI3K, LY294002, inhibited only the mAb-mediated morphogenetic response and did not affect the basal protrusive activity of MDA-MB-231 cells . In contrast, inhibitors of myosin light chain kinase (ML7), protein kinase C (bisindolylmaleimide I), and cAMP-dependent protein kinase had no appreciable effects on the morphology of the Matrigel-embedded cells . We also analyzed the effect of the drugs that affect the actin cytoskeleton. Interestingly, we found that jasplakinolide, a macrocyclic peptide that induces actin polymerization , negated the effect of the antitetraspanin mAb on cell morphology . Conversely, cytochalasin B, which destabilizes actin filaments, induced long protrusions even in the absence of the antitetraspanin mAb . Collectively, these results suggest that the elongation of the invasive protrusions involve activities of various classes of signaling enzymes (e.g., tyrosine kinases, tyrosine phosphatases, PI3K) and may be linked to destabilization of the actin cytoskeleton. We hypothesized that MMPs, a group of ECM-degrading enzymes, may play an important role in controlling cellular behavior within Matrigel. Indeed, both basal and mAb-induced protrusive activities of MDA-MB-231 cells were completely abolished when the cells were cultured in the presence of BB 3103, a wide-range MMP inhibitor . On the other hand, aprotinin, a common inhibitor of serine proteases, caused only ∼25% inhibition of the mAb-induced protrusive activities . Next, we addressed the question whether or not α3β1–tetraspanin complexes may be involved in the regulation of MMP production. When grown on tissue culture plastic MDA-MB-231 cells secrete two major gelatinolytic activities of 92 and 56 kD . In addition, we consistently detected a weaker gelatinolytic band of 72 kD. Western blotting analysis carried out with MMP-specific antibody has shown that 92-, 72-, and 56-kD proteins corresponded to MMP-9, MMP-2, and MMP-1, respectively . To examine whether clustering of the α3β1–tetraspanin complexes with mAbs can affect production of MMPs, MDA-MB-231 cells spread on tissue culture plastic were incubated with anti-α3 or various antitetraspanin mAbs for 48 h and the samples of conditioned media were analyzed by gelatin zymography. As illustrated in Fig. 8 B, treatment with anti-α3 and all four tested antitetraspanin mAbs enhanced the production of the latent form of MMP-2 by 2–15-fold. Notably, this treatment did not affect production of MMP-9 or MMP-1 . In the control experiments, anti-α2 or antiemmprin mAbs did not alter the amounts of MMP-2 and MMP-9 secreted by the MDA-MB-231 cells. Likewise, a specific increase in production of MMP-2 was observed when the cells were plated on the immobilized anti-α3 and antitetraspanin mAbs . Interestingly, we have noticed that the efficacy of the stimulatory signals is dependent on presentation of the particular antitetraspanin mAb. Indeed, clustering with soluble anti-CD81 and anti-CD151 mAbs caused only a modest increase in production of MMP-2 when compared with anti-CD9 and anti-CD63 mAbs . On the other hand, the stimulation induced by the immobilized mAbs was comparable for all four tetraspanins . Not only do these data suggest that the postclustering events that follow the binding of soluble mAbs to the cells (for example, endocytosis and/or recycling of the α3β1–tetraspanin protein complexes) are regulated in the mAb-specific manner, but they also point to another level of control over the cellular response to the clustering of the complexes. To obtain direct evidence that tetraspanins are involved in the adhesion-dependent production of MMP-2, we examined gelatinolytic activities present in culture media conditioned by HT1080/zeo and HT1080/CD9 cells. The HT1080/CD9 line has been established by expressing human CD9 cDNA in HT1080, the human fibrosarcoma cell line, whereas HT1080/zeo was developed as a control drug-resistant cell line (see Materials and Methods for details). Both cell lines expressed similar levels of β1 integrins and were comparable in their ability to attach to various ECM ligands (data not shown). When plated on collagen or laminin 5–containing ECM, both cell lines secrete comparable amounts of MMP-9 . Strikingly, the amount of MMP-2 secreted by the HT1080/CD9 cells plated on laminin 5–containing ECM was significantly higher (approximately fourfold) than that secreted by the HT1080/zeo cells . On the other hand, growth media conditioned by the cells plated on collagen contained similar amounts of MMP-2 . Given the fact that attachment of the HT1080 cells to laminin 5–containing ECM is mediated by α3β1 integrin , these results provide strong support for the idea that α3β1–tetraspanin protein complexes (and the α3β1–CD9 complex in particular) may be involved in the ECM-induced production of MMP-2. Next, we carried out two sets of experiments to address the question of whether the effect of the mAbs on the morphology of the Matrigel-embedded cells is related to the increased production of MMP-2. First, MDA-MB-231 cells were cultured in Matrigel in the presence of both anti-CD63 mAb and TIMP-2, a potent inhibitor of MMP-2. As shown in Fig. 7 , TIMP-2 only partially negated the mAb-induced protrusive activity of the MDA-MB-231 cells. Second, we analyzed the effect of pharmacological inhibitors on the mAb-induced production of MMP-2. These experiments were carried out with MDA-MB-231 cells plated on the immobilized antitetraspanin mAb. Preliminary experiments have demonstrated that the presence of pharmacological inhibitors during the time of the experiment had no significant effect on the morphology or viability of the cells attached to the immobilized antitetraspanin mAbs. However, we decided against applying the inhibitors to the mAb-treated cells grown on the uncoated glass coverslips , since some of them induced dramatic changes of cell morphology and might, therefore, affect the MMP production in a nonspecific fashion. The MDA-MB-231 cells were seeded on immobilized anti-CD151 mAb, the strongest stimulus of MMP-2 production , and the effect of the pharmacological inhibitors was analyzed 24 h after the plating. Of the various inhibitors tested, we found that only LY294002 (inhibitor of PI3K) had a negative effect on the MMP-2 production induced by the antitetraspanin mAb . Thus, we concluded that the PI3K-dependent signaling pathway makes an essential contribution in the production of MMP-2 triggered by the α3β1–tetraspanin complexes. However, increased production of MMP-2 by itself may not be sufficient to facilitate cell protrusive activity within three-dimensional ECM, as orthovanadate, a potent inhibitor of the protrusive activity , had no effect on MMP-2 production. To analyze whether antibody-induced production of MMP-2 and changes of cell morphology could affect invasiveness of MDA-MB-231 cells, we performed the Matrigel penetration assay. As shown in Fig. 11 A, continuous presence of various antitetraspanin mAbs during the time of the assay (tested separately or in combinations) increased invasiveness of MDA-MB-231 cells 1.5–3.5-fold. Similarly, the mAb to α3 integrin subunit also facilitated cell invasion through Matrigel. Interestingly, we found that the inhibitory mAb to α2 integrin subunit also increased invasiveness of the MDA-MB-231 cells (up to 1.6-fold). In the control experiments, mAbs to α6 integrin subunit or to emmprin had no effect on cell invasion . Next we investigated the effect of pharmacological inhibitors on invasiveness of MDA-MB-231 cells. As illustrated in Fig. 11 B, inhibitors to PI3K, myosin light chain kinase, and one of the protein tyrosine kinase inhibitors, genistein, efficiently blocked cellular invasion induced by the antitetraspanin mAb, with only a minimal effect on the basal level of invasion. Similarly, treatment with jasplakinolide specifically negated the effect of the mAb on invasion of MDA-MB-231 cells. On the other hand, orthovanadate, cytochalasin B, and to a certain extent protein kinase C inhibitor were effective in blocking both basal and mAb-induced cellular invasion . Interestingly, herbimycin, a more selective inhibitor of tyrosine kinases, had no effect on the mAb-induced invasion but facilitated basal invasiveness of the MDA-MB-231 cells. Taken together, these results demonstrate that the invasive process involves multiple signaling proteins, and there is only a partial correlation between the ability of MDA-MB-231 cells to develop invasive protrusions and their overall invasive potential. Finally, we investigated the contribution of MMPs in cellular invasion through Matrigel. We found that both broad-range and more selective (TIMP-2) inhibitors of MMPs decreased invasiveness of the MDA-MB-231 cells . Given a notable consistency of LY294002 inhibiting both morphological and biochemical consequences of the mAb clustering, we wanted to investigate whether there is a direct link between activation of the α3β1–tetraspanin complexes and signaling pathways involving PI3K. To this end two sets of experiments were carried out. First, we examined whether or not plating of MDA-MB-231 cells on the mAbs facilitated an interaction of PI3K with tyrosine phosphorylated cellular proteins. Lipid kinase assays performed on the anti-pTyr immunoprecipitates have shown that the activities of the associated enzyme in cells plated on different substrates (including various mAbs and ECM proteins) were unchanged relative to a control sample (e.g., cells kept in suspension) . Second, we assessed phosphorylation levels of c-Akt, a serine/threonine kinase whose phosphorylation (and activation) is regulated by PI3K . We found that plating of MDA-MB-231 cells on various antitetraspanin and particular anti-α3 mAbs induced a small (∼1.8–4-fold, in 3 separate experiments) but reproducible increase in the phosphorylation levels of c-Akt . On the other hand, c-Akt phosphorylation level in the cells attached to the antiemmprin mAb was comparable to that found in the control sample (e.g., cells kept in suspension) . Although activation of c-Akt may not be directly related to cell invasion or induction of cellular protrusions within Matrigel, these results clearly indicate the α3β1–tetraspanin protein complexes can modulate signaling pathway involving PI3K. Penetration through the basement membrane is an important step during tumor dissemination. To gain an insight into cellular and molecular mechanisms that control this process we examined invasive behavior of tumor cells in response to Matrigel, a widely used mimic of the basement membrane. Numerous earlier studies have provided a detailed morphological analysis of tumor cells migrating across planar surfaces, and uncovered a number of signaling pathways controlling cell motility. A critical novel aspect of our study is the choice to use three-dimensional Matrigel to analyze specifically invasive migration. By culturing cells within a three-dimensional ECM environment, we were aiming to observe cellular responses that would more accurately reflect invasive processes occurring in vivo. Our data clearly indicate that significant differences exist between planar and three-dimensional migration. Migration of MDA-MB-231 cells on absorbed Matrigel could be described by a well-defined three-step extension–retraction model: extension of lamellipodial protrusions → generation of traction forces, jerky cell body translocation → retraction of the trailing edge (Sugiura, T., and F. Berditchevski, unpublished results). In contrast, cells embedded in or plated on top of Matrigel remain rounded and generate highly dynamic short pseudopodial extensions that may play a major part in governing limited ability for invasive movement (mainly, wobbling, turning around, and slow directional movement). The evidence presented in this report suggests that α3β1–tetraspanin protein complexes could make two important contributions into the invasive process. First, the α3β1–tetraspanin protein complexes can control elongation of invading pseudopodia. Second, α3β1–tetraspanin protein complexes are involved in the production of MMP-2, a member of the MMP family that is associated with the invasive phenotype of tumor cells both in vivo and in vitro. Rapid retraction of the invasive pseudopodia within three-dimensional ECM environment, saturated with potential integrin-binding sites, implies that the signaling pathways triggered within extending protrusions are directed against generating strong cell–ECM interactions along the protrusion length. Given the effect of the antitetraspanin mAbs on the morphology of the Matrigel-embedded cells, we hypothesize that the function of the α3β1–tetraspanin protein complexes may be linked to stabilization of the invasive protrusions. Furthermore, our data indicate that the α3β1 integrin in complexes with tetraspanins plays a modulatory/signaling role in this process. Although it is theoretically possible that the morphogenetic effect of the mAbs is caused by direct modulation of adhesive capacity of the α3β1 integrin, two lines of evidence argue against this notion. First, treatment of MDA-MB-231 cells with anti-TM4SF mAbs did not influence adhesion of the cells to Matrigel or to any other ECM ligands that were tested in a short-term adhesion assay (Berditchevski, F.B., unpublished results). Second, the α3β1–tetraspanin complexes were clustered at the tips of short, thread-like protrusions that resembled microvilli, distinct morphological structures that are thought to mediate transient rather than stable adhesive interactions . Indeed, the α3β1–tetraspanin clusters were devoid of vinculin (Sugiura, T., unpublished results), a cytoskeletal protein associated with focal complexes and focal adhesions (both are stable adhesion complexes) but known to be excluded from microvilli . The fact that jasplakinolide blocks the mAb-induced extension of protrusions whereas cytochalasin B induces their formation even in the absence of the mAbs suggests that the actin cytoskeleton is the ultimate target for the signals triggered by the α3β1–tetraspanin complexes. Although a particular intermediary component that may be involved in the α3β1–tetraspanin-induced reorganization remains unknown, actin-binding proteins of the ezrin/radixin/moesin (ERM) family may be among the potential candidates. In this regard, it has been reported that the ectopic expression of the truncated form of ezrin and moesin could destabilize cortical cytoskeleton and induce formation of long filopodia-like extensions . Furthermore, thrombin-induced phosphorylation of moesin on threonine 558 in platelets, a modification that potentiates its binding to F-actin, closely correlated with the formation of long filopodial protrusions . Numerous earlier studies have shown that the dynamics of actin cytoskeleton can be controlled at various levels, with different tyrosine kinases, tyrosine phosphatases, and PI3K being intimately involved in this process . Which signaling pathways are utilized by the α3β1–tetraspanin protein complexes to manipulate actin cytoskeleton in the MDA-MB-231 cells? As a part of the current study, we specifically addressed the question as to whether or not activity of the α3β1–tetraspanin protein complexes is linked to the PI3K signaling pathways. Our data clearly indicate that this link is possible. First, we found that LY29004, a specific inhibitor of PI3K, has completely abolished a morphogenetic effect of the mAbs and attenuated mAb-induced invasiveness of the MDA-MB-231 cells. Second, we observed that clustering of the α3β1–integrin protein complexes stimulates phosphorylation of c-Akt, a process that is tightly dependent on the activity of PI3K . Although by itself phosphorylation of c-Akt may not be directly related to the rearrangement of the actin cytoskeleton, these data illustrate a functional connection between the α3β1–integrin protein complexes and PI3K-dependent signaling. Earlier studies have shown that PI3K activity can be stimulated by cell adhesion to ECM . Furthermore, at least two integrin receptors (e.g., α6β4 and αII B β3) were specifically implicated in activation of PI3K-dependent signaling in carcinoma cells and in platelets . However, the proximal events linking the ligation of these integrins to the PI3K signaling pathways remain unknown. Thus, our data not only indicate that another integrin, α3β1, is involved in the PI3K-dependent signaling, but also point to a specific type of the integrin accessory proteins that is required for this process (see also below). Theoretically, integrins can affect the PI3K-dependent signaling either directly (by modulating enzymatic activity of PI3K) or indirectly (by regulating activities and/or compartmentalization of other cellular proteins involved in the PI3K signaling, e.g., other phosphatidylinositide kinases, phosphatidylinositide phosphatases, cytoskeletal proteins). In turn, activation of PI3K may be linked to tyrosine phosphorylation of cellular proteins , which either directly (through Src homology 2 [SH2] domain of p85 subunit of the class I PI3K) or indirectly (through Ras-dependent activation of p110 subunit of PI3K) recruit and activate the enzyme . The fact that activity of the PI3K coimmunoprecipitated with pTyr-containing cellular proteins was not affected in cells attached to the antitetraspanin mAbs argues against the former possibility, and suggests alternative mechanisms. For example, it is possible that the function of α3β1–tetraspanin protein complexes is linked to the activation of the class II or class III PI3Ks, the enzymes that are regulated in a different fashion from well-characterized mechanisms of activation of the class I PI3Ks. A similar pathway has been shown to operate in platelets after the activation of the αII B β3 integrin . Alternatively, it is possible that the complexes can influence the PI3K-dependent signaling without directly affecting the activity of the enzyme. For example, it is feasible that the α3β1–tetraspanin protein complexes can regulate local concentration of phophatidylinositol (PtdIns)-4-P, a potential substrate for PI3K, using associated phosphatidylinositol 4-kinase . This may subsequently increase production of PtdIns-3,4-P2. Not only does this lipid specifically target various cellular proteins whose function may be linked to actin cytoskeleton , but when further converted to PtdIns-3,4,5-P3, it may facilitate additional recruitment (through the SH2 domain of its regulatory subunit) of PI3K and subsequent allosterical activation of the enzyme . Conversely, we cannot exclude a possibility that α3β1–tetraspanin protein complexes modulate activity of phosphoinositide phosphatases (e.g., SH2 domain–containing inositol 5′-phosphatase, PTEN), thus affecting local concentrations of D-3 phosphoinositides available for binding to their protein targets (including c-Akt). Distinguishing between these various possibilities represents an important challenge for future studies. An important aspect for consideration in invasive migration is the balance between the protruding forces generated by the cell and the surrounding extracellular matrix, a mechanical barrier that confronts them. Although different groups of ECM-degrading enzymes may be potentially involved, our data indicate that MMPs have a major role in both supporting protrusive activity and invasive migration of MDA-MB-231 cells. The involvement of integrins in MMP-dependent degradation of ECM occurs at various levels, including regulation of production of the enzymes (see Introduction), their activation , and site-specific targeting . Here we have shown that signaling through the α3β1 integrin, but not through α2β1 or α6β4 integrins, specifically regulates production of MMP-2. These results add to recent data that have implicated α3β1 integrin in the regulation of MMP-2 production and invasiveness of rhabdomyosarcoma and glioblastoma cells . Interestingly, in contrast to these earlier studies, treatment of MDA-MB-231 cells with anti-α3 or antitetraspanin mAbs did not induce activation of MMP-2. Furthermore, the expression level and cellular distribution of MT1-MMP, a membrane-type MMP known to be crucial for activation of MMP-2 , were not affected in the mAb-treated cells (Sugiura, T., and F. Berditchevski, unpublished results). Thus, it is possible that either α3β1-dependent activation of MMP-2 in rhabdomyosarcoma cells involves a different type of α3β1-containing protein complex(es) (that is not expressed on MDA-MB-231 cells) or the α3β1–tetraspanin-induced signaling pathway is partly deficient in the breast carcinoma cells. Although production of MMP-2 is clearly regulated in both normal and cancer tissues, surprisingly little is known about the signaling pathways that control this process. Prostaglandin E2, phorbol esters, and cAMP were shown to stimulate transcription of MMP-2 in glomerular mesangial cells and fibrosarcoma cells . Notably, we have found that the mAb-induced production of MMP-2 does not involve the activation of cAMP-dependent protein kinase, but instead, requires the activity of PI3K. This suggests that D-3 phosphoinositides may be signaling mediators in this process. Interestingly, an inhibitor of protein tyrosine phosphatases did not affect the α3β1–tetraspanin-mediated production of MMP-2. This latter observation leads to two important conclusions: (a) production of MMP-2 by itself is not sufficient to stimulate protrusive activity of tumor cells; and (b) signaling pathways leading to the rearrangement of the actin cytoskeleton and formation of long invasive protrusions bifurcate after activation of PI3K. Finally, an important conclusion that can be drawn from our study is that there is a functional diversity associated with various cell surface pools of the α3β1 receptors. Indeed, although in MDA-MB-231 cells α3β1 integrin is associated with both tetraspanin and emmprin, only the tetraspanin-containing complexes were implicated in the formation of invasive protrusions and the production of MMP-2. These observations provide strong evidence for the idea that the associated protein partners dictate signaling specificity of integrins. On the other hand, our data clearly indicate that there is a signaling redundancy between various α3β1–tetraspanin complexes, as all tested antitetraspanin mAbs induced similar morphological and biochemical responses. It has been postulated that tetraspanins form a network of various interconnected cell surface complexes, a tetraspan web, which, in fact, may be considered as one signaling entity . Thus, our results not only support this notion, but also highlight a specific signaling pathway involving the α3β1–tetraspanin protein web. It should be noted, that in spite of the apparent phenomenological similarities, there were quantitative differences (both at the morphological and biochemical levels) in cellular responses to various antitetraspanin mAbs. This may arise from the unique structural features of a particular tetraspanin (for example, CD63 possesses a lysosomal targeting signal that may specifically affect a postclustering internalization of the protein and its most proximal interacting partners) and, consequently, signaling asymmetry of the web (e.g., there may be differences in spatial proximity of a particular tetraspanin to a specific signaling protein associated with the web [for example, PI4K]). Cellular invasion is a complex process controlled by multiple interconnected signaling pathways (observed effect of various pharmacological inhibitors in this study), and may involve different members of the integrin family of adhesion receptors. Indeed, we have shown here that not only α3β1 integrin, but also α2β1 integrin may contribute to the invasive phenotype of the MDA-MB-231 cells. Importantly, in preliminary experiments we have established that although it stimulates cell invasion, the anti-α2 mAb has minimal effect on chemotactic migration of the MDA-MB-231 cells towards absorbed Matrigel (Sugiura, T., and F. Berditchevski, unpublished results). This observation implies that α2β1 integrin is specifically engaged during migration through the three-dimensional environment. Although this issue remains open for further investigation, one possibility is that the anti-α2 mAb may affect transient adhesive interactions between the embedded cells and polymerized Matrigel. As discussed above, in the absence of strong attachment points (such as focal adhesions that are assembled in cells migrating on planar surfaces), these highly dynamic interactions would have a dominating role in determining a migratory potential of the cells. Production of ECM-degrading enzymes (including MMPs), reorganization of the actin cytoskeleton (including destabilization of the cortical cytoskeleton), and the ability to generate traction forces are interdependent yet distinct cellular events that are critical for migration within three-dimensional ECM. Here we show that α3β1–tetraspanin protein complexes may play a crucial role in controlling all three constituents of the invasive process. Thus, further dissecting the signaling events associated with activation of the complexes may prove to be important for better understanding of the molecular mechanisms of tumor cell invasion and metastasis. | Other | biomedical | en | 0.999997 |
10498668 | Cyclic GMP-gated (CNG) 1 channels play a central role in vertebrate phototransduction by controlling the flow of cations across the plasma membrane of rod and cone photoreceptors. While the principal physiological properties of the rod cGMP-gated channel have been known for some time , several basic questions about its mechanism of opening and closing remain unresolved. The light-sensitive current flowing through cGMP-gated channels shows a pronounced outward rectification and is almost constant within the range of voltages at which rod photoreceptors operate (−30 to −70 mV). This outward rectification, which is greatly alleviated in the absence of divalent cations, has been attributed to the voltage-dependent blockage by external Ca 2+ and Mg 2+ . At low cGMP concentrations ([cGMP]), however, the current–voltage (IV) relation is outwardly rectifying even in the absence of divalent cations on both sides of the membrane . This effect could account for a major fraction of the outward rectification of the light-sensitive current in intact rods because, in the dark, [cGMP] is only a few micromolar and is further decreased by light . The cGMP-dependent rectification could be generated either by a voltage-dependent incidence of subconductance levels (sublevels) at unchanged open probability or by a voltage-dependent open probability at unchanged single channel conductance. The first mechanism is particularly appealing, because the cGMP-gated channel is cooperatively activated by binding of several cGMP molecules . At low [cGMP], sublevels could correspond to opening of partially liganded channels. In fact, several investigators have noticed sublevels preferentially at subsaturating [cGMP] . Presently used kinetic models for the gating of CNG channels assume either that the channel has to be fully liganded to open or that opening may already occur in the partially liganded channel according to the Monod-Wyman-Changeux model that describes allosteric transitions in proteins . In an effort to understand the cGMP-dependent rectification, we studied single-channel and macroscopic currents. In the single-channel experiments, we took advantage of the superior resolution of thick-walled glass pipettes and the slower open-close kinetics of homooligomeric α subunits compared with the fast “flickery” kinetics of the native channels . We show that subconductance states are extremely rare events at all [cGMP] and that the outward rectification is caused by an increased open probability of the channel at positive compared with negative voltages. Macroscopic currents were measured under steady state conditions and in response to voltage steps to relate the absolute open probability of the channels to both the [cGMP] and voltage. Assuming four cGMP binding reactions, the experimental data can be described adequately with both an allosteric and a sequential model with slight superiority of the allosteric model. Ovarian lobes were resected from Xenopus laevis under anesthesia (0.3% 3-aminobenzoic acid ethyl ester, Ms-222) and transferred to a Petri dish containing Barth medium containing (mM): 84 NaCl, 1 KCl, 2.4 NaHCO 3 , 0.82 MgSO 4 , 0.33 Ca(NO 3 ) 2 , 0.41 CaCl 2 , 7.5 Tris-HCl, pH 7.4. Stage V and VI oocytes were isolated mechanically or by incubation for 20–30 min in a Ca 2+ -free Barth medium containing 1 or 2 mg/ml collagenase. Within 2–7 h after isolation, RNA specific for the α subunit of the cGMP-gated channel from rod photoreceptors was injected into oocytes through glass micropipettes (15–20 μm diameter). The injected volume was 30–50 nl containing either ∼0.5 ng/μl RNA (single-channel experiments) or ∼50 ng/μl RNA (macroscopic currents). Oocytes were stored at 18°C overnight and defolliculated manually 20–28 h after isolation. Oocytes were further incubated for 3–7 d after injection at 18°C until experimental use. Before patching, the vitelline membrane of the oocytes was mechanically removed after exposing the cells to a hypertonic solution containing (mM): 200 aspartate, 20 KCl, 1 MgCl 2 , 5 EGTA, 10 HEPES-KOH, pH 7.4. Skinned oocytes were transferred to the experimental chamber, which was mounted on the stage of an inverted microscope. The patch pipettes were pulled from borosilicate glass tubing. The tips were coated with Sylgard 184 ® (Dow Corning Corp.). The bath and pipette solutions contained (mM): 140 KCl, 10 EGTA, 10 HEPES, pH 7.4. Macroscopic currents were recorded with a conventional patch-clamp technique . The glass tubing had an outer diameter of 2.0 mm and an inner diameter of 1.0 mm. The pipette resistance after fire polishing was 2–3 MΩ. The patches contained between several hundred and 2,000 active channels generating currents of as much as 2 nA at +100 mV. Currents of such magnitude may generate significant accumulation and depletion of ions near the membrane . When working with the same solution at both sides of the patch, a sensitive test for such concentration changes is to step the voltage from remote values (e.g., +100 mV) to 0 mV. Any concentration change would then cause a transient rebound current. We tried to keep the patch as close at the pipette tip as possible by minimizing suction. Under those conditions, it was possible to record ionic currents without noticeable rebound current at 0 mV. Only such patches are included in this study. Single-channel currents were recorded with a patch-clamp technique with improved resolution using short (8-mm total length) and thick-walled patch pipettes that were pulled from borosilicate glass tubing with an external and internal diameter of 2.0 and 0.5 mm, respectively. These pipettes were not fire polished. The technique of preparation of the thick-walled pipettes has been previously described in detail . The resistance of the pipettes used here was 30–50 MΩ. The thick-walled pipettes were used repeatedly by breaking the tips at the bottom of the chamber . The resistance of broken pipettes was 10–35 MΩ. All ionic currents through cGMP-gated channels were recorded in inside-out patches that were excised ∼30 s after the formation of a seal. The seal resistance exceeded 4 GΩ with the conventional patch pipettes and 50 GΩ with the thick-walled patch pipettes. cGMP-gated channels were activated by replacing the bath solution with a bath solution containing 3′,5′-cyclic guanosine monophosphate. Each excised patch was first exposed to a solution containing 700 μM cGMP to determine either the amplitude of the macroscopic current in macropatches or the number of active channels in single-channel experiments. Only patches were accepted in which removal of cGMP caused complete disappearance of channel activity. Recording was performed with an Axopatch 200A amplifier (Axon Instruments). Single-channel recordings were filtered online at a cut-off frequency of 2 or 5 kHz and were further filtered to the indicated final cut-off frequency with a Gaussian filter algorithm. Macroscopic currents were filtered at a cut-off frequency of 10 kHz. Recording and analysis of the data was performed on a PC-80486 with the ISO2 software (MFK Niedernhausen). Single-channel traces were sampled at 25 kHz, traces with macroscopic currents were sampled either at 10 kHz (20, 70, and 700 μM cGMP, 12-bit resolution) or 50 kHz (700 μM and 7 mM cGMP). Macroscopic currents were corrected for leak and capacitive components by subtracting respective currents in the absence of cGMP in the bath solution. All macroscopic currents considered herein are averages of 10–20 consecutive recordings. Amplitude histograms were built either in a conventional way, including all sampling points, or by using the variance-mean technique described by Patlak 1988 to improve resolution. In the latter case, transition points were eliminated by shifting a window of defined length along the traces, plotting the variance as function of mean current within the window, and discarding all original sampling points with variances above a defined threshold. The windows used here were 280 μs long. The threshold variance was set to the variance of the background noise. The fits were performed with a derivative-free Levenberg-Marquardt algorithm. Statistical data are given as mean ± SD. To test whether at low [cGMP] rectification is caused by a voltage-dependent incidence of sublevels at unchanged P o or by a voltage-dependent P o at unchanged single channel conductance, we recorded single-channel activity at high (70 μM) and low (7 μM) [cGMP] . At both [cGMP], the unitary current level was similar. The corresponding amplitude histograms were fitted with sums of Gaussian functions. The fit shows that at 7 μM cGMP the channels operated with the same conductance as at 70 μM. The same result was obtained in three other patches. Smaller or larger levels than the mean open level (sub- or superlevels, respectively) were observed only extremely rarely. Therefore, the higher degree of outward rectification of the IV relation at low versus high [cGMP] must be caused by a voltage-dependent open probability at unchanged single-channel current. Fig. 2 A shows representative single-channel currents at 7 μM cGMP at both −50 and +50 mV from a patch containing at least three channels. Comparison of the channel activity at the two voltages suggests two differences: at +50 mV, the frequency of openings was increased and longer events appeared more often than at −50 mV. The average currents indicate noticeable outward rectification. Amplitude histograms showed only the fully open level at each voltage (not shown). The open probability was evaluated in the following way: Gaussian curves were fitted to the amplitude histograms that contained all sampling points. The ratio between the area under the open level peak and the total area provides nP o ; for the patch in Fig. 2 , top, it was 0.041 at +50 mV and 0.026 at −50 mV. The ratio P o,rel = P o (+50 mV)/ P o (−50 mV) yields a relative measure of P o at +50 mV with respect to that at −50 mV (assuming that n is constant). P o,rel was 1.6 for the example shown in Fig. 2 A. The P o,rel at ±50 mV favorably compares with the ratio of the ensemble average currents I +50 /I −50 of 1.8. Together with the results of the previous paragraph, it has to be concluded that the pronounced outward rectification at low [cGMP] is primarily caused by an increase of P o at more positive voltages. Fig. 2 B shows recordings at 700 μM cGMP from a patch that contained only one active channel. From amplitude histograms, P o at +50 and −50 mV was calculated to be 0.97 and 0.81, respectively; hence P o,rel was 1.2. Since the single-channel current was only less different at +50 than at −50 mV (0.90 ± 0.17 vs. 0.84 ± 0.17 pA), the moderate outward rectification at saturating [cGMP] is also generated by the voltage dependence of P o . The previous section suggests that a larger P o at positive compared with negative voltages is caused by longer and more frequent opening events. We therefore analyzed the open-time distribution at ±50 mV as function of [cGMP]. At 70 and 700 μM cGMP, only patches containing one active channel were used, whereas at 7 and 20 μM cGMP, patches with several channels were used for the analysis to obtain a sufficiently large number of events. In these patches, the incidence of overlapping opening events was below 5%, which only negligibly modified the open-time distribution. Fig. 3 shows open-time histograms of recordings from two patches (7 and 700 μM cGMP; ±50 mV). All distributions were well described by the sum of two exponentials with contributions A 1 and A 2 and the time constants τ o1 and τ o2 , respectively. Consider the open time distribution at 7 μM cGMP and −50 mV. The vast majority of openings had a mean lifetime of roughly 1 ms; a small fraction [A 2 /(A 1 + A 2 ) ≈ 2%] of longer events had a mean lifetime of roughly 5 ms. At +50 mV, the mean open times of these two kinetically distinct events were either unchanged or even slightly shorter, but the channel opened more often and the longer opening events occurred more frequently [A 2 /(A 1 + A 2 ) ≈ 4%]. In the presence of 700 μM cGMP, the lifetimes of both short and long opening events were not much different when V m was −50 mV. Longer opening events occurred roughly four times more frequently [A 2 /(A 1 + A 2 ) ≈ 8%] than in the presence of 7 μM cGMP at the same voltage. However, switching the voltage from negative to positive values at 700 μM cGMP had a pronounced effect on gating kinetics: both τ o1 and τ o2 increased by roughly five- to sixfold and the contribution of the longer openings increased dramatically [A 2 /(A 1 + A 2 ) ≈ 21%]. Fig. 4 summarizes the results from nine similar experiments at [cGMP] of 7, 20, 70, and 700 μM. In Fig. 4 A, the time constants for the fast (τ o1 ) and slow (τ o2 ) exponentials are plotted as function of [cGMP]. The vertical lines connect data points of the same exponential and from the same patch at −50 (squares) and +50 (circles) mV. At 7 and 20 μM [cGMP], τ o1 and τ o2 do not significantly depend on voltage. At the higher concentrations of 70 and 700 μM, both τ o1 and τ o2 are significantly larger at +50 than at −50 mV (not clearly visible in the diagram for τ o1 at 70 μM cGMP). The voltage-dependent increase of τ o1 and τ o2 at high [cGMP] would suggest that the outward rectification becomes more pronounced at high rather than at low [cGMP], assuming an equal number of openings at both voltages. This contrasts with the experimental finding that outward rectification is steeper at low compared with high [cGMP]. The resolution of this apparent contradiction is that channels at low [cGMP] open much more often at +50 than at −50 mV. Since the evaluation of the contributions A 1 and A 2 at low [cGMP] was complicated by a variable number of channels in the patches, we evaluated for equal time intervals at each voltage the ratios A 1 (+50 mV)/A 1 (−50 mV) and A 2 (+50 mV)/A 2 (−50 mV). These ratios are independent of the number of channels. Fig. 4 B shows a plot of these ratios as function of [cGMP]. As anticipated, both ratios are largest at the lowest [cGMP] and smallest at the highest [cGMP]. At 7 μM cGMP, this implies that the channels open much more often at +50 than at −50 mV. Furthermore, the ratio A 2 (+50 mV)/A 2 (−50 mV) is approximately twice as large as the ratio A 1 (+50 mV)/A 1 (−50 mV). At [cGMP] of 70 and 700 μM, both ratios fall to values far below unity, indicative of a significantly smaller number of openings at +50 compared with −50 mV. This matches the longer open times at +50 compared with −50 mV. From Fig. 4 , three conclusions may be derived. (a) At high [cGMP], the low number of openings at +50 compared with −50 mV approximately compensates for the long openings. This result explains why rectification of the macroscopic current is weak. (b) At low [cGMP], τ o1 and τ o2 are independent of voltage, but the number of events in the distribution of both the fast and the slow openings considerably increases. The slow component A 2 is about twice as voltage dependent as the fast component A 1 . The steep outward rectification is therefore solely caused by a significant increase of the number of openings at positive voltages. (c) The fact that a large increase of τ o1 and τ o2 could be reached only at high [cGMP], and positive voltage implies a high degree of coupling between cGMP binding and voltage-dependent gating. Although the single-channel analysis provided criteria for appropriate kinetic models, the variability of the single-channel data did not allow a more precise analysis of the gating mechanism. We therefore performed experiments in macroscopic currents. Upon stepping the holding voltage of −100 mV to positive test voltages, two current components were observed at all [cGMP]: an instantaneous outwardly directed current was followed by a time-dependent current that increased to a new steady state level . When the voltage was returned to −100 mV, an inwardly directed instantaneous current was followed by a tail current that relaxed to the same current level as observed before the depolarizing step. In terms of P o , these observations may be interpreted as follows. The amplitude of the instantaneous current at the beginning of the test pulse is determined by P o at the holding voltage, whereas the amplitude of the steady state current at the end of the test pulse is determined by P o at the test voltage. In addition, the time-dependent current at the test voltage represents extent and kinetics of channel gating from P o at the holding voltage to P o at the test voltage (voltage-dependent activation). After stepping back to the holding voltage, the amplitude of the instantaneous current is determined by P o at the test voltage, whereas the amplitude of the steady state current is determined by P o at the holding voltage. Hence, the tail current at the holding voltage represents the extent and kinetics of the channel gating when changing from P o at the test voltage to P o at the holding voltage (voltage-dependent deactivation). At the test voltage, the amplitude ratio of the steady state current with respect to the instantaneous current must equal the amplitude ratio of the instantaneous tail current with respect to the steady state current at the holding voltage. This ratio is defined by how many times P o at the test voltage is larger than P o at the holding voltage. At 20 μM cGMP, the amplitude of the activating current component was clearly larger than that of the instantaneous current component, whereas at 7 mM cGMP it was much smaller. This indicates a stronger effect of voltage on channel gating at low compared with high [cGMP]. Fig. 5 also shows that the kinetics of the activating and deactivating current component became greatly accelerated at increased [cGMP], but was largely independent of voltage (see also below). To discriminate between different state models describing the gating of CNG channels, the relation between the absolute P o and [cGMP] was determined. Because in single-channel recordings determination of P o does not provide the required accuracy, we measured the dose–response relation of P o in macroscopic currents with three different methods as follows: (a) At large P o , its exact value can be determined by the analysis of stationary noise. The noise variance σ 2 is related to the amplitude of the mean current I by: 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*}{\mathrm{{\sigma}}}^{2}={iI-I^{2}}/{n{\mathrm{,}}}\end{equation*}\end{document} where i denotes the single-channel current and n the total number of channels. The single-channel current i was determined from single-channel recordings, σ 2 and I were determined from macroscopic currents. The background noise variance was subtracted. These two measurements then yield ni , the maximum current that is obtained when all channels are open. The ratio I /( ni ) gives the P o at the respective [cGMP]. The contribution of shot noise and Johnson noise to the total noise was calculated to be negligible. For large values of P o , the variability in the results was remarkably low. This is illustrated by the dose–response relationship of Fig. 6 A, where the small error bars (SD) are hidden in the respective symbols for the data at +100 mV/700 μM [cGMP], +100 mV/7 mM [cGMP], and −100 mV/7 mM [cGMP]. The data at −100 mV/700 μM [cGMP] and +100 mV/70 μM [cGMP] were also determined with the noise analysis, albeit the SD was larger. (b) The data points at +100 mV/7 μM [cGMP], −100 mV/7 μM [cGMP], and +100 mV/20 μM [cGMP] were obtained by recording first the steady state current in multichannel patches and thereafter at +100 mV/700 μM [cGMP]. The amplitudes of the currents at the lower concentrations were normalized in each individual patch with respect to the amplitude of the current at 700 μM [cGMP] measured in the same patch. The mean was calculated at each concentration and final values in the diagram were obtained by considering that P o was 0.93 at +100 mV/700 μM [cGMP]. (c) The data points at −100 mV/70 μM [cGMP] and −100 mV/20 μM [cGMP] were calculated from voltage step protocols as shown in Fig. 5 . As described in the previous section, the amplitude ratios were formed from the instantaneous current with respect to the steady state current after stepping to +100 mV and from the steady state current with respect to the instantaneous current after stepping back to −100 mV. Finally, the mean ratios at 20 and 70 μM [cGMP] were multiplied with the values of the respective data points at +100 mV. Fig. 6 A shows that at −100 mV the dose–response relation constructed in this way saturated at significantly lower P o and was slightly shifted to higher [cGMP] with respect to the dose–response relation at +100 mV. The experimental data provide five constraints for a kinetic model. (a) Even at the highest [cGMP] (700 μM or 7 mM) and the most positive voltage (+100 mV), P o does not reach unity. This observation implies that channels close by a reaction whose equilibrium is independent of [cGMP]. (b) At −100 mV, the maximal P o value at saturating [cGMP] is considerably lower than that at +100 mV. A corollary of this observation is that one voltage-dependent reaction must exist that follows the binding of cGMP. (c) The effect of voltage on P o is large at low [cGMP] and small at high [cGMP]. This result can be explained by coupling between the action of [cGMP] and voltage. (d) In a total of 24 experiments similar to those illustrated in Fig. 5 , the time course of activation did not show any delay. This observation indicates that only a single voltage-dependent reaction is rate limiting for channel opening. (e) The channels operate with only a single conductance. With these constraints, two types of models are reasonable: allosteric and sequential models. Fig. 1 illustrates an allosteric model with four cGMP binding reactions as used previously to describe gating of CNG channels . The opening reaction was assumed to be voltage dependent. The rate constant for an individual cGMP-dependent reaction is given by m[cGMP] k 1 with m = 1…4. The reverse reaction is given by (4 − m + 1) k 2 . The voltage-dependent rate constants k 3 and k 4 were assumed to be k 3,0 exp (0.5 zF V m / RT ) and k 4,0 exp (−0.5 zF V m / RT ), respectively. k 3,0 and k 4,0 are the rate constants at V m = 0 mV; F is the Faraday constant, V m the transmembrane voltage, R the gas constant, T the absolute temperature, z the effective gating charge, and f the allosteric factor. The value of 0.5 indicates a symmetric barrier for the voltage-dependent reaction. This assumption is without relevance for all fits of steady state properties. T was 298 K. For the fit of the dose–response relations, the allosteric factor was set to 10 because with our model this value predicts a spontaneous P o (in the absence of cGMP) of 10 −4 , similar to the measured value of 1.25 × 10 −4 . The three free parameters were k 1 / k 2 , k 3,0 / k 4,0 , and z . The curves in Fig. 6 A illustrate that the allosteric model describes the data well, which also confirms the assumption of four cGMP binding reactions because the number of binding sites determines the steepness of the dose–response relation. The parameters were k 1 / k 2 = 2.00 × 10 4 M −1 , k 3,0 / k 4,0 = 8.24, z = 0.21. Fig. 2 shows a corresponding sequential model with four cGMP binding reactions in which the channel has to be fully liganded before a voltage-dependent reaction causes opening (termed “G 4 O model”). The meaning of all parameters and constants is the same as in the allosteric model, apart from the absence of the allosteric factor f . The sequential model equals the allosteric model in the limit that the allosteric factor reaches infinity. We tried to fit the dose–response relations with sequential models including between two and five cGMP binding reactions. Fits with similar quality as with the allosteric model were obtained with the binding of three cGMP molecules (termed “G 3 O model”) and four cGMP molecules, whereas the fits were worse with two and five cGMP molecules. The parameters from the fit with the G 4 O model are k 1 /k 2 = 2.65 × 10 4 M −1 , k 3,0 / k 4,0 = 7.95, z = 0.22 (fit not shown). To test whether the allosteric, the G 3 O, and the G 4 O models correctly predict P o for voltages between −100 and +100 mV, we evaluated the amplitude of deactivating tail currents at −100 mV, following respective test pulses. P o was determined by relating the tail current amplitude at +100 and −100 mV to the respective P o values obtained above. This analysis was performed at 20, 70, 700 μM, and 7 mM cGMP . The plot of the data points as function of the test pulse voltage shows that the voltage dependence was steepest in the negative branch of the voltage range at high [cGMP] and in the positive branch at low [cGMP]. These four P o V relations were fitted simultaneously with each of the three models. The allosteric model , which included the allosteric factor f as fourth free parameter, yielded k 1 / k 2 = 1.9607 × 10 4 M −1 , k 3,0 / k 4,0 = 7.84, z = 0.23, f = 10.24 . Interestingly, the fit converged with an allosteric factor close to the value assumed above. A similarly reasonable fit was obtained with the G 3 O model ( k 1 / k 2 = 1.63 × 10 4 M −1 , k 3,0 / k 4,0 = 7.77, z = 0.23). In contrast, with the G 4 O model the predicted P o value was too small at positive voltages and 20 μM cGMP. Assuming independent binding of four cGMP molecules, this result shows that the allosteric model is superior over the corresponding sequential G 4 O model. The fit of the dose–response relations yielded only values for the ratios k 1 / k 2 and k 3,0 / k 4,0 . The individual rate constants of the voltage-dependent reaction ( k 3,0 and k 4,0 ) were determined as follows: at the saturating [cGMP] of 7 mM , it is reasonable to assume that all channels are fully liganded. In our models, voltage-dependent activation should then obey a monoexponential time course with the time constant τ = 1/( k 3 + k 4 ). Fig. 7 shows normalized time courses of the activating component of current at +40 and +100 mV and 7 mM [cGMP] together with theoretical curves of best fits with a single exponential. Monoexponential functions described the measured time courses adequately. In all experiments, these time course were slower at +40 mV compared with +100 mV. As time constants τ, we obtained 535 ± 116 μs and τ = 436 ± 41 μs (mean ± SD; n = 4), respectively. For the allosteric model, the values of k 3,0 and k 4,0 were then calculated with k 3,0 / k 4,0 = 7.84 and z = 0.23 according to: 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_{3,0}=\; \left {\mathrm{{\tau}}} \right \left \left[{\mathrm{exp}} \left \left({0.115{\times}\;F{\mathrm{V}}_{{\mathrm{m}}}}/{{\mathrm{RT}}}\right) \right \;+{\mathrm{exp}}{ \left \left(-{0.115{\times}\;F{\mathrm{V}}_{{\mathrm{m}}}}/{{\mathrm{RT}}}\right) \right }/{ \left \left 7.84\right] \right \right ^{-1}{\mathrm{.}}} \right \end{equation*}\end{document} All symbols have the same meaning as described above. From the τ value at +100 mV, we calculated k 3,0 = 1.39 × 10 3 s −1 and k 4,0 = 1.67 × 10 2 s −1 . Similar values of the rate constants k 3,0 and k 4,0 were obtained from the τ value at +40 mV. Similar values were also obtained for the G 3 O and G 4 O models. Knowing k 3,0 and k 4,0 , we attempted to determine the parameters k 1 and k 2 , rather than their ratio, by simultaneously fitting the allosteric model to current traces after voltage steps at three different [cGMP] . Although there was only one free parameter left, the fit did not converge and therefore did not allow us to derive a unique set of rate constants. However, it did allow us to estimate a lower limit for the rate constants k 1 and k 2 . A reasonable description of the measured traces was obtained with k 1 > 3 × 10 7 M −1 s −1 and k 2 > 1.5 × 10 3 s −1 (with k 1 /k 2 < 22 × 10 4 M −1 ). The consequence is that for all tested [cGMP] between 20 μM and 7 mM, neither the rate of cGMP binding nor that of cGMP unbinding is limiting for the time course of voltage-dependent activation and deactivation. Fig. 8 B shows theoretical currents calculated with the allosteric model; k 1 and k 2 were set to 3 × 10 7 M −1 s −1 and 1.5 × 10 3 s −1 , respectively. The theoretical currents were scaled with respect to the measured single-channel current i at ±100 mV (dotted lines). Simulation of current traces as shown in Fig. 8 A with both the G 3 O and the G 4 O models yielded similarly reasonable results (not shown). Previous studies reported the occurrence of sublevels . In our experiments, the incidence of sublevels was very low at all [cGMP]; hence, sublevels cannot significantly contribute to macroscopic currents through the rod cGMP-gated channel α subunit. This conclusion also holds for the salamander rod cGMP-gated channel, which maximally spends 5–10% of its time in a state of small conductance of <5–6 pS . Sublevels in the salamander channel occurred more frequently at low [cGMP] and were virtually absent at [cGMP] > 50 μM . The native rod cGMP-gated channel is composed of two distinct subunits, a smaller α subunit and a larger β subunit . The β subunit is also engaged in ligand binding , pore formation, and gating , suggesting that cGMP-dependent sublevels observed in the native channel are imparted by the β subunit. Ruiz and Karpen 1997 , however, observe a high incidence of two sublevels that contribute roughly 50% to P o of the heterologously expressed α subunit . These authors also show that sublevels result from channel species that have fewer than the maximal number of four cGMP molecules bound. The reason for the significant differences between their and our observations is not clear. Changing the voltage at low [cGMP] primarily affects the frequency of opening and the relative abundance of longer open periods, whereas the two mean open times are largely equal. The finding of two components in the open-time distribution implies the existence of at least two open states. In the modeling strategy, these open states were lumped to one open state because of a lack of stringent criteria on how to include these open states in the model (see below). At low [cGMP], and correspondingly small P o , the open times were independent of voltage . This result does not conflict with the voltage-dependent gating described in the macroscopic currents because the relatively low degree of voltage dependence found in the macroscopic currents ( z = 0.22) is hidden in the variability of the open time histograms. At high [cGMP], P o increased to large values. Changing then the voltage from negative to positive values increased the mean open times dramatically and, concomitantly, decreased the frequency of openings. The increase of the open times at high [cGMP] and positive voltage cannot be explained by an unbinding reaction of cGMP from the channel, which should be independent of the [cGMP]. The most likely interpretation of the prolonged open times at elevated [cGMP] is that they result from unresolved closures due to an immediate reopening when the channel is fully liganded. Hence, the rate constant of the voltage-dependent opening reaction must be very rapid. In conclusion, the single channel experiments suggest a kinetic model in which channel opening is the result of one or more cGMP binding steps followed by at least one voltage-dependent step. However, the variability of the single-channel data did not allow us to derive stringent criteria for modeling of the cGMP- and voltage-dependent gating. This was only possible with macroscopic currents. A plot of the normalized current through CNG channels (response) as function of the [cGMP] (dose) is the standard and most simple procedure to determine both the [cGMP] of half maximum activation and the number of the cGMP molecules binding to a channel . The exact measurement of P o is not trivial. In the single channel experiments, the accuracy is limited by the typical variability inherent in single-channel measurements. In macroscopic currents, determination of P o requires knowledge of the limiting P o value at saturating [cGMP]. We determined P o at saturating (and also slightly lower) [cGMP] with stationary noise analysis because at high P o its value does not critically depend on the exact value of the single-channel current I. The position of the dose–response relation at −100 mV was determined with respect to that at +100 mV by evaluating the instantaneous and steady state currents at one voltage (either −100 or +100 mV). In this way, the error introduced by the uncertainty in the determination of the IV relation of the single-channel current was avoided. As a consequence, our dose–response relations for the absolute value of P o at +100 and −100 mV provided more constraints to discriminate among kinetic models than normalized relations used previously. In our modeling strategy, we tested an allosteric model with four cGMP binding reactions and two sequential models containing three (G 3 O model) or four cGMP binding reactions. All three models fitted the steady state dose–response relations of the absolute P o at +100 and −100 mV and they produced reasonable time-dependent currents in response to voltage steps . However, when fitting the P o V relations , the allosteric and G 3 O models were superior to the G 4 O model. Including furthermore that CNG channels may open even in the absence of cGMP and that four α subunits, containing one cGMP-binding site each , form one functional CNG channel , the allosteric model seems to be more adequate than each of the sequential models. Interestingly, the allosteric factor f was determined to be very similar to that obtained from measurements in the absence of cGMP. With respect to the rate constants describing cGMP binding ( k 1 ) and unbinding ( k 2 ), our models allowed us only to determine the ratio k 1 / k 2 and to estimate lower limits for the absolute values. The reason for this indeterminateness is that in the case of rapid binding and unbinding the activation time course is defined by the relative occupancy of the last closed states before opening in conjunction with the kinetics of the C ⇔ O transition. The finding of two open times, suggesting at least two open states, was not included in our models because we did not have sufficient criteria to specify a model with two open states. In all our models, τ o calculates to be 1/ k 4 . At +50 and −50 mV, the predicted values for τ o are 7.2 and 4.8 ms. Interestingly, these values approximately match the range of the slow mean open time τ o2 in the single-channel recordings. It may therefore be speculated that the C ⇔ O transition, whose closing reaction generates τ o2 , notably contributes to the voltage-dependent activation. With respect to the fast closing reactions corresponding to τ o1 , one possibility is that the open channel closes to an additional closed state, similar to a channel block. For the allosteric model, we can exclude that the rate constants of the transitions O 4 ⇒ C 4 and O 5 ⇒ C 5 correspond directly to 1/τ o1 and 1/τ o2 , respectively, because at saturating [cGMP], when only C 5 ⇔ O 5 is occupied, the channel should open predominantly with long openings, also at negative voltages. For the fit of the open time histograms , this would mean that A 2 × τ o2 >> A 1 × τ o1 . This relation was not observed. Linear state models were repeatedly used to interpret gating of CNG channels . Voltage dependence of this gating was studied in channels of retinal rods by Karpen et al. 1988 who analyzed relaxation kinetics of macroscopic currents in response to voltage steps. These authors proposed a linear state model similar to the G 3 O model used here: during the process of activation, three cGMP molecules sequentially bind to the channel and only the fully liganded channel may open by a voltage-dependent reaction. Both the experimental results and several assumptions used for modeling were different between the results of Karpen et al. 1988 in native channels of salamander rods and the present results in homooligomeric channels formed from α subunits only. (a) At all [cGMP], the activation time course in the native channels was severalfold faster than that in our currents. The reason for this difference is not clear. Possible explanations are: different subunit compositions (heterooligomeric channels formed by α and β subunits versus homooligomeric channels formed by the α subunit only) and different species (salamander retinal rods versus bovine α subunit). (b) We related our models to measured absolute values of steady state P o , whereas Karpen et al. 1988 used relative currents with respect to the current at saturating concentrations. (c) In the model of Karpen et al. 1988 , cGMP binding to the three binding sites was not assumed to be independent. In contrast, our model was based on the assumptions of independent cGMP binding. To our knowledge, more detailed experimental data to differentiate between the cGMP-binding reactions are not available. (d) In the model of Karpen et al. 1988 , the entire voltage sensitivity was attributed to the closing reaction, whereas we used models with a symmetric barrier for the voltage-dependent reaction. In the case of experimental evidence for opening of partially liganded channels, allosteric models are favored for interpretation . These models generally allow opening from all closed states and the probability for opening increases in proportion to the number of ligands bound. A special case of such a model was used by Taylor and Baylor 1995 to interpret single-channel data of CNG channels of salamander rods: during the process of activation, three cGMP molecules sequentially bind to the channel and the channel may open in a voltage-dependent reaction with either two or three cGMP molecules bound. In contrast to Karpen et al. 1988 , the entire voltage sensitivity was attributed to the rate constant of the opening reaction. It is noteworthy that the time constant of relaxation at saturating [cGMP] was only 22 μs . This value basically agrees with the fast kinetics reported by Karpen et al. 1988 in the same channels, but is much faster than the corresponding activation kinetics observed in our homooligomeric channels (τ = 535 μs at +40 mV). An explanation for the faster activation time course in the native cells is that they contain additional factors (possibly subunits) that are not present in our experiments. Interestingly, an allosteric model of the type used herein was also successful to describe the gating of large conductance Ca-activated K + channels . These channels show several structural and functional similarities with the CNG channels: they belong to the same S4 helix–containing superfamily and they are activated by both voltage and the binding of a ligand to the COOH terminus. Though there is also an essential difference in function (Ca-activated K + channels can be activated by voltage nearly maximally even in the absence of Ca 2+ , whereas CNG channels can practically not be activated in the absence of cGMP), the usefulness of the allosteric model to describe gating properties in both channel types suggests a uniform gating process. | Study | biomedical | en | 0.999996 |
10498669 | The so-called persistent sodium current ( I NaP ) 1 is known to be expressed, together with the classical, transient sodium current ( I NaT ), by numerous mammalian neuronal types. Its basic features include persistence during prolonged depolarizations, lower threshold of activation than I NaT , and low amplitude (the underlying conductance normally representing 0.2–2% of the total sodium conductance; French and Gage 1985 ; French et al. 1990 ; for review see Taylor 1993 ; Crill 1996 . After the initial descriptions of the actions of sustained Na + currents on neuronal electroresponsiveness , I NaP was first demonstrated with voltage-clamp studies in neocortical neurons , and biophysically characterized in the hippocampus . I NaP has also been observed, directly or indirectly, in an increasing number of neuronal structures including basal ganglia , amygdala , thalamus , hypothalamus , cerebellum , and peripheral ganglia . Due to its voltage-dependent properties, I NaP can contribute to important integrative functions such as amplification of excitatory postsynaptic potentials , generation of pacemaker activity , and firing-pattern shaping . Since I NaP can generate membrane bistability and plateau potentials, it has also been implicated in the pathogenesis of some forms of epilepsy . In addition, the ability of I NaP to sustain long-lasting Na + influxes, and therefore to steadily increase intracellular Na + concentration, has raised interest as to its possible role in mechanisms of neurodegeneration . The entorhinal cortex (EC) has proven to be a particularly interesting neuronal system for the study of I NaP functions. In the stellate cells of EC layer II, which give rise to the main cortical projection to the hippocampus , I NaP has been shown to critically participate in the generation of subthreshold membrane-potential oscillations in the theta-rhythm range . This particular intrinsic subthreshold activity is considered to be a critical determinant for the generation of the population theta oscillations generated by the EC network . The theta rhythm has been shown to contribute to synaptic-plasticity processes , and it is thus believed to play a major role in temporal lobe learning and memory functions . On the other hand, since the EC is also known to play a crucial role in temporal lobe epileptogenesis , it has been hypothesized that the presence of a robust I NaP in EC neurons may contribute to epileptogenic processes . For the above considerations, a detailed knowledge of the biophysical properties of I NaP expressed by EC layer II neurons seems of great interest since it would help to understand how this current specifically influences the physiological behavior of these cells. In this study, we have characterized I NaP in both acutely isolated and in situ EC layer II neurons. Our results revealed the existence of some interesting biophysical properties of I NaP that had not been thoroughly investigated yet, including: (a) a slow voltage- and time-dependent inactivation occurring with voltage-dependent time constants in the order of seconds; (b) a full inactivation at sufficiently positive potentials; and (c) a truly persistent, or “window,” current arising from the particular activation and steady state inactivation properties of the corresponding conductance. Moreover, we describe the single-channel events that account for all of the above-mentioned macroscopic phenomena. Young-adult Long-Evans rats (P25–P35) were killed by decapitation. The brain was quickly removed under hypothermic conditions, blocked on the stage of a vibratome (Pelco), and submerged in an ice-cold cutting solution containing (mmol/liter): 115 NaCl, 5 KCl, 4 MgCl 2 , 1 CaCl 2 , 20 PIPES, and 25 d -glucose, pH 7.4 with NaOH, bubbled with pure O 2 . Osmotic pressure (π) of the latter solution, as measured with a Micro Osmette 5004 osmometer, was typically 320 mOsm. Horizontal slices of the retrohippocampal region were cut at 350–400 μm. For in situ recordings, slices were stored at room temperature in the above solution until use. For recordings on isolated neurons, the layer II of medial entorhinal cortex was dissected from each slice . Neurons were acutely isolated from the tissue fragments thus obtained following an enzymatic and mechanical dissociation procedure described elsewhere . The recording chamber was mounted on the stage of an upright microscope (see below). Slices were laid onto the bottom of the chamber and perfused with an extracellular solution containing (mmol/liter): 34 NaCl, 26 NaHCO 3 , 80 tetraethylammonium (TEA)-Cl, 5 KCl, 3 CsCl, 2 CaCl 2 , 3 MgCl 2 , 2 BaCl 2 , 2 CoCl 2 , 0.4 CdCl 2 , 4 4-aminopyridine (4-AP), 10 glucose, pH 7.4 when bubbled with 95% O 2 , 5% CO 2 (π ≈ 320 mOsm). The association of Co 2+ and Cd 2+ was found to depress residual inward rectification insensitive to the application of tetrodotoxin (TTx; Sigma-Aldrich Canada Ltd.) more effectively than Cd 2+ alone in slices (but not in freshly dissociated neurons; see below). Preliminary experiments were performed in in situ neurons in the absence of extracellular Co 2+ and Ba 2+ and in the presence of 0.2 rather than 0.4 mM Cd 2+ . Both average peak amplitude and current density of ramp (50 mV/s)-evoked, TTx-subtracted I NaP s recorded under the former and latter ionic conditions (−179.3 ± 120.7 vs. −195.8 ± 100.0 pA, respectively, and −17.7 ± 29.1 vs. −12.9 ± 10.6 pA/pF, respectively, n = 54 and 8, respectively) were not significantly different ( P = 0.68 and 0.63, respectively). Hence, possible enhancing effects of nonphysiological extracellular divalent cations on I NaP amplitude did not significantly affect measures in our experimental conditions. Patch pipettes were fabricated from thick-wall borosilicate glass capillaries by means of a P-97 horizontal puller (Sutter Instruments Co.). The intrapipette solution contained (mmol/liter): 110 CsF, 10 HEPES-Na, 11 EGTA, 2 MgCl 2 , pH 7.25 with CsOH (π adjusted to ∼290 mOsm with mannitol). When filled with the above solution, the patch pipettes had a resistance of 3–5 MΩ. Slices were observed with an Axioskop microscope (Carl Zeiss, Inc.) equipped with a 40× water-immersion objective lens and differential-contrast optics. A near-infrared charge-coupled device camera (XC-75; Sony Corp.) was also connected to the microscope, and used to improve cell visualization for identification of neuron types and during the approaching and patching procedures. With this equipment, the principal cells of EC layer II were easily distinguished based on their somato-dendritic shape , size, and position . Patch pipettes were brought in close proximity to the selected neurons while manually applying positive pressure inside the pipette. Tight seals (>100 GΩ) and the whole-cell configuration were obtained by suction . Series resistance ( R s ) was always compensated by ∼55% with the amplifier's built-in compensation section. R s , as estimated off-line from the peak amplitude of averaged capacitive transients evoked by −5-mV voltage square pulses (with the low-pass filter set at 10 kHz), was on average 8.5 ± 2.1 MΩ ( n = 54). Cell capacitance was evaluated online by canceling the fast component of whole-cell capacitive transients evoked by −10-mV voltage steps with the amplifier compensation section, and reading out the corresponding value. Voltage-clamp recordings were performed at room temperature (∼22°C) using an Axopatch 1D amplifier (Axon Instruments). The general holding potential was −80 mV. The recording chamber was mounted on the stage of an inverted microscope (see below). After seeding into the chamber, dissociated cells were perfused with a standard HEPES buffer containing (mmol/liter): 140 NaCl, 5 KCl, 10 HEPES (free acid), 2 CaCl 2 , 2 MgCl 2 , 25 glucose, pH 7.4 with NaOH, bubbled with pure O 2 (π ≈ 320 mOsm). After wash-out of cell debris, cell perfusion was switched to a solution suitable for Na + -current isolation containing (mmol/liter): 100 NaCl, 40 TEA-Cl, 10 HEPES (free acid), 2 CaCl 2 , 3 MgCl 2 , 0.2 CdCl 2 , 5 4-AP, 25 glucose, pH 7.4 with NaOH, bubbled with pure O 2 (π ≈ 318 mOsm). The intrapipette solution was the same as described in the previous paragraph. Cells were observed at 400× with an Axiovert 100 microscope (Carl Zeiss, Inc.). After tight-seal formation (>100 GΩ) and the establishment of the whole-cell configuration, series resistance was on average 12.0 ± 4.5 MΩ ( n = 38), and was always compensated by ∼70%. The remaining procedures and experimental conditions were the same as described in the previous paragraph. Single-channel, cell-attached experiments were performed in acutely isolated neurons. After seeding into the recording chamber, cells where initially perfused with the same solution as described in the previous paragraph. The pipette solution contained (mmol/liter): 130 NaCl, 35 TEA-Cl, 10 HEPES-Na, 2 CaCl 2 , 2 MgCl 2 , 5 4-AP, pH 7.4 with HCl (π ≈ 338 mOsm). Single-channel patch pipettes had resistances ranging from 10 to 35 MΩ when filled with the above solution, and were always coated with Sylgard ® (Dow Corning Corp.) from the shoulder to a point as close as possible to the tip so as to minimize stray pipette capacitance. After obtaining the cell-attached configuration, the extracellular perfusion was switched to a high-potassium solution containing: 140 K-acetate, 5 NaCl, 10 HEPES (free acid), 4 MgCl 2 , 0.2 CdCl 2 , 25 glucose, pH 7.4 with KOH (π ≈ 320 mOsm) so as to hold the neuron resting membrane potential at or near 0 mV. Recordings were performed at room temperature with an Axopatch 200B amplifier (Axon Instruments). Capacitive transients and linear current leakage were minimized online by acting on the respective built-in compensation sections of the amplifier. Long-duration (20-s) depolarizing voltage steps were delivered one every 40 s from a holding potential of −80 or −100 mV. Voltage protocols were commanded and current signals were acquired with a Pentium PC interfaced to an Axon TL1 interface, using the Clampex program of the pClamp 6.0.2 software (Axon Instruments). Current signals were filtered online (using the amplifier's built-in low pass filter) and digitized at different frequencies according to the specific experimental aim. Filtering and acquisition frequencies were 5 and 20 kHz, respectively, for I NaT recordings; 0.1–1 and 0.67–10 kHz (depending on the protocol duration), respectively, for I NaP recordings; 1 and 2 kHz, respectively, for single-channel recordings. In all of the voltage protocols applied, cell-membrane potential was kept at the holding level for 15 (in whole-cell experiments) or 20 s (in single-channel experiments) between the end of each sweep and the beginning of the subsequent sweep (or of the conditioning prepulse preceding it, when applied). This avoided the development of cumulative voltage-dependent inactivation of I NaP during consecutive acquisition cycles. Whole-cell recordings were analyzed by means of the Clampfit program (Axon Instruments). Offline leak subtraction was performed on I NaT - (but not I NaP -) protocol traces. Current density was calculated by dividing the peak current amplitude by cell capacitance, estimated as explained above. Conductance values were calculated from Na + -current amplitudes by applying the extended Ohm's law in the form: G Na = I Na /(V – V Na ), where V Na is the nominal (Nernst) Na + reversal potential. Data were fitted with exponential functions, I = ( A i · exp(− t /τ i ) + C , using Clampfit, or with Boltzmann functions, G = G max /{1 + exp[(V − V 1/2 )/ k )]}, using Origin 3.06 (MicroCal Software). Single-channel recordings were analyzed using Clampfit, Fetchan, and pStat (Axon Instruments). Residual capacitive transients were nullified by offline subtracting fits of average blank traces. Residual leakage currents were carefully measured in every single sweep at trace stretches devoid of any channel openings, and digitally subtracted. Channel dwell times were determined using a standard threshold routine of the Fetchan program. Ensemble-average traces were fitted with single exponential functions, I = A · exp(− t /τ) + C , using Clampfit. Dwell-time histograms were fitted with double exponential functions, N = A 1 · exp(− t /τ 1 ) + A 2 · exp(− t /τ 2 ), using pStat. Average values were expressed as mean ± SD, unless otherwise explicitly stated. Statistical significance was evaluated by means of the two-tail Student's t test for unpaired data. For a phenomenological description of I NaP activation and slow inactivation, a simple Hodgkin-Huxley model was assumed. We applied the basic relationship: 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*}I_{{\mathrm{NaP}}} \left \left({\mathrm{V}},t\right) \right =G_{{\mathrm{NaP}}} \left \left({\mathrm{V}},t\right) \right {\cdot} \left \left({\mathrm{V}}-{\mathrm{V}}_{{\mathrm{Na}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where 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*}G_{{\mathrm{NaP}}} \left \left({\mathrm{V}},t\right) \right =G_{{\mathrm{NaP}} \left \left({\mathrm{max}}\right) \right }{\cdot}m_{{\mathrm{{\infty}}}} \left \left({\mathrm{V}}\right) \right {\cdot}h \left \left({\mathrm{V}},t\right) \right {\mathrm{,}}\end{equation*}\end{document} and m and h are the probabilities of the activating and inactivation particles, respectively, to be in the permissive position. I NaP activation was assumed to be instantaneous, and m ∞ (V) was derived directly from the G NaP activation curve. h ∞ (V) was derived directly from the G NaP steady state inactivation curve. The transitions of the inactivating particle, h , were modeled according to the following first-order kinetic scheme: \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}{\mathrm{{\alpha}}}\\ 1-h\end{matrix}{\Leftrightarrow}\begin{matrix}h\\ {\mathrm{{\beta}}}\end{matrix}\end{equation*}\end{document} from which it follows: 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*}h \left \left({\mathrm{V}},t\right) \right =h_{{\mathrm{{\infty}}}} \left \left({\mathrm{V}}\right) \right - \left \left[h_{{\mathrm{{\infty}}}} \left \left({\mathrm{V}}\right) \right -h_{0}\right] \right {\cdot}{\mathrm{exp}} \left \left(-{t}/{{\mathrm{{\tau}}}_{{\mathrm{h}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} where 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*}{\mathrm{{\tau}}}_{{\mathrm{h}}} \left \left({\mathrm{V}}\right) \right ={1}/{ \left \left[{\mathrm{{\alpha}}} \left \left({\mathrm{V}}\right) \right +{\mathrm{{\beta}}} \left \left({\mathrm{V}}\right) \right \right] \right }\end{equation*}\end{document} and 5 \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*}h_{{\mathrm{{\infty}}}} \left \left({\mathrm{V}}\right) \right ={{\mathrm{{\alpha}}} \left \left({\mathrm{V}}\right) \right }/{ \left \left[{\mathrm{{\alpha}}} \left \left({\mathrm{V}}\right) \right +{\mathrm{{\beta}}} \left \left({\mathrm{V}}\right) \right \right] \right }{\mathrm{.}}\end{equation*}\end{document} Numerical values for the rate constants, α and β, were derived from the experimental values of time constants of inactivation and recovery from inactivation (τ h ) and from the h ∞ curve by applying and . After obtaining the analytical functions describing the voltage dependence of the rate constants (see results ), the time course of I NaP s activated in response to various voltage protocols was numerically reconstructed on the basis of the and , and in its differential form. The simulation programs were compiled using QuickBASIC 4.5 (Microsoft Corp.). In acutely dissociated EC layer II principal neurons, a prominent I NaP could be evoked by delivering long-lasting depolarizing pulses. Fig. 1 A illustrates current traces from a representative neuron. I NaP was blocked by 1 μM TTx, and was therefore routinely isolated via TTx subtraction. I NaP threshold of activation was at about −65 mV, and peak at −30 mV. Average I NaP absolute amplitude (derived by averaging the data points between 400 and 500 ms from the pulse onset) and current density (calculated as explained in materials and methods ) were −96.5 ± 61.5 pA and −12.0 ± 7.0 pA/pF, respectively, at the peak of the current-voltage (I–V) relationship ( n = 5). The average I NaP I–V relationship showed a linear region from −30 to −5 mV, the linear best fitting of which returned a zero-current level at +63.0 mV (not shown). This value compares favorably with the theoretical Nernst Na + reversal potential calculated for our ionic conditions (V Na = +61.0 mV). The voltage dependence of the conductance underlying I NaP ( G NaP , calculated as explained in the materials and methods ) is shown in the average plot of Fig. 1 A, inset. Boltzmann fitting to data points returned a half-activation voltage, V 1/2 , of −44.4 mV, and a slope factor, k , of −5.2 mV. The ratio between the peak G NaP value found in each cell and the maximal value of the conductance underlying the transient Na + current ( I NaT ) expressed by the same cell was also calculated, and averaged 0.0187 ± 0.0097 ( n = 5). To quickly explore the whole voltage range of I NaP activation, ramp protocols were then used. Slow ramps at 50 mV/s were initially selected since they allowed full inactivation of fast-decaying Na + -current component(s). Fig. 1 B shows the currents evoked by such a protocol in a representative acutely dissociated neuron, both in control conditions and in the presence of 1-μM TTx (inset). Offline digital subtraction returned the TTx-sensitive I NaP in isolation . The continuous I–V relationship thus obtained showed a threshold at −70/−60 mV and a peak at −40/−30 mV. Noteworthy in both pulse and ramp protocols, I NaP activation was accompanied by an evident increase in current noise, especially at voltage levels close to the peak of the I–V relationship , consistent with the relatively high conductance (∼20 pS) characterizing the channels responsible for I NaP generation in EC layer II neurons . Ramp protocols were also used in experiments performed in in situ neurons. In this situation, TTx subtraction always returned prominent I NaP s in isolation, whose I–V relationship closely resembled that of I NaP s in acutely dissociated neurons . I NaP amplitude, measured at the peak of the I–V relationship, was significantly higher in in situ than in isolated neurons (−179.3 ± 120.7 pA, n = 54, vs. −65.6 ± 37.9 pA, n = 38; P < 5 × 10 −7 ), whereas the current density did not significantly differ in the two situations (−17.7 ± 29.1 pA/pF, n = 54, vs. −16.5 ± 8.6 pA/pF, n = 38; P = 0.8). These findings strongly suggest that the channels responsible for I NaP are located not only on the soma, but also on neuronal processes severed by the dissociation procedure. Activation curves of I NaP s recorded in both in situ and isolated neurons were also constructed. Conductance values were derived from I NaP s by applying the extended Ohm's law (see materials and methods ), and the resulting activation curves were fitted with single Boltzmann functions . Average half-activation potentials and slope factors were very similar in in situ neurons (V 1/2 = −51.3 ± 3.9 mV, k = −4.0 ± 0.7 mV, n = 39) and isolated neurons (V 1/2 = −48.7 ± 4.7 mV, k = −4.4 ± 0.9 mV, n = 19). These values compare favorably with those obtained from step protocols (see above), and are also in good agreement with the activation parameters previously reported for I NaP s expressed in other neuronal systems . Given the effectiveness of TTx subtraction in isolating I NaP s in both isolated and in situ neurons, this procedure was routinely used in our study. All of the data presented from this point on are from TTx-subtracted currents. It is well known that a noninactivating, “window” current ( I NaTW ) can be generated by the gating properties of fast, transient Na + channels . To address the issue of whether the I NaP expressed by EC layer II neurons can be accounted for by a classical window conductance, we analyzed the voltage-dependence properties of I NaT in acutely dissociated neurons. Fig. 2 A shows Na + -current traces recorded in a representative neuron in response to an activation–inactivation pulse protocol. Peak-current amplitudes were measured and used for deriving conductance values ( G NaT ); normalized activation and steady state inactivation plots were then constructed , and fitted with Boltzmann functions. The optimal approximation to data points returned by single Boltzmann functions suggested the existence of functionally homogeneous, transient Na + channels in the neuronal preparation under examination. In eight cells, average V 1/2 and k were −32.5 ± 6.5 mV and −3.6 ± 0.9 mV, respectively, for the activation function, and −59.8 ± 5.2 mV and 4.5 ± 0.9 mV, respectively, for the steady state inactivation function. These values are similar to those reported in a number of other studies on neuronal I NaT voltage dependence . The product of the activation and steady state inactivation functions was then calculated in individual cells to derive the theoretically predicted voltage dependence of the window conductance ( G NaTW ) arising from transient Na + channels . Fig. 2 C illustrates the I NaP evoked by a standard ramp protocol, and isolated via TTx subtraction, in the same cell as in A and B. The conductance underlying I NaP ( G NaP ) was calculated and compared with the predicted G NaTW ; an evident discrepancy in the voltage dependence of the two conductances could be observed at potentials positive to about −30 mV, where G NaTW rapidly fell towards zero, whereas G NaP maintained relatively high values, though it also showed a characteristic decline from its maximum (see below). The same discrepancy between G NaTW and G NaP was observed in four other acutely dissociated neurons, in which both well-clamped I NaT s and sizable I NaP s could be recorded. In a broader cell population, the amplitudes of the reconstructed G NaTW and of G NaP were measured both at the peak [ G (max) ] and at a voltage point positive by 20 mV to that of the peak [ G (+20) ]. The average ratios G NaTW(+20) / G NaTW(max) and G NaP(+20) / G NaP(max) were 0.076 ± 0.057 ( n = 8) and 0.715 ± 0.102 ( n = 12), respectively ( P < 5 × 10 −12 ). In addition, in those neurons in which both I NaT and I NaP were quantified, the size of the predicted I NaTW was much smaller than that of the observed I NaP (see Table ). These data clearly indicate that by far most of the I NaP expressed by EC layer II neurons is not a classical I NaTW , similar to what has been previously observed in hippocampal neurons . Whereas a depolarization-activated conductance, once maximally recruited, would be expected to maintain a steady value at more positive voltage levels, G NaP , as mentioned above, consistently showed some degree of decline from its maximum. A possible explanation for this observation is the existence of a time-dependent inactivation of G NaP acting during the ramp . Under this hypothesis, G NaP should inactivate more when elicited with increasingly slow ramps. To address this issue, we performed a series of experiments in which the amplitude of the I NaP s evoked by voltage ramps was analyzed as a function of the ramp slope. Fig. 3 A shows the protocols applied and the TTx-subtracted currents thereby obtained in a representative in situ neuron. I NaP amplitude appeared to markedly depend on the depolarization rate. The average, normalized I NaP amplitude measured at the peak of the I–V function was then plotted as a function of the inverse of the ramp slope, a quantity directly related to ramp duration ( n = 12). The resulting plot demonstrated a biexponential decay, with a fast “slope constant” and an ∼15-fold slower one. These data strongly suggest that at least two kinetic components exist in I NaP , each characterized by a different inactivation rate. The ratio between the two slope constants and that between their relative amplitude coefficients were such that, with 50-mV/s ramps, >96% of the ensuing I NaP 's peak amplitude was accounted for by the slow component. Since we were interested in the slowest Na + -current components, which more closely approach the notion of “persistent” Na + current, we decided to employ 50-mV/s ramps in the rest of our study so as to maintain the peak value of “true” I NaP s relatively unaffected by time-dependent inactivation, while ruling out most of the “intermediate” kinetic components. Moreover, our observations confirmed the validity of applying 50-mV/s ramps for obtaining data on I NaP voltage dependence of activation (see previous paragraphs). The above data clearly pointed to the existence of a time- and voltage-dependent inactivation of I NaP . However, inactivation properties of voltage-dependent channels have been shown to be possibly affected by the composition of intracellular milieu, and in particular by intracellular nonphysiological halogenic anions . Since the main anion in the intracellular solution used in our experiments was fluoride (F − ), we performed control experiments in which internal F − was substituted with other molecules, namely sulphate (SO 4 2− ; n = 4), and methanesulphonate (MeSO 3 − ; n = 3). The same ramp protocols as described in the previous paragraph were applied under these ionic conditions. In no case did we observe significant differences in I NaP amplitude and its ramp–slope dependence as compared with F − experiments. In particular, peak amplitude and current density of ramp (50 mV/s)-evoked, TTx-subtracted I NaP s were −155.4 ± 58.9 pA and −9.8 ± 3.6 pA/pF in SO 4 2− or MeSO 3 − experiments (data pooled together), not significantly different from the control values reported above ( P = 0.66 and 0.55, respectively). The ratio between the amplitudes of I NaP s evoked by 6.25- vs. 50-mV/s ramps was 0.497 ± 0.134 in SO 4 2− /MeSO 3 − experiments, again not significantly different from that found when using F − (0.51 ± 0.08, n = 12, P = 0.79). These observations indicate that the above-described phenomena are indeed of physiological relevance. We then investigated the issue of I NaP inactivation in further detail by analyzing the effects of variable prepulse potentials on ramp-activated I NaP s. The protocol employed is illustrated in Fig. 4 A1. 50-mV/s voltage ramps were preceded by very-long-lasting (15 s) conditioning prepulses at various voltage levels (V cond ). When V cond s of −90 to −50 mV were used, the ramp started from the same voltage level as V cond itself, rather than from a fixed, negative voltage level: in this way, the possible occurrence of recovery from inactivation during the initial part of the ramp was avoided. At more positive V cond s, the ramp started from −50 mV, so as to preserve the voltage region of I NaP peak. Currents recorded with the above protocol in a representative in situ neuron are shown in Fig. 4 A2. I NaP peak amplitude turned out to markedly depend on the conditioning potential. Average, normalized current traces obtained from seven in situ neurons are depicted in Fig. 4 B1. It can be observed that voltage-dependent steady state inactivation of I NaP was nearly complete at about −20 mV. The average plot of I NaP 's voltage dependence of inactivation could be fitted by a single Boltzmann function, with V 1/2 at about −49 mV and a slope factor, k , of ∼10 mV. In addition, we also constructed an activation plot from the average I NaP derived from the same neuron pool, and fitted it with a single Boltzmann function . Note that, importantly, G NaP activation and steady state inactivation functions overlapped over a wide voltage range. Due to this phenomenon, a significant window conductance ( G NaPW ) is expected to arise from G NaP . The predicted voltage dependence of G NaPW is depicted in Fig. 4 B2 (dotted line), and will be compared with relevant experimental data later on in the paper. The kinetic properties of I NaP voltage-dependent inactivation were then further characterized. Time dependence of inactivation was first analyzed by means of prepulse-ramp protocols ; the voltage ramp eliciting I NaP was preceded by a prepulse at various voltage levels (from −60 to −20 mV), which was made to vary in duration from 0 to up to 20 s. Currents recorded in response to such a protocol in a representative in situ neuron are shown in Fig. 5 A. Average, normalized peak-current amplitudes were used for constructing plots of time dependence of inactivation, each one referring to a specific conditioning potential . These plots could be best fitted with single exponential functions; the time constants were slow and ranged from ∼6.8 to ∼2.6 s, depending on the conditioning potential. The time course of I NaP recovery from inactivation was also investigated. The voltage protocols applied consisted of a first 10-s prepulse at −30 mV that substantially inactivated I NaP , followed by a second prepulse at −90 or −80 mV of variable duration (from 0 to 10 s), and by the standard voltage ramp. Currents recorded in response to such a protocol in a representative in situ neuron are shown in Fig. 6 A. Average, normalized peak-current amplitudes were used for constructing plots of time dependence of recovery from inactivation, for both recovery potentials . These plots could be best fitted with single exponential functions, with time constants of ∼5.2 (−80 mV), and 4.7 s (−90 mV). As mentioned above and illustrated in Fig. 4 B2, the wide overlapping of I NaP activation and steady state inactivation curves is expected to result in a prominent window current ( I NaPW ) distinct from the classical window current ( I NaTW ) predicted on the basis of the gating properties of the fast, transient Na + conductance. To test this prediction, voltage protocols consisting of very-long-lasting depolarizing pulses were applied to in situ neurons so as to try to uncover steady current components in I NaP . Fig. 7 A1 shows average, TTx-subtracted Na + currents obtained from five neurons in response to 15-s voltage steps at −60 to −10 mV. After an initial phase displaying fast and intermediate-speed decay components, a slower decaying current component, which was identified as the I NaP under study, became evident. When the last 14 s of the current trace were considered, the decay phase of I NaP could be best fitted by a single exponential function, with voltage-dependent time constants very similar to those determined for the time-dependent inactivation of I NaP revealed by ramp protocols (see above). In addition to the decaying component, fittings returned a steady (offset) component ( I ss ) whose amplitude also displayed a marked voltage dependence. When plotted as a function of test potential , the normalized I ss amplitude closely paralleled that of the expected I NaPW . Therefore, a steady current component of I NaP can be directly demonstrated whose voltage-dependent behavior fits that predicted for the time-independent I NaPW . On the basis of the above data, we then estimated the relative contribution of I NaPW and I NaTW to the total, noninactivating Na + current generated by EC layer II cells in a subthreshold region of membrane voltages, where it is known to sustain theta-like membrane-potential oscillations lasting for indefinitely long periods . Our measurements indicate that at −50 mV, a level close to that at which the maximal amplitude of subthreshold oscillations is observed , <20% of the total, persistent Na + current is accounted for by I NaTW , whereas the remaining part must derive from the window current generated by the true I NaP ( Table ). To further clarify the basis of the experimentally observed decline in G NaP at positive potentials , a theoretical reconstruction of the biophysical properties of I NaP inactivation was carried out. A simple Hodgkin-Huxley model, considering a single inactivation gate switching between two energy states, was considered in order to give account for the monoexponential time course of I NaP decay and recovery from inactivation. This reconstruction was merely phenomenological and was given no mechanistic meaning since single-channel data clearly indicated different features of the underlying elementary events (see below). The time constants of I NaP inactivation and recovery from inactivation and the data on voltage dependence of I NaP steady state inactivation were processed to derive numerical values for the rate constants of the inactivation-gate transitions, as explained in materials and methods . Fig. 8 B shows the voltage dependence of the values thus obtained for the two rate constants, α and β. The plots were then best fitted with the empirical function, α (or β) = ( a · V m + b )/{1 − exp[(V m + b / a )/ k ]}, where V m is the membrane voltage. The numerical values returned by the fittings for the a , b , and k coefficients, in both the α and β plots, are indicated in the legend to Fig. 8 . The voltage-dependence functions thus obtained for α and β were then used to derive the predicted voltage dependence of I NaP inactivation-gating time constants (see materials and methods ). The concordance between the reconstructed, theoretical function and the real data is shown in Fig. 8 A. The kinetic parameters obtained in the above manner were then used to verify the possible effects of I NaP slow inactivation on the results of voltage-clamp ramp protocols. Reconstructed I NaP s evoked by simulated voltage ramps of variable slope are illustrated in Fig. 8 C. It is apparent that progressively reducing the depolarization rate causes a decrease in I NaP peak amplitude, and the appearance of increasing discrepancies between the true I NaP voltage dependence and that measured in the late part of the ramp protocol. Fig. 8 E shows the voltage dependence of G NaP as obtained in response to a simulated 50 mV/s-ramp protocol: the reconstructed G NaP declined at voltages positive to about −30 mV very similarly to the experimentally observed G NaP , whereas G NaP voltage dependence of activation, as measured by considering only the early part of the ramp protocol, was negligibly affected. Moreover, the plot of reconstructed I NaP 's peak amplitude as a function of the inverse of ramp slope could be fitted, with some approximation, with a single exponential function, with a slope constant (ς) similar to the slow slope constant observed in experimental plots , and no sign of any fast, early component. The latter results further confirm the adequacy of the ramp protocol we routinely used for characterizing the biophysical properties of I NaP , and in particular the slow inactivation of this current. Finally, a simulated protocol of steady state inactivation revealed that, during a ramp preceded by a prepulse that fully inactivates I NaP , a significant recovery from inactivation can occur provided that the ramp starts at sufficiently negative levels and is sufficiently slow. For instance, during a 25-mV/s ramp starting at −80 mV, 17% of the total current can recover from inactivation (not shown). By contrast, voltage protocols similar to those we employed for the study of I NaP voltage-dependent inactivation (see above) determined no appreciable ramp-dependent recovery from inactivation of I NaP . The single-channel basis of I NaP in EC layer II neurons has been described elsewhere . In that study, we found that, after membrane step depolarization, I NaP is generated by early as well as late single Na + -channel openings of much more prolonged duration and of significantly higher conductance than the usual, transient Na + -channel openings responsible for I NaT generation. Here, we report how the same persistent Na + -channel activity also accounts for some particular biophysical properties described for macroscopic I NaP , namely I NaP time-dependent inactivation and I NaPW generation. Recordings from cell-attached patches in acutely isolated EC layer II neurons frequently revealed the presence of a persistent Na + -dependent channel activity that proved to remain stable even over prolonged periods of time (tens of minutes). When very-long-lasting (20 s) depolarizing steps at −10 mV were commanded from a holding potential of −80 mV, multiple, repetitive single-channel openings were observed that tended to cluster preferentially at the beginning of the test pulse. Typical examples of this channel activity for a series of consecutive 20-s test pulses is shown in Fig. 9 A1. A detail of some prolonged, late channel openings is also provided by Fig. 9 A1, inset. Ensemble averaging of multiple traces was then carried out for each patch . Due to the very long overall duration of the recording cycle required for every single sweep (40 s), only a limited number of traces could be recorded in each patch (15 on average), what explains the low signal-to-noise ratio of ensemble-average traces. In all cases, however, the averaged currents showed a noticeable trend to decay towards zero, and this decay could be properly fitted by a single exponential function . The time constant of average-current decay was 2.66 ± 0.52 s in six patches at −10 mV, a value that compares favorably with those found in whole-cell protocols on macroscopic I NaP inactivation at the most positive voltage levels tested . Ensemble averaging of all of the available sweeps recorded from the same six patches returned a better signal-to-noise ratio . The decay time constant of the resulting average current was 2.46 s, again in good agreement with the data obtained from the analysis of both individual patches and whole-cell recordings. The open-time distribution of the single-channel activity evoked by 20-s depolarizing steps at −10 mV was then investigated. Fig. 10 A shows the open-time distribution found in the same patch as illustrated in Fig. 9 A. As in this case, all plots were best fitted by double-exponential functions, with average time constants of 3.35 ± 0.86 and 21.07 ± 17.74 ms, and an average ratio of the slow vs. fast exponential-component weight ( W = A · τ) of 0.118 ± 0.048 ( n = 6). It seems important to point out at this time that: (a) even the faster of the two time constants exceeds by more than six times the mean open time found in classical, transient Na + channel openings at approximately the same test voltage level ; yet (b) the values of both time constants were much smaller than those of the time constants of inactivation of ensemble-average currents as well as whole-cell I NaP . This specific issue will be further addressed below. Long-lasting depolarizing protocols at more negative test-voltage levels were also applied in cell-attached recordings so as to investigate the possible bases of I NaPW generation. A typical example of the recordings obtained at the test voltage of −40 mV is shown in Fig. 11 A. Again, multiple, repetitive single-channel openings were observed and these were able to generate a measurable inward current in ensemble-average traces . Note, however, that at these more negative voltage levels channel openings were more widely distributed over the entire 20-s sweeps. Consequently, at −40 mV the ensemble average-current decayed at a slower rate (with a time constant of 4.33 ms) than at −10 mV. In addition, this decay was towards a steady value ( C in exponential fittings; see materials and methods ) that was higher than zero and represented 25.1% of the current's total amplitude coefficient (namely A + C ). These data are in good agreement with the whole-cell data on I NaP inactivation and I NaPW generation illustrated above. They are also consistent with the idea that the same single-channel events can account for I NaP as well as a steady, nondecaying Na + -current component (namely I NaPW ) generated within a limited voltage window, where it represents a substantial fraction of total I NaP . Finally, the analysis of open-time distribution at −40 mV also revealed the existence of two exponential components , with average time constants of 1.33 ± 0.14 and 6.63 ± 3.47 ms, and an average W 2 / W 1 ratio of 0.069 ± 0.042 ( n = 3). At this potential, therefore, the discrepancy between mean open times and time constants of inactivation of both ensemble-average currents and macroscopic I NaP was even bigger than at −10 mV. The observation that the channel mean open times are far exceeded by the time constants of ensemble-average current inactivation clearly indicates that the latter do not reflect average channel-opening lifetimes in a simple model considering two open states each undergoing one single closure process (with rate constant b ) towards an absorbing state, like: \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}a_{{\mathrm{i}}}\\ C\end{matrix}{\rightarrow}\begin{matrix}b_{{\mathrm{i}}}\\ O_{{\mathrm{i}}}\end{matrix}{\rightarrow}I_{{\mathrm{i}}}{\mathrm{,}}\end{equation*}\end{document} with the index i being either 1 or 2, and with a i >> b i . Rather, alternative models considering late first openings and/or late reopenings must be taken into account. In an extreme situation, late channel openings such those of Fig. 9 A1 might all be first openings of as many distinct channels inactivating towards an absorbing state, in a model qualitatively similar to that depicted in Scheme I, but in which the kinetics of the macroscopic inactivation process would rather reflect the rate constants of the closed-to-open reactions, a i . This interpretation, which resembles the classical Aldrich-Corey-Stevens model of the kinetics of transient Na + channels , seems very unlikely since, in patches containing most likely only one “persistent” channel, delayed first openings occurred very infrequently, whereas late reopenings were often observed . An alternative possibility is that the channel, once open, can reach multiple nonconductive states, one of which is virtually absorbing at positive potentials, thus causing a true channel inactivation. Under this assumption, a channel behavior such as that in Fig. 9 A1 could be accounted for by repetitive reopenings of a small number of persistent channels. The most economical kinetic scheme of such a behavior is seen in Fig. 2 . A nonnegligible rate constant (δ) for the reaction I i → O i should also be considered for more negative voltages, at which a major I NaPW is produced. The analytical derivation of relaxation time constants from rate constants reveals that if α i >> γ i , and γ 1 ≈ γ 2 (or if only one inactivated state exists, communicating with one of the two open states), one slow time constant of ensemble-average current inactivation would be produced, which would faithfully reflect 1/γ. Other possible kinetic schemes consider that an inactivated state is reached from a closed rather than a conducting state. The evaluation of the relationships between transition rate constants in such a scheme and the time constants of ensemble-average current inactivation would require the study of closed-time distribution. This could not be reliably accomplished from our data since in no patches in which slow inactivation was studied could the presence of one single channel be assumed. Despite these limitations in providing precise kinetic schemes, our data clearly demonstrate the importance of late channel (re)openings, rather than of early, very-long-lasting openings, for the generation of slow kinetic components in macroscopic I NaP . The present study provides a biophysical characterization of the I NaP expressed by rat EC layer II principal neurons. The major issues we addressed deal with the mechanism of generation of I NaP , the possible influence of the voltage protocols employed for the study of I NaP biophysical properties, and the real degree of I NaP persistence over time and specific voltage windows. In general, the hypotheses on generation of persistent Na + currents consider two main possibilities: (a) I NaP simply derives from an incomplete steady inactivation of transient Na + channels over a narrow voltage window, due to the partial superimposition of activation and steady state inactivation voltage-dependence curves of the corresponding conductance; (b) I NaP is the result of channel openings that do not functionally behave according to the properties of transient Na + channels, but derive from a rare and atypical gating modality of the same channels , possibly under modulatory control by G proteins or other factors, or, alternatively, from a different channel species. To discriminate between these two possibilities, it is necessary to accurately determine the properties of the window current generated by transient Na + channels and compare them with those of I NaP present in the same cell preparation. The study of acutely dissociated EC neurons allowed us to perform careful measurements on I NaT , and therefore to make reliable predictions on the resulting I NaTW . The comparison between I NaTW and I NaP was done both on a statistical basis and, in some cases, in the same cells. Our data on the voltage dependence and amplitude of these currents indicate that I NaTW , in contrast with what was reported in a previous study , cannot be a major source of the I NaP expressed by EC principal neurons. An alternative, specialized mechanism must be implied in the generation of the large I NaP s found in these neurons. An exciting possibility, already suggested elsewhere on the basis of whole-cell data , is that I NaP is generated by a specialized Na + channel at least biophysically distinct from those generating the fast transient Na + current . Indeed, single-channel, patch-clamp experiments in EC layer II neuron somata indicate that while I NaT is generated by a typical ∼15-pS channel with fast activation and inactivation kinetics, I NaP is due to an ∼20-pS channel activity with a 10-mV lower threshold of activation, and a sustained, high open probability during prolonged depolarizations . The issue regarding the molecular diversity of Na + channels expressed by EC layer II neurons is beyond the scope of the present study and will be discussed elsewhere. Another interesting issue raised by our data relates to the definition itself of I NaP . Due to their practicality, ramp protocols are the most widely used means for eliciting and isolating I NaP . However, the employment of such protocols presupposes that they can adequately reproduce the voltage dependence of I NaP without significantly recruiting any other kinetically different (i.e., faster-decaying) Na + -current component. This is often implicitly assumed rather than demonstrated. Moreover, the notion of “slow voltage ramp” suitable for I NaP activation varies considerably among different experimental works . The experiments we carried out by running voltage ramps of variable slopes clearly indicate that the amplitude of the ensuing I NaP strictly depends on the depolarization rate applied. Our data show how, when ramps of progressively decreasing slopes (from 100 to 6.25 mV/s) are commanded, I NaP amplitude decreases in a roughly biexponential fashion. The existence of a faster exponential component may be due to the presence of Na + -current components kinetically intermediate between classical, “fast” Na + currents and the persistent Na + current. These intermediate kinetic components were easily observed in our step-protocol recordings , and their properties in EC layer II stellate cells will be described in detail elsewhere. The measurements on I NaP s evoked with “slow” voltage ramps may therefore be contaminated by the superimposition of such current components, unless the commanded ramp is slow enough. Moreover, our data show that, importantly, the process of I NaP slow inactivation is a potential source of distortions in the measurements of I NaP biophysical parameters (amplitude, voltage dependence of activation, reversal) when this current is elicited by voltage ramps. All these considerations point to the importance of accurately choosing the voltage protocol applied for the study of I NaP in each specific experimental situation. We propose that some previously reported, atypical biophysical features of I NaP , particularly regarding its nonsigmoidal voltage dependence of activation , are the result of the interaction between multiple Na + -current slow decay components and the ramp protocols employed. In our study, the ramp protocols routinely used were chosen so as to minimize both the possible contribution of intermediate-kinetics Na + current components and the effects of voltage- and time-dependent inactivation of I NaP . The inactivation properties of what, according to our operative definition, can be considered as I NaP have been characterized in detail in our study. Our experiments indicate that, in our preparation, the steady state inactivation of the conductance underlying I NaP ( G NaP ) (a) has a voltage dependence that extends over a wide voltage window, and (b) reaches a nearly complete level at −20 to −10 mV. The former of these features, together with the relative position of the G NaP activation curve along the voltage axis, is expected to give rise to a major, time-independent, “window” current over a limited voltage range. This window current ( I NaPW ), clearly different from that arising from the voltage-dependence properties of the transient Na + conductance ( I NaTW ), could also be directly demonstrated both as nonzero baselines at the beginning of ramp protocols on voltage dependence of inactivation , and as offsets, or pedestals, in exponentially decaying I NaP s elicited by long-lasting voltage steps . The peak of the observed I NaPW occurred at voltage levels very close to those at which I NaP -dependent, theta-like subthreshold membrane-potential oscillations are generated by EC stellate cells . Our data indicate that the contribution of I NaPW to the total persistent Na + current over the voltage range of subthreshold-oscillation generation must be substantial, since the peak I NaPW amplitude was estimated to exceed that of the predicted I NaTW by more than four times. Since the subthreshold oscillations generated by the stellate cells can last indefinitely, our observations also imply that only a fraction of the total I NaP , namely I NaPW itself, is sufficient for sustaining them. I NaP slow inactivation and recovery from inactivation were found to occur with voltage-dependent time constants in the order of a few seconds. We worked out an analytical reconstruction of the kinetics of these processes which, if introduced into suitable neuronal models, should allow us to make predictions on the effects of slow voltage-dependent inactivation on I NaP modulation of membrane-voltage events. We demonstrated that I NaP inactivation can considerably affect the apparent current maximal amplitude during the delivery of slow depolarizing ramps. Therefore, it is conceivable that the recruitment of I NaP and its impact onto membrane-voltage dynamics can be significantly influenced by the speed of membrane depolarization. For instance, the ability of I NaP to bring the membrane potential towards threshold for action-potential firing may be expected to be higher in response to a step depolarizing current injection than to slower or sustained depolarizations. This is consistent with the role of I NaP , demonstrated in various central nervous system neurons including EC stellate cells in promoting transient low-threshold spikes and sustaining phasic firing in response to fast depolarizations. On the other hand, I NaP inactivation, which we found to be eventually complete above a physiologically interesting range of membrane potentials, may have an important role in limiting detrimental Na + influx during pathological conditions such as seizures and ischaemia . The question can then be raised whether the biophysical properties we describe here for the I NaP expressed by EC stellate cells represent a general feature of this current in various neuronal populations. Slow time-dependent inactivation of I NaP has been previously reported in neocortical pyramidal neurons , although in that case the voltage dependence of the process was not investigated in detail, whereas a nondecaying “offset” I NaP component corresponding to ∼30% of the total was found in experiments on time dependence of inactivation even after inactivating prepulses at +20 mV. This is in contrast with our finding of a virtually complete inactivation at approximately −20/−10 mV. The discrepancy may imply the existence of interesting functional differences among I NaP s expressed by different neuronal populations, which encourages further studies in other cell systems; alternatively, it may be the consequence of the inactivation protocol employed in Fleidervish and Gutnick 1996 , in which the depolarizing ramp after the inactivating prepulse started from a fixed, negative voltage level (−80 mV): this may allow some degree of recovery from inactivation during the early phase of the ramp, as also suggested by the output of our modeling study ( results ). Finally, the present study provides an insight into the fine mechanisms underlying the complex biophysical features displayed by I NaP . Our previous work has already clarified the nature and elementary properties of the single-channel events responsible for I NaP generation in EC principal neurons . The data we report here clearly point to the importance of late Na + -channel (re)openings, many times longer in duration than those generating classical, transient I NaT in central neurons, for determining the behavior of slow kinetic components of macroscopic I NaP . Late Na + -channel (re)openings have already been identified as a possible source of I NaP in neocortical neurons and ventricular myocytes , but those we observed were comparatively much longer-lived. The slow inactivation of whole-cell I NaP we characterized was paralleled by that of ensemble-average traces from single-channel, cell-attached recordings. In turn, the latter appeared to be the consequence of the slow delivery of channels to an inactivated state. This can be the consequence of either late first openings or, much more probably, low-rate transitions from open states that are more likely to switch to a close, re-recruitable state, or vice versa. The inactivated state was nearly absorbing at −10 mV, but not at −40 mV, where the existence of a major window current generated by the voltage-dependent properties of G NaP had been predicted and demonstrated (see above). Again, whole-cell I NaPW had its single-channel correlate in late, repetitive openings able to generate measurable net inward currents after as long as 20 s of membrane depolarization. In conclusion, the I NaP expressed by EC layer II principal neurons is a prominent current operating in a subthreshold range of membrane potentials, most of which is generated by a process independent of the classical gating behavior of the transient Na + conductance. It also displays complex and previously nonrecognized biophysical characteristics that appear to be tailored to the specific role this current is known to play in the generation of the sub- and near-threshold membrane-potential events typical of the same neurons. The concept of “persistent Na + current” may turn out to be susceptible to some critical revision also in other experimental situations, with reference to both the existence of multiple and heterogeneous functional components, and the expression of kinetic and voltage-dependence properties that might influence their impact onto neuronal function in previously unforeseen ways. | Review | biomedical | en | 0.999997 |
10498670 | Most cells in the visual system have receptive fields with antagonistic surrounds. Bipolar cells are the first cells in the visual system with this organization. The antagonistic surround of these cells is generated for a large part by feedback connections in the outer retina. In general, antagonistic surrounds play a role in contrast enhancement and in edge detection. In addition, these feedback connections control the size of the horizontal cell (HC) 1 receptive fields , play a prominent role in the generation of spectral opponent HCs , and form the neural basis of the color constancy . The strength of the feedback signal from HCs to cones seems to vary strongly with the adaptation state of the retina. For instance, in the light adapted retina, two types of spectrally opponent HCs can be found, whereas in the fully dark adapted retina, spectrally opponent HCs are completely absent . Changes in response properties of ganglion cells can also be found. In a fully light-adapted condition, these cells have antagonistic surrounds. These surrounds are absent in the dark-adapted condition . Given this important role of negative feedback in the signal processing in the retina, variations of the strength of negative feedback will have strong impact on the whole visual system. For goldfish, the negative feedback pathway from HC to cones has been described in detail . Horizontal cells feed back to cones by modulating the cone Ca-current directly in a γ-aminobutyric acid (GABA)–independent way. Hyperpolarization of the HCs results in a shift of the Ca-current activation function to more negative potentials, yielding an increase in Ca influx in the cone synaptic terminal and thus an increase in glutamate release . The neurotransmitter involved is presently unknown. Although the properties of the negative feedback pathway are beginning to be resolved, the way this pathway is modulated during light/dark adaptation is not clear at all. In this study, we describe a mechanism by which the efficiency of the feedback signal is changed during adaptation. To change the adaptation state of the retina, a train of bright white flashes was used. It will be shown that during this stimulus protocol, the efficiency of the feedback signal increases strongly due to a slight depolarization of the cones. This cone depolarization is due to intrinsic photoreceptor adaptation. This study illustrates that local photoreceptor adaptation yields a strong change in the network properties of the retina. Goldfish, Carassius Auratus , (12–16 cm standard body lengths) were kept at 18°C under a 12-h dark/12-h light regime. Before the experiment, the fish was kept in the dark for 7 ± 1 min. The fish was decapitated, and an eye was nucleated. This eye was hemisected and most of the vitreous was removed with filter paper. The retina was isolated, placed receptor side up in a superfusion chamber and superfused continuously (1.5 ml/min) with oxygenated Ringer's solution (pH 7.8, 18°C). This procedure was done, using infrared (λ = 920 nm) illumination. A 450-W Xenon lamp supplied two beams of light. These were projected through Uniblitz VS14 shutters (Vincent Associates), neutral density filters (NG Schott), lenses, and apertures. For the patch clamp measurements, the spots, 20 μm in diameter, were projected through a 40× water immersion objective (N.A. = 0.55) of the microscope and the spots, 3,000 μm in diameter, were projected through the condenser (N.A. = 1.25) of the microscope. The full-field white light stimuli used for intracellular recordings were projected onto the retina through a 2× objective lens (N.A. = 0.08) of the microscope. For all experiments, only white light stimuli were used. The light intensities are expressed in log units relative to 4 × 10 3 cd/m 2 . Since we used white light stimuli in our experiments, we compared the size of the cone light responses to white light with the responses of M cones to 550-nm light flashes and calculated the amount of effective quanta in our white light to be 1.0 × 10 5 s −1 μm −1 (550 nm). In goldfish, Malchow and Yazulla 1988 calculated that 50% of the pigment was bleached when the retina was stimulated with 3.16 × 10 9 photons s −1 μm −1 , left on for 4 min. The amount of photons they used exceed our stimulus condition by six log units. This is an indication that in our experimental conditions, bleaching hardly plays a role. The patch pipettes were pulled from borosilicate glass (GC150TF-10; Clark) and the intracellular microelectrodes were pulled from aluminosilicate glass (o.d. = 1.0 mm, i.d. = 0.5 mm; Clark) with a P-87 micropipette puller (Sutter Instruments Co.). Patch pipettes had impedances between 5 and 10 MΩ when filled with pipette medium and measured in Ringer's solution. The series resistance during the whole cell recording was <12 MΩ. Electrodes were mounted on an MP-85 Huxley/Wall-type micro manipulator (Sutter Instruments Co.) and connected to a 3900A Integrating Patch Clamp (Dagan Corp.). Microelectrodes had impedances ranging from 100 to 200 MΩ when filled with 4 M KAc. The intracellular voltages were measured with an electrometer and recorded on paper (Graphtec Linearcorder). Data acquisition, control of the optical stimulator, and control of the patch clamp were done with a 1401 AD/DA convertor (Cambridge Electronic Design Ltd.) and an MS-DOS–based computer system. The Ringer solution contained (mM): 102.0 NaCl, 2.6 KCl, 1.0 MgCl 2 , 1.0 CaCl 2 , 28.0 NaHCO 3 , 5.0 glucose, and was continuously gassed with ∼2.5% CO 2 and 97.5% O 2 , yielding a pH of 7.8. Some Ringer solutions contained drugs as indicated in the text, and in the figure legends: flupentixol (50 μM; Research Biochemicals, Inc.), niflumic acid (100 μM; Sigma-Aldrich Chemical Co.), CoCl 2 (2 mM; Merck). In tetraethylammonium (TEA)-Cl Ringer's solution, 15 mM of the NaCl was replaced by 5 mM CsCl and 10 mM TEACl. The standard patch pipette medium contained (mM): 12.0 KCl, 61.0 d -gluconic-K, 1.0 MgCl 2 , 0.1 CaCl 2 , 1.0 EGTA, 5.0 HEPES, 5.0 ATP-Na 2 , 1.0 GTP-Na 3 , 0.2 3′:5′-cGMP-Na, 20 Phosphocreatine-Na 2 , 50 U/ml creatine phosphokinase. To change the Cl-equilibrium potential, KCl was exchanged for equimolar d -gluconic-K. The pH of the pipette medium was adjusted to 7.25 with KOH. Cesium pipette mediums contained equimolar d -gluconic-Cs and 12.0 CsCl instead of the KCl and d -gluconic-K. All chemicals were obtained from Sigma-Aldrich Chemical Co. The liquid junction potential was measured with a patch pipette filled with the pipette medium, and positioned in a bath filled with pipette medium. The reference electrode was filled with 3 M KCl. After the potential was adjusted to zero, the bath solution was replaced with Ringer's solution. The resulting potential change was considered as the junction potential. The liquid junction potential was determined for the various pipette solutions and all data were corrected accordingly. Dopaminergic interplexiform cells were destroyed with intraocular injections of 5 μl Ringer solution containing 6-hydroxy-dopamine (5 μl) and paragyline (10 μl; both from Sigma-Aldrich Chemical Co.), administered on two successive days . These fish were used 14–33 d after injection. All dopamine-depleted retinas used for intracellular recordings were tested afterwards for the presence of dopamine-containing cells using the tyrosine hydroxylase method . Data were taken only from retinas that did not contain any tyrosine hydroxylase–positive cells. Cones were selected under visual control and spectrally classified . Only L, M, and S cones were found. HCs were classified on their spatial and spectral properties . Of the three classes of HCs, only monophasic HCs were used in the present study. Since the large modulation of the light-sensitive conductance masks the much smaller presynaptic calcium current of the cone, it is not possible to measure light-induced changes in presynaptic calcium currents of cones directly. To overcome this problem, previously recorded light responses of a cone to spot stimuli (20 μm diameter) of 500-ms durations were “played back” as command voltages in a condition where the light conductance of the cone is blocked with an intense (−0.15 log) white spot (20 μm diameter). In this way, “light-induced” changes in presynaptic calcium current can be measured without the interference of the light-sensitive conductance. Fig. 1 explains the voltage clamp protocol used to isolate light-induced changes in calcium current. The insert displays the original hyperpolarizing response of a cone to a 500-ms flash that was used to construct the voltage clamp protocol. Trace 1 consists of this hyperpolarizing response repeated nine times. Trace 2 is similar to trace 1, but now with a depolarization of 4 mV superimposed, applied in discrete 1-mV steps just before the second, third, fourth, and fifth hyperpolarizing responses. Because the 1-mV voltage steps were taken in between the “light flashes,” the whole light response is shifted to a 1-mV more-depolarized level. During the last four hyperpolarizing steps, this final 4-mV depolarized level was maintained. For trace 3, a similar protocol as for trace 2 was generated, but the depolarizing steps were twice as large, yielding a total depolarization of 8 mV. The final amount of depolarization is indicated by the numbers to the right of the voltage traces. After the “flashes,” a 100-ms step to −7 mV was applied, followed by a 1,500-ms step to −77 mV. Finally, the cone membrane potential was stepped back to the initial potential. This last part of the protocol was used to study the tail currents. During the step to −77 mV, a 50-ms step to −87 mV was applied. This step was used for leak subtraction to isolate the presynaptic currents of the cone. The part of the protocol used for determining the tail and leak currents are not shown in the figures. To measure feedback from HCs in cones, the cone light responses were saturated with a spot of white light (−0.15 log) with a 20-μm diameter. Stimulating the cones with light in this saturated condition does not lead to a light response (not shown). Cones were clamped at the potential indicated in the text and figure legends and 10 white, full field flashes (intensity −1.0 log) of 500-ms durations were delivered to the retina. First we will show that the kinetics of the HC light response changes during a train of repetitive flashes. After excluding all other possibilities, we will show that this can be accounted for by an adaptation-induced depolarization of the cone. Furthermore, we will show how this adaptive depolarization of the cones causes changes in the synaptic transmission between cones and HCs. Finally, a simple simulation will show that a small depolarization of the cone can account for the complex changes in HC response kinetics observed during light adaptation. HCs feed back to cones by shifting the calcium current activation function of the cones using an unknown neurotransmitter. Because we cannot measure the amount of this neurotransmitter, we will define the feedback signal as the size of the shift of the calcium current activation function of the cone. The resulting change in the calcium influx will be called the efficiency of the feedback signal. Fig. 2 A shows HC responses to a train of 10 white light flashes of 500-ms durations with an inter-stimulus interval of 200 ms for three different intensities (−2.0, −1.0, and 0.0 log). For the −2.0 log intensity, the responses are small and sustained. For the −1.0 log intensity, the responses are larger and show a pronounced secondary depolarization; i.e., the difference between peak and plateau value. This secondary depolarization has been shown to be the consequence of negative feedback from HCs to cones . The highest intensity shows round, saturated responses without the secondary depolarization. Of the three intensities, only −1.0 log shows a change in kinetics of the HC responses during the flash train. Comparison of the response to the first and last flashes reveals that the kinetics of the light response has changed considerably . The gray bar in Fig. 2 B marks the size of the secondary depolarization in response to the first flash, and the dashed lines that to the last flash. Thus the secondary depolarization, which is due to negative feedback from HCs to cones, has increased during the flash train. This increase in the size has a time constant of 2.7 ± 1.1 s ( n = 7). It is known that dopamine modulates the feedback signal from HCs to cones . To test whether dopamine can account for the observed changes of HC light responses during the flash train, the experiment of Fig. 2 was repeated in dopamine-depleted animals. Fig. 3 A shows the first and last light responses of a HC from a retina, without dopaminergic interplexiform cells (IPCs). Like Fig. 2 , the gray bar and the dashed lines indicate the amount of secondary depolarization of the first and last flash, respectively. The retina was isolated 14 d after intraocular injection of 6-hydroxy-dopamine, which is known to kill the IPCs. In these dopamine-depleted retinas, the changes in HC kinetics during the flash train remain present. This result was found in eight retinas that did not stain for tyrosine hydroxylase. Furthermore, blocking the D1 and D2 receptors in control retinas with the antagonist flupentixol did not influence the changes in response shape during the flash train. These experiments show that dopamine, the main neuromodulator involved in light–dark adaptation, is not involved in the changes in HC kinetics during the flash train. The next step was to determine whether the changes of HC responses are due to changes in (a) the cones, (b) the HCs, or (c) the reciprocal cone/HC synapse. One possibility is that changes in the cone light response during the flash train can account for the effects observed in the HC response. Fig. 4 A shows the voltage responses of a cone under whole-cell configuration to the same flash train as used for the HCs. Cones hyperpolarize in response to repetitive stimulation, but, unlike HCs, the kinetics of the responses show a decrease in transientness . This result was obtained for both full-field as well as small-spot stimulation (20 μm in diameter; not shown). However, the mean membrane potential of the cone depolarizes with the number of flashes. In 12 cells tested, the mean depolarization was 3.3 ± 1.4 mV (31% of the amplitude of the light response of each cone). The time constant of this depolarization was 3.3 ± 0.5 s, which is of the same order as the time constant of the change of the secondary depolarization of the HCs. Since the kinetics of the cone responses show a decrease in transientness, it cannot account for the increase in transientness of the HC light responses. However, during the stimulation protocol, the cone calcium current might have changed, leading to a change in the Ca-dependent glutamate release, and thus to a change in the synaptic output of the cone. The next step was therefore to measure changes in presynaptic currents of the cone during the flash train. Since the conductance modulated by light is so large that it completely masks the presynaptic currents of the cone, voltage light responses of a cone were recorded and used as a command voltage in a condition that the cone light response was saturated with an intense small white spot (for details of the protocol, see materials and methods ). Fig. 5 shows the presynaptic currents generated in a cone, clamped at its resting membrane potential, in response to a voltage protocol that simulates “light responses.” Fig. 5 (top trace) shows that each hyperpolarizing light response causes an outward current in the cone, and this does not change with repetitive light responses. However, when the voltage protocol includes a stepwise depolarization of the cone (second and third current traces) the outward current elicited by each light response is increased in amplitude. The final amplitude of the step-wise (four steps) applied depolarization is indicated by the numbers to the right of the traces. The middle current trace (4-mV depolarization) mimics the physiological condition . In that condition, the light-induced change in presynaptic currents of the cone increase with the number of flashes with a similar time constant as the increase in rollback in the HC response; i.e., the modulation depth of a light flash increases during the stimulation protocol. This increased modulation depth of a light flash could be due to two features of the command voltage protocol: (a) the repetitive light-induced hyperpolarizations or (b) the gradual “adaptation-induced” depolarization. In the upper trace , the cone does not depolarize, although the same “flashes” are presented. In this condition, no changes in presynaptic currents of the cone are seen. This result suggests that the gradual depolarization, instead of the repetitive hyperpolarizations, is the basis of the increase in response size. Fig. 5 (bottom trace) confirms that the increase in presynaptic currents of the cone are due to the depolarization of the cone because an 8-mV depolarization increases the light-induced changes in the presynaptic currents even further than a 4-mV depolarization. These experiments ( n = 14) show that the currents elicited in the absence of any depolarization (0 mV) do not change over the period of stimulation, whereas the presence of depolarization (4 or 8 mV) clearly potentiates the amplitude of the presynaptic currents of the cone. In the next section, we will show that these presynaptic outward currents elicited by hyperpolarizing light responses in the cone are due to reductions in inward calcium current, the presynaptic calcium current in the cone terminal. Since the cone in Fig. 5 was voltage clamped at its resting membrane potential and the light-sensitive conductance is saturated, four different currents will be active ; i.e., the inward current activated by hyperpolarization (I h ), the calcium current (I Ca ), the calcium-dependent chloride current (I ClCa ), and the delayed rectifier (I Kdr ). Blocking the I Kdr with extracellular TEA and intracellular Cs 2+ did not change the increase in current during the voltage protocol (not shown). This result excludes the possibility that I Kdr is modulated during this protocol. The increase in the modulation depth of a light flash due to depolarization of the cone is not likely to be due to modulation of I h because depolarization of the cone would then decrease the amount of current. The two remaining currents, I Ca and I ClCa , are located in the terminal of the cone and could therefore be involved in synaptic transmission. It is known that large divalent cations like cobalt (Co 2+ ) block Ca-dependent synaptic transmission. To determine the contribution of I Ca to the presynaptic currents, the effect of Co 2+ on the experiments of Fig. 5 was studied. To check the effectiveness by which we could block I Ca , we first determined the effect of Co 2+ on the current–voltage relations of the cones. Fig. 6 (top) shows the leak-subtracted current responses to a voltage ramp protocol from −70 to 20 mV before (1), during (2), and after (3) cobalt. Co 2+ shifts the calcium current activation function to more positive potentials ( n = 4). This is a general feature of large divalent cations . This shift will block the synaptic transmission from cones to HCs because I Ca is shifted out of the operating range of the cone. Fig. 7 shows that the presynaptic currents are almost completely blocked with Co 2+ . Fig. 7 (top, Control) shows the increase in light-induced changes in the presynaptic currents due to the voltage trace that depolarizes to 8 mV. This trace is similar to the 8-mV trace, presented in Fig. 5 . Fig. 7 (middle) is the response of the same cell, but now in the presence of 2 mM Co 2+ . Almost no modulation of the presynaptic currents is present ( n = 4). After washing out the Co 2+ , the presynaptic currents reappear. Co 2+ shifted I Ca to positive potentials. If the presynaptic currents are mainly carried by Ca, then depolarizing the cone by an equal amount as I Ca has shifted to positive potentials would prevent Co 2+ from block the presynaptic currents. This assumption is confirmed in Fig. 8 A. A current trace to the same 8-mV part of the protocol as Fig. 5 is shown in the presence of Co 2+ when the cone is clamped at a potential 20-mV depolarized from the resting membrane potential. In the presence of Co 2+ , almost no modulation of the presynaptic currents is present when the cone is clamped at its resting membrane potential, and gradually depolarizes to a level 8-mV more depolarized . Depolarizing the cone 20 mV reveals the presynaptic currents again ( n = 3). If I Ca is the main source of the presynaptic currents, hyperpolarizing the cone would have an equal effect as the application of Co 2+ . In Fig. 8 B, the response of a cone in control Ringer's solution to a similar protocol (only the 8-mV trace) as used in Fig. 5 is shown, but now at a clamp potential of −87 mV. At that potential, no light-induced changes in presynaptic currents are present. Together, these experiments show that I Ca forms the basis of the presynaptic currents. I ClCa is known to generate slow tail currents. To investigate whether the I ClCa also plays a role in the increase in presynaptic currents during the flash train protocol, tail currents were measured before and directly after the presynaptic current protocol. No changes in tail currents were found during these experiments (not shown), indicating that this current stays unmodulated during the flash train. In addition, we tested the role of I ClCa by blocking this current with niflumic acid . Fig. 9 B shows that application of 100 μM niflumic acid did not block the light-induced increase in synaptic currents ( n = 4), whereas tail currents present in control disappeared in the presence of niflumic acid (2). Furthermore, changing the calculated equilibrium potential for chloride from −47 to −28 mV did not change the depolarization-induced increase of the presynaptic currents (not shown). These experiments show that I ClCa is not involved in the increase in presynaptic currents. Therefore, we can conclude that the increase in outward current, observed when the cone is allowed to depolarize is actually a decrease in the standing inward I Ca . Since I Ca is directly related to the glutamate release, changes in I Ca will result in changes in HC input. The next section will show that the change in HC response kinetics during the flash train is also due to changes in the cones. One of the striking changes in the HC response properties is the change in the secondary depolarization. This secondary depolarization is due to negative feedback from HCs to cones. If the changes in HC kinetics are due to changes in HC properties, one should expect that the output of the HCs would also change. Because we cannot measure the amount of neurotransmitter released by the HCs, we looked at the effect of feedback on the cones; i.e., the change in calcium influx in the cone terminal. This effect of the feedback signal on the cone calcium current can be measured most effectively by clamping the cone at one potential, saturating the cone with a small spot and stimulating the retina with a full field flash stimulus (for details of the feedback protocol, see materials and methods ). The result of this feedback protocol is that the cone recorded from responds only to the signal it receives from the HCs. In this way, the feedforward signal can be separated from the feedback signal, which cannot be done measuring HCs themselves. Fig. 10 shows a voltage-clamped cone stimulated with the feedback protocol. It is obvious from this figure that the feedback-induced inward current shows no pronounced changes during the flash train ( n = 4). So, contrary to the increase in secondary depolarization, the feedback signal as measured in the cones has not increased during the flash train. Thus, after a train of flashes, the secondary depolarization has increased. However, the feedback signal received by cones has remained equal. Presynaptic calcium currents also remained constant when the cone was clamped at one potential and showed only an increase when the cone was led to depolarize . Therefore, it was tested whether a similar 4-mV depolarization of the cone increased the feedback-induced current in cones. Fig. 11 shows the feedback responses of a cone when it is depolarized, in four 1-mV steps during the protocol. Whereas the feedback-induced currents to the first three light flashes are small, the gradual depolarization of the cone results in a dramatic increase of the feedback-induced currents ( n = 5). These experiments show that the depolarization of the cone makes feedback from the HCs to cones more efficient. The main result of this paper is that stimulating the retina with a train of white light flashes can lead to a doubling or even a tripling of the amount of calcium flowing into the cone synaptic terminal due to the feedback signal from HCs to cones, while the feedback signal itself does not increase during the flash train. This increase in calcium influx is independent of dopamine and can be attributed to adaptation-induced depolarization of the cone membrane potential. In the next sections, we will discuss this mechanism in detail and present results of a quantitative computer simulation showing that cone polarization alone is enough to generate the observed results. Finally, we will discuss the relevance of this mechanism in relation to the literature concerning adaptational changes in retinal processing. Two things happen with the HC responses during the adapting flash train used in this study that need to be accounted for: (a) the onset response becomes faster and (b) the secondary depolarization becomes larger. The only significant change found in the cones during the flash train is that the mean cone membrane potential slightly depolarized during the flash train. Both the transient character of the first light response of the cone and the depolarization of the mean membrane potential are presumably due to the Ca-dependent feedback on the phototransduction pathway . The question now is, can the changes in HC kinetics during the flash train be accounted for by only a slight depolarization of the cone membrane potential? Fig. 12 A shows the current-voltage relation of the I Ca of a cone. Since there is a linear relation between I Ca of the cone and the amount of glutamate release, the I Ca can be taken as the amount of glutamate release . In the dark, cones rest at about −40 mV. At this potential, there is a certain amount of glutamate release . Hyperpolarization of the cone by light results in a reduction in I Ca ; i.e., in a decrease in glutamate release . Due to this decrease in glutamate release, HCs will hyperpolarize, leading to a change in feedback signal to the cones. By some yet unknown mechanism, the change in feedback shifts the calcium activation function to more negative potentials (dashed curve). This in turn causes an increase in I Ca , which leads to an increased glutamate release, leading to the secondary depolarization of the HC responses. The flash train gradually depolarizes the mean membrane potential of the cone by ∼4 mV, so the cone rests at −36 mV. Because the cone gradually becomes depolarized during the train of flashes, the range of its light response is shifted to a steeper part of the calcium activation function. Thus, the same hyperpolarizing cone light response will cause a larger decrease in calcium current, and a larger reduction in glutamate release, accounting for the increase in the feedforward HC response. The gradual depolarization of the cone during light adaptation can also explain the increase in the secondary depolarization in HCs. During the flash train (i.e., during light adaptation), the feedback signal from HCs to cones does not change. This means that in the light-adapted state the feedback from the HCs during each flash causes the same leftward shift of the calcium activation function as in the dark-adapted state. However, since the cone potential is in a steeper part of the calcium-activation function, this same leftward shift now causes a larger increase in presynaptic calcium current, a larger increase in glutamate release, and a larger secondary depolarization in HCs. So, now that we have discussed the increase in secondary depolarization of the HC light response due to the small depolarization of the cone, we can discuss how both the increase in feedforward signal and the increase in feedback efficiency can lead to a faster time to peak of the HC light response. The feedforward signal increases less than the increase in feedback efficiency . The reason for this is that the 4-mV depolarization is a small depolarization relative to the cone light response, whereas it is a large depolarization relative to the feedback-induced shift in calcium activation function. Since feedback is slower than feedforward, and since the feedback efficiency increases more than the feedforward signal, the result is that the time to peak of the HC responses decreases. Now the question arises why this decrease in time to peak of the HC response and the increase in rollback cannot be seen in the feedback signal received by the cones. Since feedback is slow, the peak of the HC light response might be cut off. Furthermore, the rollback is small, relative to the total response of the HCs, which actually generates the feedback signal. Together, they may explain why the changes in HC kinetics cannot be seen in the feedback signal received by the cones. To test whether the effect of the depolarization on the I Ca of the cone was enough to account for the observed changes in the kinetics due to this increase in synaptic efficiency, a quantitative model simulation was performed. The model consists of a cone and a HC. The cone projects via a Ca-dependent glutamatergic pathway to the HC and the HC feeds back to the cone via modulation of the Ca current by an unknown neurotransmitter . Cones are modelled using a simple resistive network consisting of a voltage-sensitive calcium conductance, the light conductance, and a passive potassium conductance. HCs are modelled with a glutamate conductance and a passive potassium conductance. A detailed description of the model is given in the . With the model, three simulations were performed. (a) The response of the model was studied using a similar flash train as used for the physiological experiments. To mimic the adaptation-induced cone depolarization as found experimentally, the cone membrane potential was depolarized by 1, 2, 3, and 4 mV during the second, third, fourth, and fifth and the rest of the flash train, respectively. The results are shown in Fig. 13 A. It is clear from this figure that the secondary depolarization increases with depolarization of the cone. This is a similar behavior as found in the physiological cone/HC system. Note that only the mean cone membrane potential is changed in the model during the adaptation by the flash train, showing that this small change is enough to induce the changes in kinetics of the HC response. With the model behaving similarly as the physiological cone/HC network, the mechanism responsible for the changes in HC kinetics during the flash train can be evaluated. First the effect of the depolarization on the presynaptic calcium current was studied just as was done in Fig. 5 . In this simulation, the HC membrane potential was held constant. This mimics the experimental condition where the cones were voltage clamped and polarized according to the prerecorded light responses. Due to the extensive electric coupling of HCs, polarization of one cone does not lead to a change in HC membrane potential. Fig. 13 B shows that due to the polarization of the cones, the light-induced change of the presynaptic calcium current almost doubles. This result is similar to the result obtained with the physiological cone/HC system and is due to the fact that depolarization shifts the membrane potential of the cone to a steeper part of the Ca current. Finally, the feedback signal in the model cones was studied . This was done in two conditions: (a) when the model cone was clamped at −45 mV and (b) when the cone was initially clamped at −60 mV and subsequently depolarized during the first five flashes by a total of 4 mV. Fig. 13 C shows that when the cone is clamped at −45 mV, feedback-induced currents remain nearly equal in size throughout the protocol. This result is similar to the physiological result. However, when the cone is clamped at the light membrane potential (−60 mV) and is depolarized by a total of 4 mV, the feedback-induced currents are almost doubled at the end of the stimulus train, just as found physiologically. These simulations show that a very simple model can account for the complex change in the HC response characteristics observed during adaptation to a flash train. Since this behavior can be generated with a model that only includes a Ca current and cone adaptation, it indicates that no other pathways or processes are involved. The relative position of the cone membrane potential to the Ca-current activation function is essential for the size and the kinetics of the HC response. Depolarization of the cone membrane potential due to adaptation of the cone leads to an increase in efficiency of the feedback pathway of the HCs to the cones. Many papers have reported changes in the cone/HC network as a consequence of light adaptation. The mechanisms underlying these changes can be separated into two broad categories: “local” and “network” adaptive mechanisms. Local mechanisms are intrinsic to photoreceptors such as bleaching adaptation and Ca-dependent feedback on the phototransduction cascade, which modulates the cGMP-gated channels in the cone outer segment . Both local mechanisms tend to depolarize the mean membrane potential of the cone when the retina becomes light adapted. The time constant of this process is between 0.1 and 100 s . Network adaptation refers to mechanisms outside the photoreceptor, such as modulation of the electric coupling between HCs by dopamine , the change in sensitivity of the glutamate and GABA c receptors on the HCs , and the change in the amount of GABA released by the HCs . These dopaminergic processes function on a time scale of minutes to hours . GABA also plays an important role in determining the network properties in the outer retina by modulating both the size and kinetics of the HC responses by means of a mechanism intrinsic to the HCs. The present paper describes changes in HC kinetics without a change in the intrinsic HC properties, indicating that GABA does not play a role. The described mechanism is also present in dopamine-depleted animals, indicating that dopamine does not play a role in this short-term change of the HC kinetics. Other groups have also reported changes in HC kinetics due to background illumination or repetitive stimulation without the interference of GABA or dopamine. Akopian et al. 1991 reported changes in the responses of turtle HCs during repetitive stimulation with saturating white light flashes. These HCs show an increase of the Off overshoot, while the On response remained unaffected. Since it has been reported that the HC Off overshoot is mediated by feedback , the findings of Akopian et al. 1991 could be accounted for by the mechanism described in this paper. The absence of any effect on the On response in their experiments can be justified by the fact that they used saturating light stimuli. That condition might be similar to the HC responses to 0.0 log intensity light flashes shown in Fig. 2 . For that intensity, we also did not find any effect on the On responses of HCs. Normann and Perlman 1990 showed that background illumination reduces the sensitivity of the HC responses in turtle. High intensities of background light did not change the kinetics of the HC light response, whereas lower intensities lead to a reduction of the time to peak and an increase in secondary depolarization of the HC light response. Their lower intensities are comparable with the intensities in our experiments, eliciting the changes in HC response kinetics. Their results are similar to the results described in this paper despite their use of background light instead of a train of white light flashes. In their paper, no explanation was given why the time to peak of the HC response decreased when they used moderate light intensities. In light of our experiments, their results can be accounted for by the light adaptation–induced depolarization of the cone, which will make the synaptic efficiency between cones and HCs higher, leading to a reduction of the time to peak of the HC response. This study illustrates that the resting membrane potential of the cone can strongly modulate the size and kinetics of the light responses of neurons in the retina. Small changes in this potential can lead to drastic changes in synaptic transmission, and feedback efficiency in particular, influencing possibly all kinds of visual processing. As mentioned in the introduction , several papers have reported changes in the amount of feedback during light adaptation. Both the surround of ganglion cells and the spectral opponent responses of HCs disappear when a retina is dark adapted. Light adapting these retinae results in the reappearance of these feedback-mediated responses. Most of these changes during light–dark adaptation are assumed to be mediated by GABA or dopamine. Although the mechanism described in this paper is not GABA or dopamine mediated, it could very well play a role in the above-mentioned GABA- and dopamine-mediated processes. For instance, GABA is known to modulate the membrane potential of cones. GABA slightly hyperpolarize cones in goldfish, tiger salamander, and turtle by activating GABA A receptors . By hyperpolarizing the resting membrane potential of the cone, GABA could decrease the feedback efficiency and thereby inhibit feedback-mediated responses. So, in conclusion, the mechanism to modulate the synaptic transmission between HCs and cones described in this paper can be driven by a variety of pathways. The effect will always be a strong modulation of feedback. | Study | biomedical | en | 0.999998 |
10498671 | Osmotic cell swelling leads to extrusion of a number of intracellular inorganic and organic osmolytes, thereby accomplishing the regulatory volume decrease (RVD) 1 . Recently, release of intracellular ATP was found to be induced by osmotic swelling in human , bovine , and mouse epithelial cells, as well as in rat hepatoma cells . Since ATP exists in the cytoplasm at millimolar concentrations, opening of the large pore may induce a passive efflux of ATP. Actually, it is known that a mitochondrial outer membrane large-conductance voltage-dependent anion channel is permeable to anionic ATP . However, the plasma membrane ATP channel has not been identified. Several studies have suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel can act as an ATP-permeable channel . The CFTR-mediated ATP release hypothesis has now been challenged by many contradictory reports . On the other hand, swelling-activated Cl − channels are known to show considerable permeability to large organic anions . Thus, it is possible that the volume-sensitive Cl − channel may serve as a pathway for ATP release. As proposed by Wang et al. 1996 and Roman et al. 1997 , there is the possibility that activation of the volume-sensitive Cl − channel is induced by released ATP through stimulation of P2 receptors. In the present study, therefore, we addressed the question of whether the volume-sensitive Cl − channel serves the pathway of swelling-induced ATP release or its activity is a prerequisite to swelling-induced ATP release, and whether activation of the volume-sensitive Cl − channel is dependent on swelling-induced ATP release in a human epithelial cell line that lacks CFTR expression. Human epithelial Intestine 407 cells were cultured in Fischers' medium supplemented with 10% newborn calf serum, as described previously . For the luminometric ATP measurements, the cells were plated on 48-well plastic plates. The ATP assay was made at the density of 4 × 10 5 cells per well. For the biosensor ATP measurements, Intestine 407 cells were cultured on glass coverslips, and then transferred to the experimental chamber. For the patch-clamp whole-cell recordings, Intestine 407 cells were cultured in suspension with agitation for 10–120 min, and then plated on the experimental chamber. PC12 cells that express the P2X 2 receptor channel were cultured, as reported previously . The extracellular bulk ATP concentration was measured by luciferin-luciferase luminometry using an AF-100 ATP analyzer and AF-2L1 reagents (TOA Electronics). The culture medium was totally replaced by 300 μl of isotonic PBS containing (mM): 137 NaCl, 2.7 KCl, 1 CaCl 2 , 1 MgCl 2 , 8.1 Na 2 HPO 4 , and 1.5 KH 2 PO 4 (pH 7.4, 300 mosmol/kg H 2 O). It is known that ATP release is dramatically stimulated by mechanical stimulation, such as a medium displacement and tilting the monolayer cells or touching to the cells . Preliminary experiments showed that this is the case for Intestine 407 cells. However, ATP release induced by replacement of the entire bathing solution subsided to the background level after 30–60 min. Also, mechanical ATP release was found to be avoided by gentle replacement of one third to one half of the solution by a new solution without touching and tilting the monolayer cells. Thus, after a 60-min equilibration, 100 μl of the extracellular isotonic solution (300 μl) was collected as a control sample and the ATP concentration was measured. Then, 200 μl of hypotonic solution was gently added to the remaining 200 μl of isotonic solution to reduce the extracellular osmolality down to the desired level (56% osmolality, unless indicated). The hypotonic solutions were prepared by mixing the isotonic PBS with a solution containing 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , and 10 mM HEPES/NaOH (pH 7.4, 35 mosmol/kg H 2 O). The test samples (100 μl) were collected after incubation for the indicated times (usually 15 min) at 37°C and the ATP concentration was measured at room temperature (24–26°C). When necessary, extracellular CaCl 2 was removed and 0.1 mM EGTA was added during a hypotonic challenge. The effects of Cl − channel blockers were observed by adding the drug to the hypotonic solution. Only glibenclamide (500 μM) among the employed drugs was found to suppress (by 20–25%) the luciferase detection of ATP, as reported previously . Thus, the data of glibenclamide effect were corrected by the calibration curve obtained by reactions with known amounts of ATP added to cell-free isotonic or hypotonic solution with or without 500 μM glibenclamide. The effect of pretreatment with antibodies was observed after incubating in the isotonic solution supplemented with 12.5 μg/ml antibodies for 60 min, and then applying the hypotonic solution in the absence of antibodies. After incubating Intestine 407 cells in the above isotonic or hypotonic solution for 30 min at 37°C, cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using the Cell Counting Kit (Dojindo) according to the manufacturer's instructions. This method is based on the ability of viable (but not dead) cells to cleave the tetrazolium ring of the MTT into a dark blue formazan reaction product. The local concentration of released ATP at the single cell surface was measured by a biosensor technique on single Intestine 407 cells, using PC12 cells expressing ligand-gated cation channels, P2X 2 receptors, as described previously . In brief, after attaining the whole-cell configuration with a PC12 cell, the cell was lightly attached to the surface of an Intestine 407 cell under a microscope. The whole-cell currents were recorded at a holding potential of −50 mV using an Axopatch 200A amplifier (Axon Instruments) at room temperature. From the whole-cell current density thus recorded, the local ATP concentration was estimated by the calibration curve obtained by the response of PC12 cells alone to known amounts of ATP, as described previously . To apply hypotonic stimulation, the bathing solution was changed from isotonic to hypotonic solution by perfusion. The isotonic solution contained (mM): 100 NaCl, 4.2 KCl, 100 mannitol, 8 HEPES, 6 HEPES-Na, 1 CaCl 2 , and 1 MgCl 2 (320 mosml/kg H 2 O, pH 7.4). The hypotonic solution (220 mosml/kg H 2 O, pH 7.4) was prepared by removing mannitol from the isotonic solution. The pipette (intracellular) solution was composed of (mM): 150 CsCl, 1 MgCl 2 , 10 EGTA, and 10 HEPES (280 mosml/kg H 2 O, pH 7.4 with CsOH). Volume-sensitive Cl − channel currents were recorded by the whole-cell patch clamp technique, as reported previously . The time course of current activation and recovery was monitored by repetitively applying alternating step pulses of ±40 mV (2-s duration) from a holding potential of 0 mV. To monitor the voltage dependence of the current, stepping pulses (2-s duration) were sometimes applied from a prepotential at −100 mV (0.6-s duration) to test potentials of −80 to +100 mV in 20-mV increments. Currents were recorded using an Axopatch 200A amplifier, filtered at 1 kHz and digitized at 4 kHz. Cs-rich solutions were employed. Isotonic CsCl bathing solution contained (mM): 110 CsCl, 10 HEPES, 8 Tris, 5 MgSO 4 , and 100 mannitol (320 mosml/kg H 2 O, pH 7.5). Cell swelling was induced by hypotonic (260 mosml/kg H 2 O) CsCl solution in which mannitol was reduced to 40 mM. The pipette (intracellular) CsCl solution contained (mM): 110 CsCl, 2 MgSO 4 , 1 Na 2 ATP, 15 Na-HEPES, 10 HEPES, 1 EGTA, and 50 mannitol (290 mosml/kg H 2 O, pH 7.3). Since the molecular pathway for swelling-induced ATP release has not as yet been identified, we attempted to produce antibodies against unidentified extracellular epitopes of plasma membrane–associated protein involved in swelling-induced ATP release. Monoclonal antibodies were obtained according to the method described elsewhere . In brief, BALB/c mice were immunized with 10 6 Intestine 407 cells. Peripheral lymphocytes were fused with myeloma NS-1 cells using 50% polyethylene glycol . The supernatant from the resulting hybridoma cells was screened to find colonies that had inhibitory effects on ATP release from swollen Intestine 407 cells. The wells that showed positive activity (15 of 288 wells) were immediately expanded and plated in 98-well plates. Positive colonies were found in 12 of 1,470 wells. The positive hybridoma cells were injected to the mice to produce ascites culture. One of these, termed H2D2-5, was employed for the present experiments after purification by using the E-Z-SEP™ antibody purification reagent (Pharmacia Fine Chemicals). By Western blot analysis using the anti–ATP release antibodies, no specific antigen was detected as a single band (data not shown). Affinity-purified mouse IgG (Zymed Laboratories) was used as a negative control. The following agents were added to bathing solutions: 0.1 mg/ml apyrase, 10–30 μM 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), 100 μM 4-acetamido-4′-isothiocyanostilbene (SITS), 500 μM glibenclamide, 100 μM arachidonic acid, and 5–30 μM GdCl 3 . Data are given as means ± SEM of observations ( n ). Statistical differences of the data were evaluated by paired or unpaired Student's t test and considered significant at P < 0.05. Luminometric ATP assay showed that Intestine 407 cells respond to a hypotonic challenge not only with cell swelling but also with significant release of ATP. The swelling-induced ATP release was found to start within a minute and continued for over 10 min . From the results collected at 1 min after a hypotonic challenge, the initial rate of swelling-induced ATP release from a single Intestine 407 is estimated to be ∼2,500 ATP molecules/s under the assumption that degradation or reuptake of released ATP was negligible. ATP release increased with decreasing extracellular osmolality . The cell viability was not essentially affected by the hypotonic challenge . The mean values of bulk ATP concentration in 400 μl ambient solutions of 4 × 10 5 cells were 0.47 ± 0.08 nM ( n = 39) after a 60-min exposure to isotonic solution and 2.63 ± 0.44 nM ( n = 53) after a 15-min exposure to hypotonic (56% osmolality) solution. The mean rate of ATP release from a single Intestine 407 cell during a 15-min exposure to a hypotonic solution is estimated to be ∼1,700 ATP molecules/s, and that during a 60-min exposure to isotonic solution is ∼60/s. A biosensor ATP assay method was employed to assess the ATP concentration at the outer surface of swollen Intestine 407 cells. Hypotonic stimulation failed to evoke currents in PC12 cell alone (data not shown, n = 11). However, when a voltage-clamped PC12 cell was placed close to an Intestine 407 cell, a series of spiky and sustained inward currents were induced within several minutes after exposure to hypotonic solution (69%) at room temperature . ATP release was found to be always coupled to cell swelling under a microscope. In the presence of an ATP-hydrolyzing enzyme, apyrase, in contrast, the inward current response to hypotonic challenge was virtually abolished, as shown in Fig. 2 B. In the absence of apyrase, the average peak response was 7.9 ± 1.2 pA/pF ( n = 20), which corresponds to 13.1 μM ATP on a calibration curve of ATP-induced PC12 responses . A possible relation of swelling-induced ATP release with the activity of volume-sensitive Cl − channel was investigated by applying a high concentration of Cl − channel blockers. Luminometric ATP assay showed that swelling-induced ATP release was not blocked by 100 μM SITS , as found previously . Glibenclamide and arachidonic acid were also ineffective at the concentrations by which volume-sensitive Cl − channel currents are known to be almost completely abolished in Intestine 407 cells . Glibenclamide insensitivity is in good agreement with a previous observation of swelling-induced ATP release from rabbit ciliary epithelial cells , but is in contrast to those of mechanical stress–induced ATP release from rabbit red blood cells or Ehrlich ascites tumor cells and cAMP-activated ATP release from CFTR-expressing human epithelial cells . Since swelling-induced ATP release was reported to be largely inhibited by a Cl − channel blocker, NPPB, in rabbit ocular ciliary epithelial cells at 100 μM , we then examined the NPPB effect. As shown in Fig. 4 A, NPPB was found to inhibit ATP release from swollen Intestine 407 cells at lower concentrations. The concentration required for 50% inhibition (IC 50 ) was 6.3 ± 0.3 μM . Cell swelling activated outwardly rectifying whole-cell Cl − currents in Intestine 407 cells, and this volume-sensitive outwardly rectifying (VSOR) Cl − current was inhibited by NPPB, as reported previously . The IC 50 value for Cl − current inhibition was 16.0 ± 0.2 μM , which is significantly different from that for ATP release inhibition (0.01 < P < 0.05). These results suggest that swelling-induced ATP release is independent of the activity of volume-sensitive outwardly rectifying Cl − channels in Intestine 407 cells. Recently, Taylor et al. 1998 showed that swelling-induced ATP release from human airway epithelial cells was inhibited by a trivalent lanthanide, Gd 3+ , which is the most commonly used blocker of mechanogated ion channels . Luciferin/luciferase ATP assay showed that ATP release from swollen human epithelial Intestine 407 cells was also sensitive to Gd 3+ . The Gd 3+ effect was concentration dependent, with IC 50 of ∼10 μM. Since Intestine 407 cells are known to have Gd 3+ -sensitive, stretch-activated, Ca 2+ -permeable cation channels , there is a possibility that the Gd 3+ effect on ATP release had been mediated by the inhibiting effect on Ca 2+ influx through this stretch-activated cation channel. However, swelling-activated ATP release was not inhibited, but rather enhanced [from 1.18 ± 0.12 nM ( n = 11) to 3.02 ± 0.27 nM ( n = 20); P = 0.000002], by Ca 2+ removal from the extracellular solution. A similar enhancing effect of Ca 2+ removal was previously observed on cAMP-activated ATP release from CFTR-expressing cells and was explained by a cofactor role of extracellular Ca 2+ for ecto-ATPase activation . Gd 3+ (30 μM) also inhibited swelling-activated ATP release in the Ca 2+ -free conditions [to 1.80 ± 0.39 nM ( n = 19); P = 0.014]. The effect of Gd 3+ on the volume-sensitive Cl − current was then examined in Intestine 407 cells. Gd 3+ (30 μM), which was applied 10–20 min before and during hypotonic challenge, failed to affect swelling-induced activation of the Cl − current . Neither time- and voltage-dependent inactivation kinetics nor outwardly rectifying current–voltage relation was affected by Gd 3+ . These results clearly indicate that the activity of volume-sensitive outwardly rectifying Cl − channels is totally independent of the swelling-induced ATP release in Intestine 407 cells. To assess the molecular nature of swelling-induced ATP release pathway, we raised antibodies that can block ATP release upon osmotic swelling in Intestine 407 cells. As summarized in Fig. 7 , pretreatment with the H2D2-5 antibodies at 12.5 μg/ml for 60 min markedly inhibited swelling-induced ATP release and slightly suppressed basal background ATP release. In contrast, mouse IgG failed to affect significantly the ATP release. Similar inhibiting effects were also obtained by pretreatment with the antibodies at 25 μg/ml for 30 min (data not shown, n = 13). As shown in Fig. 8 , VSOR Cl − currents were not significantly affected by pretreatment with H2D2-5 antibodies (25 μg/ml, 30–50 min). The mean peak current densities with pretreatment with the antibodies ( n = 9) and the negative control IgG ( n = 10) were 274.3 ± 32.6 and 281.4 ± 46.1 pA/pF ( P = 0.45) at +60 mV and 166.1 ± 20.7 and 165.8 ± 28.0 pA/pF ( P = 0.25) at −60 mV, respectively. These results also show that the swelling-induced ATP release is not a prerequisite to activation of the volume-sensitive Cl − channel in Intestine 407 cells. Also, it is likely that the swelling-induced ATP release pathway is distinct from the pore of volume-sensitive outwardly rectifying Cl − channels in the human epithelial cell line. Extracellular ATP at low micromolar concentrations is known to influence activities in a number of muscular and neuronal cells as well as platelets . Recently, ATP release has been found to be induced by stimulation with cAMP , by removing extracellular Cl − , by mechanical perturbation , and by osmotic perturbation in nonneuronal, nonmuscular cells. CFTR was reported to be associated with ATP release induced by these maneuvers by some investigators . However, the pore properties of the CFTR Cl − channel and the ATP channel were found to be distinct . Other investigators showed that the ATP release is not specifically associated with CFTR . The present study demonstrated that reduction of extracellular osmolality can induce ATP release in a dose-dependent manner in a human epithelial cell line that lacks CFTR expression . The possibility that ATP release induced by hypotonic perturbation was an artifact of cell lysis or damage could be ruled out because (a) a hypotonic challenge did not essentially affect the cell viability , and moreover, (b) the swelling-induced ATP release could be inhibited by NPPB , Gd 3+ , and anti–ATP-release antibodies . Upon swelling-induced ATP release, the extracellular ATP concentration in the immediate vicinity of the outer cell surface was found to reach 13 μM . This value would be an underestimate because most cells express ecto-nucleotidases that rapidly hydrolyze ATP present at the cell surface . This concentration is much higher than that needed to stimulate P2-purinergic receptors . Since Intestine 407 cells have purinergic receptors , it is possible that released ATP has some biological functions. There have been two pieces of evidence that extracellular ATP is involved in the RVD after osmotic swelling: (a) addition of ATP to the bathing solution facilitates RVD in Intestine 407 cells , and (b) removal of released ATP with apyrase retards RVD in rat hepatoma cells and Intestine 407 cells (Maeno, E., and Y. Okada, unpublished observations). This ATP action would be mediated by stimulation of P2-purinergic receptors because a blocker of the receptor, suramin, inhibited RVD in both cell species . Roman et al. 1997 and Wang et al. 1996 proposed the hypothesis that the released ATP is involved in activation or upregulation of volume-sensitive Cl − channels, which are associated with Cl − efflux during RVD, in rat hepatoma cells. However, addition of extracellular ATP did not activate inward Cl − currents (Cl − efflux), but inhibited outward Cl − currents (Cl − influx) under hypotonic conditions in human epithelial Intestine 407 cells and rat glioma C6 cells . Furthermore, the present study showed that Gd 3+ (30 μM) virtually abolished swelling-induced ATP release without affecting activation of swelling-induced VSOR Cl − currents in Intestine 407 cells . In addition, even when the ATP release had been blocked by anti–ATP release antibodies , the activity of the VSOR Cl − channel was normally induced upon osmotic swelling . In Intestine 407 cells, therefore, it appears that ATP released upon osmotic swelling facilitates RVD by a mechanism other than activation of volume-sensitive, outwardly rectifying Cl − channels or regulation of the volume-regulatory Cl − efflux. Intestine 407 cells are known to respond to extracellular ATP with an increase in cytosolic Ca 2+ and Ca 2+ -activated K + conductance . Ca 2+ -activated K + conductance was shown to be responsible for volume-regulatory K + efflux during RVD of Intestine 407 cells . Taken together, it can be speculated that swelling-induced ATP release brings about autocrine stimulation of Ca 2+ -activated K + efflux by stimulation of purinergic receptors, thereby facilitating RVD. The route of swelling-induced ATP release is not yet identified, although some investigators have suggested that CFTR and MDR1 are involved in the route. The involvement of CFTR can be excluded in the present study because Intestine 407 cells lack expression of CFTR and failed to respond with ATP release to stimulation with 1 mM dibutyryl cyclic AMP (Tanaka, S., and Okada, Y., unpublished observations). In fact, a blocker of CFTR Cl − channel, glibenclamide, failed to suppress swelling-induced ATP release in the present study . Since Intestine 407 cells are known to express MDR1 , the involvement of MDR1 in swelling-induced ATP release seems possible. However, the transporter function of P-glycoprotein, the product of the MDR1 gene, has recently been shown to be inhibited by glibenclamide . Also, our recent studies have demonstrated that osmotic swelling induced ATP release to an essentially similar degree in both human epidermoid KB cells lacking MDR1 expression (KB-3-1) and a MDR1-transfected KB cell line (KB-G2) (Hazama, A., and Y. Okada, unpublished observations). It seems unlikely that the pore of the volume-sensitive outwardly rectifying Cl − channel can provide a pathway of swelling-induced ATP release in human epithelial Intestine 407 cells because: (a) blockers of the Cl − channel, SITS, arachidonate and glibenclamide, did not abolish swelling-induced ATP release , (b) a potent blocker of swelling-induced ATP release, Gd 3+ , did not inhibit the Cl − currents , and (c) antibodies that inhibit swelling-induced ATP release never affected the Cl − currents . However, the possibility remains that the ATP release represents an ATP efflux through ATP-permeable anion channels that can be activated by cell swelling, but is distinct from the volume-sensitive outwardly rectifying Cl − channel. Since the rate of swelling-induced ATP release seems very low (on the order of 10 3 s −1 per cell) compared with the transporting rate of many types of ion channels (10 6 –10 8 s −1 per channel), it is also possible that the ATP release is mediated by transporter or exocytosis, but not by ATP-selective channels. | Study | biomedical | en | 0.999996 |
10498672 | Inactivation of Ca 2+ channels is thought to be of two types—voltage-dependent inactivation, like that of Na + channels , and Ca 2+ -dependent inactivation, which is determined by the intracellular Ca 2+ concentration . Ca 2+ -dependent inactivation is an important example of negative feedback through which the Ca 2+ levels inside the cell can regulate the amount of Ca 2+ influx. It is, as yet, unknown whether Ca 2+ merely influences the rate constants of entry to and departure from the inactivated state, or if it actually causes the channels to enter an entirely different inactivation state. In this paper, we have studied the inactivation of Ca 2+ channels in Lymnaea neurons. Ca 2+ -dependent inactivation in molluscan neurons has received considerable attention; it was in these neurons that this phenomenon was first characterized . However, it is necessary to reexamine the inactivation of molluscan Ca 2+ channels because the original studies did not take into account the outward proton current, which was discovered later in snail neurons and can easily be misinterpreted as Ca 2+ -current inactivation. Also, the early studies were inconclusive about the amount of voltage-dependent inactivation present in molluscan neurons . These studies, and others established that Ca 2+ channel inactivation under some conditions has a bell-shaped voltage dependence; i.e., depolarizations to potentials that elicit large Ca 2+ currents also cause maximal amounts of inactivation. This is consistent with the idea that inactivation is caused by Ca 2+ influx, and thus a bell-shaped inactivation curve is often interpreted to indicate the presence of Ca 2+ -dependent inactivation. In this study, we show that Ca 2+ channel inactivation in Lymnaea neurons has both Ca 2+ - and voltage-dependent components, and that both of these components have a bell-shaped voltage dependence. From the kinetics of the development of and the recovery from inactivation, we infer that there are two distinct inactivation states, even in the absence of Ca 2+ -dependent inactivation, and an increase in Ca 2+ causes a greater occupancy of the longer-lived inactivation state. We find that while Ca 2+ -dependent inactivation is influenced by Ca 2+ influx, its magnitude does not depend linearly on the magnitude of the influx, as was shown previously , but instead saturates at relatively low levels of Ca 2+ influx. Intracellular EGTA (5 mM) can completely suppress Ca 2+ -dependent inactivation, suggesting that Ca 2+ -dependent inactivation is not caused by Ca 2+ ions binding to the channel protein itself, as proposed by earlier models . We focus our attention on other models that propose that the cytoplasmic Ca 2+ levels control Ca 2+ -dependent inactivation through enzymatic actions , or by modulating the polymerization state of the cytoskeleton . We find no evidence to support that serine/threonine phosphorylation controls Ca 2+ -dependent inactivation in Lymnaea neurons. Cytochalasin B, a disrupter of actin filaments, causes a large increase in inactivation of Ca 2+ channels. However, it appears that the increases in inactivation do not result from a disruption of actin filaments by cytochalasin B. Neurons were dissociated from the pedal, parietal, and visceral ganglia of adult Lymnaea stagnalis, and prepared for patch clamp experiments as previously described . The cells used for this study were nearly spherical, and their diameters ranged from 50 to 75 μm. The Axopatch 200A patch clamp amplifier (Axon Instruments) was used in this study to measure currents. pClamp software (version 6.0) was used for data acquisition (Clampex) and analysis (Clampfit). The patch clamp electrodes typically had resistances of 1 MΩ and tip diameters of 12–16 μm. Series resistance (usually ∼2–4 MΩ) was electronically compensated to >90%. Inactivation measurements were taken at least 10 min after entering the whole-cell configuration, unless otherwise noted, to allow for the diffusion of the electrode solution into the cell. Junction potential errors have not been corrected for in these experiments and are expected to be approximately −10 to −15 mV . Linear leak currents and capacitive transients are subtracted using a P/4 protocol. Currents recorded in the standard solutions are comprised of voltage-gated Ca 2+ and H + currents . These currents are not contaminated with outward Cs + or Cl − currents, since replacing internal Cs + with N -methyl- d -glucamine + , or external Cl − with methanesulfonate − (CH 3 SO 3 − ) does not produce any change in the shape of the recorded currents. Internal perfusion experiments were done following the method described by Neher and Eckert 1988 . The intracellular solution of the cell could be completely exchanged in <6 min, as estimated from measuring outward K + currents while replacing intracellular K + with Cs + . The Lymnaea saline used for dissociation and storage of cells contains 50 mM NaCl, 2.5 mM KCl, 4 mM MgCl 2 , 4 mM CaCl 2 , 10 mM HEPES ( N -[2-hydroxyethyl] piperazine- N ′-[2-ethane sulfonic acid]), adjusted to pH 7.4 with NaOH. The standard extracellular saline used for recording Ca 2+ or Ba 2+ currents is composed of 76 mM TrisCl and 10 mM CaCl 2 or BaCl 2 , and is adjusted to pH 7.4. In some experiments where the concentration of Ca 2+ in the external solution is reduced to 1 mM, 9 mM MgCl 2 is added to keep the total concentration of divalent ions constant. All of the intracellular solutions contain 50 mM HEPES, 0.5 mM MgCl 2 , 3–12 mM CsCl, 15–20 mM aspartic acid, and 2 mM Mg-ATP, with varying amounts of calcium buffers, adjusted to pH 7.3 with CsOH, thus making Cs + the main intracellular cation. The different levels of intracellular Ca 2+ buffers used in this study are 0.1 mM EGTA, 5 mM EGTA, 5 mM EGTA with 2.5 mM CaCl 2 , 5 mM EGTA with 4.5 mM CaCl 2 , and 11 mM 1,2-bis(2-amino phenoxy) ethane- N, N, N ′, N ′-tetraacetic acid (BAPTA) with 1 mM CaCl 2 . These solutions adjusted to various free Ca 2+ levels were used for calibrating Fura-2. H-7 [1-(5-isoquinolinylsulfonyl)-2-methyl piperazine], cyclosporin A, and colchicine are readily soluble in water and their stock solutions were made in distilled water. Although phalloidin is not highly soluble in water, its aqueous solubility is adequate to form a 5-mM stock solution in distilled water. The stock solution for okadaic acid (K + salt) was only three times more concentrated than the final concentrations required and was made directly in the extracellular saline. Stock solutions for calmidazolium, cytochalasin B, and cytochalasin D were made in DMSO. Appropriate volumes of these stock solutions were then added to the external solution in the bath to bring the bath concentration of these compounds to the required levels. Free Ca 2+ levels in cells were measured by loading the cells with an intracellular solution containing 10 μM Fura-2, a Ca 2+ -sensitive ratiometric dye . Fura-2 fluorescence was measured in a confocal arrangement using pinholes as previously described . The calibration solutions described above were used for calibrating Fura-2 in microcuvettes (borosilicate microslides with an optical path length of 50 μm; VitroCom Inc.). The UV exciting Fura-2 was controlled by a Lambda 10-2 filter wheel (Sutter Instrument Co.) and limited to 200-ms pulses of 380 and 360 nm radiation for each measurement of Ca 2+ . Background fluorescence measurements were made before the patch membrane was ruptured, and later subtracted from all records. In the studies reported in this paper, inactivation has been measured using mainly a three-pulse protocol . First a short test pulse (10 ms) to +40 mV is applied, then a conditioning pulse (150 ms) of variable amplitude, followed by a gap (20 ms) at the holding potential (−60 mV), and, finally, a second test pulse to +40 mV is applied. The inactivation caused by the conditioning pulse is calculated as the percent reduction in the test pulse current after the conditioning pulse. In an earlier study , we have shown that this method of measuring Ca 2+ channel inactivation avoids errors caused by H + currents, which can be prominent in snail neurons under the conditions used to record Ca 2+ currents . This is primarily because H + current activates slowly at the test pulse potential, and any H + current activated by the conditioning pulse is given time to deactivate completely during the 20-ms gap, before the second test pulse is applied. Some recovery from inactivation also takes place during the 20-ms gap; consequently, our measurements of inactivation reflect the fraction of Ca 2+ channels in the inactivated state 20 ms after the end of the conditioning pulse and not the total inactivation caused by the conditioning pulse. The length of this gap is determined by the time that H + and Ca 2+ currents, activated by the conditioning pulse, take to completely deactivate. While H + currents deactivate relatively fast, Ca 2+ tail currents after conditioning pulses to large voltages can take 20 ms to decay to baseline. Thus, the gap length has been chosen to be 20 ms to minimize the contamination of the test pulse current by conditioning pulse tail currents, while maximizing the amount of inactivation that can be accurately measured. Since large, positive pulses longer than 150 ms cause a rapid rundown of Ca 2+ current in Lymnaea neurons, we have restricted the study presented here to inactivation caused by 150-ms long conditioning pulses. Consequently, the inactivation levels we measure here are not steady state. In some experiments, tail currents have been used to measure inactivation , using a protocol similar to the one described above. A short test pulse (3 ms) to +120 mV is applied before and after the conditioning pulse, and inactivation is calculated as the percent reduction in the tail current (measured at −40 mV) elicited by the termination of the test pulse. Tail currents typically reached their peak magnitude in 100 μs. We have previously shown that this protocol gives a valid measure of inactivation and also avoids errors due to H + currents. These tail-current measurements are also not contaminated by gating currents or other currents not flowing through Ca 2+ channels. This is shown by reducing the concentration of permeant ions in the extracellular solution and observing that as the permeant ion concentration approaches zero, the tail current measurement also approaches zero . Statistical analyses were performed using Systat software (Version 6; SPSS Inc.) and the corrected R 2 parameter was used to determine the quality of fit of a model to the experimental data. Inactivation is measured using a three-pulse protocol . In this protocol, a short test pulse is applied before and after a conditioning pulse to variable potentials, and inactivation is measured as the percentage reduction in the test pulse current due to the conditioning pulse. There is a 20-ms gap at the holding potential between the conditioning pulse and the second test pulse, which is necessary to allow background currents activated during the conditioning pulse to completely deactivate. However, it also results in some Ca 2+ channels recovering from inactivation during the gap. Thus, our measurement of inactivation relates to the fraction of Ca 2+ channels that are still inactivated at the end of a 20-ms gap. Inactivation measured this way in cells containing a high level of intracellular Ca 2+ buffer (5 mM EGTA) is relatively small (the peak inactivation being ∼0.25), and exhibits a bell-shaped voltage dependence . We show below that the inactivation measured in these conditions is independent of Ca 2+ influx. A common test for the presence of Ca 2+ -dependent inactivation is to compare inactivation of Ca 2+ currents with that of Ba 2+ currents. The underlying assumption is that the intracellular site, which mediates Ca 2+ -dependent inactivation, is less sensitive to Ba 2+ ions than to Ca 2+ ions. Therefore, the current-dependent component of inactivation during Ba 2+ influx would be reduced, as compared with that during Ca 2+ influx. Exchanging external Ca 2+ with Ba 2+ does not cause a reduction in the levels of peak inactivation measured in Lymnaea neurons containing 5 mM EGTA , suggesting that there is very little Ca 2+ -dependent inactivation in this case. The leftward shift in the inactivation curve observed with external Ba 2+ can be explained on the basis of the shift of Ba 2+ current activation to potentials lower than those for Ca 2+ -current activation. Ca 2+ -dependent inactivation also implies that inactivation should depend on the amount of Ca 2+ influx. This is not true for Lymnaea neurons containing 5 mM EGTA. The standard extracellular solution in our experiments contains 10 mM Ca 2+ ; reducing this concentration 10-fold to 1 mM does not cause a significant reduction in the amount of inactivation measured , even though the current magnitude decreases fourfold. We also compared inactivation in cells dialyzed with a 5 mM EGTA solution to that in cells dialyzed with an 11 mM BAPTA solution. In the second case, not only is there a higher concentration of a Ca 2+ buffer, but the buffer used (BAPTA) is also substantially faster. Consequently, the Ca 2+ transients in cells with 11 mM BAPTA should be substantially smaller than those in cells with 5 mM EGTA . However, the inactivation measured in the two cases is similar , showing that the Ca 2+ transients in 5 mM EGTA are already too small to affect inactivation. Another way of assessing the contribution of Ca 2+ influx in Ca 2+ -channel inactivation is to eliminate all Ca 2+ influx, and measure the inactivation of the Ca 2+ channel current carried by monovalents . However, replacing permeant divalent cations with impermeant ones (such as Mg 2+ ) in the extracellular solution causes Lymnaea neurons to develop a large leak current, possibly because of loss of K + channel selectivity . As an alternative approach, we have used 100 μM Cd 2+ , a Ca 2+ channel blocker, to eliminate Ca 2+ influx. Cd 2+ was chosen because it is known to block Ca 2+ channels in a voltage-dependent manner ; it is a more effective blocker at positive potentials than at negative ones, and, consequently, currents during a positive pulse are blocked more than the tail currents (measured at negative potentials). Thus, using 100 μM Cd 2+ in the external solution, we can block 90% of the Ca 2+ influx during a conditioning pulse and still measure inactivation using the tail currents, which are only blocked by 40% (see materials and methods ). We find that for cells containing 5 mM EGTA, the amplitude of peak inactivation does not change when 100 μM Cd 2+ is added to the external solution , which supports our conclusion that inactivation in 5 mM EGTA is independent of Ca 2+ influx. (We consistently observe that Cd 2+ also causes a change in the shape of the inactivation curve, which we do not understand.) We conclude from the four types of experiments described above that Ca 2+ -channel inactivation in cells containing 5 mM EGTA is entirely voltage dependent and is independent of Ca 2+ influx. Ca 2+ channels in Lymnaea neurons are capable of exhibiting Ca 2+ -dependent inactivation when the intracellular Ca 2+ buffering is lowered (shown below). Thus, we conclude that 5 mM EGTA reduces intracellular Ca 2+ transients to a size where they are incapable of activating the site that mediates Ca 2+ -dependent inactivation. To demonstrate that Ca 2+ channels in Lymnaea neurons are capable of exhibiting Ca 2+ -dependent inactivation, we compared Ca 2+ -channel inactivation in two identical populations of neurons loaded with different amounts of intracellular Ca 2+ buffer. Cells containing 0.1 mM EGTA show substantially more inactivation than those containing 5 mM EGTA , indicating that increased levels of intracellular Ca 2+ lead to an increase in Ca 2+ channel inactivation. This result was confirmed with internal perfusion experiments in which intracellular solutions were changed while recording from one cell. Inactivation was first measured when the cells were perfused with a solution containing 5 mM EGTA, and was found to increase if the intracellular solution was changed to one containing only 0.1 mM EGTA . In control experiments, in which the second intracellular solution perfused into the cells was the same as the first, inactivation was not affected by the exchange (data not shown). To ensure that it is indeed the Ca 2+ -buffering properties of EGTA and not some other pharmacological property that contributes to Ca 2+ -channel inactivation, we measured inactivation in cells where the intracellular solution contained 5 mM EGTA, loaded with different amounts of CaCl 2 . Cells containing 5 mM EGTA alone show smaller magnitudes of inactivation than those in which 5 mM EGTA has been loaded with 2.5 mM CaCl 2 or 4.5 mM CaCl 2 , suggesting that it is the concentration of free Ca 2+ buffer that is important for determining the amount of inactivation. These solutions of a fixed amount of Ca 2+ buffer and variable amounts of CaCl 2 also contain slightly different levels of free Ca 2+ . But the free Ca 2+ levels in all these solutions are low (<10 −6 M) and do not appear to determine the amount of Ca 2+ -dependent inactivation . The increased Ca 2+ channel inactivation in Lymnaea neurons containing 0.1 mM EGTA is dependent upon Ca 2+ influx. In these cells, exchange of an external Ca 2+ -containing solution with a Ba 2+ -containing solution causes a significant decline in peak inactivation , even though the magnitude of Ba 2+ current is two to three times larger than that of Ca 2+ current. Similarly, changing the external Ca 2+ concentration from 10 to 1 mM also leads to a decrease in the total Ca 2+ -channel inactivation measured in cells containing 0.1 mM EGTA . Furthermore, 100 μM Cd 2+ in the extracellular solution causes a substantial decrease in inactivation measured in cells containing 0.1 mM EGTA ; the remaining inactivation is not significantly different from that in cells with 5 mM EGTA under similar conditions . From these experiments, we conclude that Lymnaea neurons containing 0.1 mM EGTA have both Ca 2+ - and voltage-dependent components, while those with 5 mM EGTA exhibit only the voltage-dependent component of Ca 2+ channel inactivation. For the purposes of this study, we define Ca 2+ -dependent inactivation in these cells as the difference in inactivation observed with cells containing 0.1 and 5 mM EGTA. We measured the rates with which the Ca 2+ channels recover from inactivation in cells containing 5 or 0.1 mM EGTA. This was done by varying the lengths of the gap after the conditioning pulse in a protocol that measures inactivation using tail currents (see materials and methods ). Using this protocol, we find that the recovery of Ca 2+ channels from inactivation has a biexponential time course at −60 mV . After a conditioning pulse to +120 mV, the rate of recovery in 0.1 mM EGTA is not substantially different from that in 5 mM EGTA. However, after conditioning pulses to +40 and +60 mV (which cause maximal inactivation) the slow component of recovery is much larger for 0.1 mM EGTA compared with that for 5 mM EGTA. This is accompanied by a modest decrease in the magnitude of the fast component of recovery in 0.1 mM EGTA. The fast component of recovery decays rapidly in the first 20 ms; thus, most of the difference observed between inactivation measured in 0.1 and 5 mM EGTA measured using the 20-ms gap is due to the differences in the magnitude of the slow component of recovery in the two conditions. The two separate time constants for the rate of recovery from inactivation indicate that there are two different inactivated states from which the channels are recovering—at negative potentials, recovery from one inactivated state takes place at a considerably faster rate than from the other inactivated state. The effect of Ca 2+ is to increase the occupancy of the latter state. We also measured the development of inactivation during conditioning pulses to +60 and +120 mV. The inactivation measurements were made after allowing the channels to recover for 20 ms after conditioning pulses of variable length, and thus are not independent of the recovery rates. During a pulse to +120 mV, inactivation develops with a single time constant of 250 ms in both 0.1 and 5 mM EGTA . However, when the conditioning pulse is to +60 mV, there is an additional faster component (τ = 50 ms), and the amplitude of this component is three times as large in 0.1 as in 5 mM EGTA. These experiments also indicate that the Ca 2+ influx during the tail currents at the end of the conditioning pulse does not contribute significantly towards inactivation, since inactivation approaches zero as the conditioning pulse becomes very short. In the discussion, we develop a model of Ca 2+ -induced inactivation that can account for our observations regarding the kinetics of inactivation in 5 and 0.1 mM EGTA. We have shown that for cells containing 0.1 mM EGTA, the Ca 2+ -dependent component of inactivation can be reduced by reducing Ca 2+ influx . The magnitude of Ca 2+ -dependent inactivation, however, is not linearly related to the amount of Ca 2+ influx. This conclusion is demonstrated by the experiment in which the Ca 2+ channel blocker Co 2+ was used to reduce the influx of Ca 2+ during conditioning pulses. This experiment is analogous to the one described above using Cd 2+ to block the Ca 2+ current, but Co 2+ is a weaker Ca 2+ channel blocker than Cd 2+ , and exerts a simpler, non–voltage-dependent block of current . In these experiments, 1 mM Co 2+ was used to replace 1 mM of the 10-mM external Ca 2+ , resulting in a >50% block of Ca 2+ currents; yet it was found to have very little effect on Ca 2+ -channel inactivation . Therefore, relatively small amounts of Ca 2+ influx during the conditioning pulse may be sufficient to cause maximal Ca 2+ -dependent inactivation. We also observe that Ca 2+ -dependent inactivation reduces to a half when the external Ca 2+ is reduced from 10 to 1 mM , even though the peak Ca 2+ current is reduced to a fourth. Ca 2+ -dependent inactivation, in this case, is the difference between the inactivation measured in cells with 0.1 mM EGTA and that in cells containing 5 mM EGTA . Our conclusion that the amount of Ca 2+ -dependent inactivation is not simply related to the magnitude of Ca 2+ influx during the conditioning pulse is also supported by other observations. In cells perfused with 0.1 mM EGTA, the decrease in Ca 2+ current over time (due to rundown) is not accompanied by any significant changes in inactivation. Also, there is no obvious correlation between the peak inactivation and the current density measured in the 53 cells containing 0.1 mM EGTA that we studied . It should be noted, however, that peak inactivation is the sum of voltage- and Ca 2+ -dependent inactivation. It is possible that in the absence of a strong dependence of Ca 2+ -dependent inactivation on Ca 2+ current density, the random variation in voltage-dependent inactivation obscures any weaker correlation between the two quantities in Fig. 6 B. We investigated the possibility that the basal (i.e., steady state) levels of Ca 2+ within the cell may affect Ca 2+ -channel inactivation. Fura-2 measurements indicated that steady state Ca 2+ levels in cells containing 0.1 mM EGTA are much higher (70–300 nM) than those in cells containing 5 mM EGTA (5–20 nM). This difference occurs despite the fact that the two intracellular solutions, containing 5 and 0.1 mM EGTA, respectively, have similar low levels of free Ca 2+ (2–10 nM, according to Fura-2 measurements in microcuvettes). The increase in free Ca 2+ levels in cells perfused with a poorly buffered solution is probably due to a high rate of Ca 2+ influx through the plasma membrane. Thus, it is possible that increased Ca 2+ -channel inactivation observed in cells with 0.1 mM EGTA results from higher basal levels of free Ca 2+ inside the cells. To examine whether differences in basal Ca 2+ levels can account for the variations in inactivation measured in different cells, we measured free Ca 2+ levels (using Fura-2) and inactivation in cells perfused with an intracellular solution containing either 0.1 mM EGTA or 5 mM EGTA /2.5 mM Ca 2+ . These two intracellular solutions were chosen since they result in comparable values of free intracellular Ca 2+ levels, but have very different Ca 2+ -buffering capacities. We find that while cells with 0.1 mM EGTA consistently show more inactivation than cells with 5 mM EGTA/2.5 mM Ca 2+ , there is no correlation between peak inactivation and steady state Ca 2+ levels for the 0.1 mM EGTA data . This leads us to believe that the site that mediates Ca 2+ -dependent inactivation is not sensitive to resting levels of Ca 2+ (≤300 nM); instead, intracellular domains of high Ca 2+ that are transiently set up when Ca 2+ channels are activated must be mediating Ca 2+ -channel inactivation. Recent studies have shown that, in ventricular myocytes, an influx of Ca 2+ through voltage-gated Ca 2+ channels triggers a release of Ca 2+ from the sarcoplasmic reticulum, and it is this release of Ca 2+ from intracellular stores that is largely responsible for Ca 2+ channel inactivation in these cells . Such a scenario could potentially explain the nonlinear dependence of Ca 2+ -dependent inactivation on Ca 2+ influx (described above). Therefore, we investigated if Ca 2+ -induced Ca 2+ release (CICR) may also play a role in the Ca 2+ -dependent inactivation of Lymnaea neurons. Ryanodine is known to be an inhibitor of CICR . Ryanodine (10 μM), applied extracellularly, does not produce any substantial change in inactivation in cells containing 0.1 mM EGTA (peak inactivation is 0.57 ± 0.07 in the control solution and 0.61 ± 0.08 in 10 μM ryanodine, n = 4). Low caffeine (1 mM) is thought to potentiate CICR, while 10 mM caffeine depletes the internal stores of calcium . However, neither concentration of caffeine has any effect on Ca 2+ channel inactivation: peak inactivation is 0.56 ± 0.03 (control), compared with 0.63 ± 0.05, n = 2 (1 mM caffeine), and 0.53 ± 0.03 (control), compared with 0.58 ± 0.05 ( n = 3) in 10 mM caffeine. While intracellular Ca 2+ levels in Lymnaea neurons were not monitored during these experiments, similar applications of ryanodine and caffeine are effective in changing intracellular Ca 2+ levels in other molluscan neurons . Therefore, we conclude that CICR is not involved in Ca 2+ -current inactivation in Lymnaea neurons, and that influx of extracellular Ca 2+ through the voltage-gated Ca 2+ channels is wholly responsible for the Ca 2+ -dependent component of inactivation. Different mechanisms for Ca 2+ -dependent inactivation have been proposed in the literature. Some researchers have concluded that Ca 2+ may bind directly to the Ca 2+ channel , or to a protein, such as calmodulin, that is closely associated with the Ca 2+ channel , causing Ca 2+ -dependent inactivation. The result that 5 mM EGTA can completely suppress Ca 2+ -dependent inactivation leads us to question this model in Lymnaea neurons (see discussion ). We think it is likely that intracellular Ca 2+ binds to some other cytoplasmic protein at some distance from the channel, which in turn influences the inactivation of Ca 2+ channels. This intermediate protein could be involved in phosphorylation or be a component of the cortical cytoskeleton . We examine these hypotheses below. It has been proposed that an increase in cytoplasmic Ca 2+ levels may lead to an activation of a Ca 2+ -dependent phosphatase (or a kinase) that may alter the phosphorylation state of the Ca 2+ channel leading to an increase in inactivation . In support of this theory, Schuhmann et al. 1997 have shown that cyclosporin A, an inhibitor of Ca 2+ -activated protein phosphatase 2B (calcineurin), can substantially reduce the magnitude of Ca 2+ -dependent inactivation of L-type Ca 2+ channels in smooth muscle cells. However, Ca 2+ channel inactivation in other preparations has been shown to be insensitive to phosphorylation . We tested for an effect of phosphorylation in Lymnaea Ca 2+ -channel inactivation by extracellularly applying the drug H-7, a broad-range serine/threonine kinase inhibitor. We find that H-7 has no significant effect upon inactivation in cells perfused with 0.1 mM EGTA solution (peak inactivation goes from 0.42 ± 0.04 in control external solution to 0.45 ± 0.04 in the presence of 250 μM H-7, n = 4). Okadaic acid, a phosphatase inhibitor, also has no effect upon inactivation in cells with 0.1 mM EGTA (peak inactivation is 0.60 ± 0.08 in the control solution and 0.61 ± 0.07 in the presence of 5 μM okadaic acid, n = 3). Since okadaic acid is not effective against calcineurin, we also tested for an effect of Cyclosporin A (CsA), a specific inhibitor of calcineurin, on inactivation of Ca 2+ channels. Neither acute application of 10 μM CsA during a whole-cell experiment, nor pretreatment of cells with 10 μM CsA for at least 20 h, causes any change in the inactivation of Ca 2+ channels (peak inactivation is 0.50 ± 0.02 for cells pretreated with CsA, n = 4, and is 0.52 ± 0.04, n = 7, for cells in the control saline under similar conditions). While we don't have any direct evidence for an effect of these drugs on phosphorylation in Lymnaea neurons, they have been shown to inhibit phosphorylation, or dephosphorylation in similar preparations of other molluscan neurons . Therefore, we believe that serine/threonine phosphorylation does not play any role in the inactivation of Ca 2+ channels in Lymnaea neurons; however, our experiments leave open the possibility that tyrosine phosphorylation may be involved. Previous experiments done in our lab have shown that the rundown process of Ca 2+ channels in giant inside-out patches is accelerated by the disruption of cytoskeleton . To address the question of whether Ca 2+ -channel inactivation in a whole-cell preparation is influenced by the cytoskeleton, we studied the effects of acute bath application of colchicine and cytochalasin B on Ca 2+ -channel inactivation. We find that 100 μM colchicine, a microtubule disrupter, has no effect upon inactivation (peak inactivation, in cells containing 5 mM EGTA, is 0.26 ± 0.02 in the control solution and 0.27 ± 0.02 upon addition of 100 μM colchicine, n = 5). However, cytochalasin B, a disrupter of actin microfilaments, causes a large increase in inactivation of Ca 2+ channels. This effect of cytochalasin B (which was applied extracellularly, being membrane permeant) is extremely robust and reproducible . Addition of the vehicle by itself produces no change in inactivation. Cytochalasin B causes a rapid and stepwise increase in Ca 2+ channel inactivation . The effect of cytochalasin B is selective for inactivation, since it has very little effect on the magnitude of Ca 2+ current , and its rate of rundown. Furthermore, the recovery of Ca 2+ channels from inactivation is also much slower in the presence of cytochalasin B, primarily because of the increased amplitude of the slow component of recovery , which is similar to the effect of reducing intracellular EGTA concentration. Surprisingly, the effect of cytochalasin B is readily reversible; the Ca 2+ -channel inactivation returns to its pre–cytochalasin B levels after a few minutes of perfusion with the control external saline. To quantify the effect of cytochalasin B on inactivation, we calculated an f parameter, the fractional increase in inactivation. The f parameter is defined as: \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*}f=\frac{{\mathrm{{\Delta}}}I}{{\mathrm{{\Delta}}}I_{{\mathrm{max}}}}{\mathrm{,}}\end{equation*}\end{document} where Δ I is the change in inactivation upon the addition of cytochalasin B, and Δ I max is the maximal possible change in inactivation. We chose f , calculated for a conditioning pulse to +40 mV, f 40 mV , to measure the effect of different concentrations of cytochalasin B upon inactivation in cells with 5 mM EGTA, and obtain the dose–response curve. The concentration of cytochalasin B that causes half the maximal effect on inactivation is ∼100 μM. The effect of cytochalasin B in increasing inactivation in cells with 5 mM EGTA was compared with that for cells with 0.1 mM EGTA. Surprisingly, f 40mV , which is measured at +40 mV, where Ca 2+ influx is maximal, is not significantly different in the two cases , suggesting that the effect of cytochalasin B in increasing inactivation is independent of the Ca 2+ -dependent inactivation. While cytochalasin B is known to disrupt the actin cytoskeleton , we could not confirm a role of actin filaments in Ca 2+ -channel inactivation using other cytoskeletal agents. Cytochalasin D, another actin filament disrupter, has no significant effect on Ca 2+ -channel inactivation in Lymnaea neurons . Also, internally applied phalloidin, a stabilizer of microfilaments, does not decrease inactivation in cells with 0.1 mM EGTA (data not shown), and is unable to block the effect of extracellularly applied cytochalasin B in cells with 5 mM EGTA . Moreover, cytochalasin B (50 μM), applied intracellularly, does not increase inactivation (data not shown). It is possible that phalloidin and cytochalasin B, added to the cell through the pipette solution, are unable to reach the cortical cytoskeleton, which alone may be involved in regulating inactivation. However, these results are also consistent with an extracellular effect of cytochalasin B on Ca 2+ -channel inactivation, and suggest that the effect of cytochalasin B on Ca 2+ -channel inactivation may be mediated by mechanisms independent of actin microfilaments. In this study, we have characterized the Ca 2+ and voltage-dependent inactivation of Ca 2+ channels in Lymnaea neurons. Many of the earliest studies describing Ca 2+ -dependent inactivation of Ca 2+ channels were done in molluscan neurons . However, these early studies were done before outward H + currents had been identified in snail neurons , and this may have resulted in erroneous measurements of Ca 2+ -channel inactivation. Outward H + currents cannot be pharmacologically isolated from inward Ca 2+ currents and can give the impression of enhanced inactivation of inward currents. We have shown earlier that the three-pulse protocol used to measure inactivation in this study avoids errors due to H + currents (see materials and methods ). For some experiments, we have used tail currents to measure inactivation. This method is also independent of H + currents because the H + tail currents deactivate faster than the Ca 2+ tail currents at negative potentials , and therefore do not contribute significantly to the measurement of peak Ca 2+ tail current . Much of the recent work in Ca 2+ -channel inactivation has focussed on the study of recombinant channels expressed in heterologous systems , and as such has been successful in avoiding the problems of overlapping currents. However, Ca 2+ -channel inactivation, especially the Ca 2+ -dependent component, is greatly influenced by the intracellular environment of the channel , which may be very different in a heterologous system compared with the native cell. Hence, study of Ca 2+ channels in their native environments provides a necessary complement to the molecularly defined study of these channels in heterologous systems. Ca 2+ -channel inactivation in Lymnaea neurons perfused with 5 mM EGTA solution is not affected by replacing external Ca 2+ by Ba 2+ , reducing the Ca 2+ influx , or by increasing the level of intracellular Ca 2+ buffering . We conclude from this that Ca 2+ channels in Lymnaea neurons containing 5 mM EGTA exhibit only voltage-dependent inactivation, even though the inactivation curve in 5 mM EGTA is bell shaped. A bell-shaped inactivation curve has often been taken as evidence for the presence of Ca 2+ -dependent inactivation, though previous studies of native Ca 2+ channels in bullfrog sympathetic neurons and of recombinant Ca 2+ channels in HEK 293 cells have also shown that purely voltage-dependent mechanisms can yield bell-shaped inactivation curves. Our results extend those of the earlier studies, for we show that even in Ca 2+ channels capable of Ca 2+ -dependent inactivation, a bell-shaped inactivation curve is not always correlated with the presence of Ca 2+ -dependent inactivation. Jones and Marks 1989 have proposed a model where the inactivated state of the channel is reached with highest probability from the open state, but the probability of this transition decreases with increasing voltage. Patil et al. 1998 have proposed a different model where the inactivated state is reached only from intermediate closed states. This model predicts that one long depolarizing step causes less inactivation than the cumulative inactivation caused by a number of shorter steps to the same voltage. However, experiments in Lymnaea neurons failed to conform to this prediction, but this model cannot be ruled out altogether. The model proposed by Correa and Bezanilla 1994 for Na + channel inactivation in squid giant axons predicts the existence of another open state that is preferentially reached from the inactivated state, and such a model also yields a bell-shaped inactivation curve. Another possibility is that the bell-shaped inactivation curve results from an inactivation process that increases monotonically with voltage combined with a “facilitation” process that increases at higher voltages. Such a facilitation of L-type current by very positive voltages has been reported, and is accompanied by a slowing of deactivation kinetics after long, positive prepulses ; this slowing of deactivation kinetics is also observed in Lymnaea channels . However, we believe that the process that underlies the slowing of deactivation kinetics in Lymnaea neurons is of a different nature than the one that leads to the bell-shaped inactivation curve. This is because the change in deactivation kinetics lasts only a very short time after the conditioning pulse, and the 20-ms gap before the second test pulse eliminates it. However, the inactivation curve measured is consistently bell shaped. It is possible, though, that some other facilitatory process not involving a change in deactivation kinetics may be involved. While a number of models are adequate for explaining the bell-shaped inactivation curves that we obtain, we do not have any evidence that strongly favors one model over another. We show that increased intracellular Ca 2+ concentration increases the time that Ca 2+ channels require to recover from inactivation. The effect of Ca 2+ in inhibiting the recovery of channels from inactivation at negative potentials has been observed before ; however, our results differ from those of the previous studies in that we do not observe a change in the time constants of inactivation due to Ca 2+ influx, but only a change in the relative amplitudes of the fast and slow components. It is more difficult to interpret our results pertaining to the development of inactivation since the measurements of inactivation are dependent on the rate of recovery. To explain these observations, we have developed a simple model of the inactivation kinetics of Ca 2+ channels at different voltages that fits the data shown in Fig. 5A and Fig. B . In this model, there are two inactivated states, I FR and I SR , that can be reached from the noninactivated states (which have been lumped together as N–I ). At negative potentials, recovery from I FR is considerably faster than that from I SR . Ca 2+ influx during a conditioning pulse increases the occupancy of the inactivated state I SR , and thus increases the amplitude of the slow component of recovery at negative potentials. Our data can be fit by making only the forward rate constant (α SR ) Ca 2+ dependent, but we cannot rule out the possibility that the reverse rate constant (β SR ) may also be dependent on Ca 2+ influx. After repolarization, I FR is depleted rapidly, while I SR is not greatly affected within the first 20 ms. Hence, the difference in inactivation that we measure between 0.1 and 5 mM EGTA with the three-pulse protocol is largely the difference in the I SR components under the two conditions. Also, the model predicts that the actual difference in inactivation between 0.1 and 5 mM EGTA during the course of a conditioning pulse is smaller than the difference we measure at the end of the 20-ms gap . This is so because the proportion of channels in I FR is larger in 5 than in 0.1 mM EGTA; however, the difference measured after a 20-ms gap reflects primarily the difference in the I SR components in the two cases. Since inactivation in 5 mM EGTA is completely voltage dependent, these results imply that α SR is not zero at positive voltages even in 5 mM EGTA, and a conditioning pulse to +60 mV causes some occupancy of I SR . In this analysis, we have assumed that there is only one class of Ca 2+ channels in Lymnaea neurons. Pharmacological and kinetic studies done in our lab have failed to resolve more than one component of Ca 2+ current, though multiple types of Ca 2+ channels cannot be ruled out. In such a case, is it possible that Ca 2+ - and voltage-dependent inactivation are due to different channel types that gate independently of each other? The results from our kinetic analyses show that the magnitude of voltage-dependent inactivation is reduced in the presence of Ca 2+ -dependent inactivation (since the fast component of recovery is smaller in 0.1 than in 5 mM EGTA), which indicates that these phenomena are not independent of each other. It is, therefore, unlikely that different channel types underlie voltage- and Ca 2+ -dependent inactivation. Our results with the Ca 2+ channel blocker Co 2+ indicate that just half of the Ca 2+ influx under standard conditions may be adequate to almost saturate the Ca 2+ -dependent component of inactivation . To illustrate the relation between Ca 2+ -dependent inactivation and Ca 2+ influx, we plotted Ca 2+ -dependent inactivation against Ca 2+ influx for a typical cell in Fig. 10 . Fig. 10 (▪, connected by a continuous line) shows the relation between these two quantities for each of the conditioning pulse potentials with 10 mM external Ca 2+ . The general shape of these points shows that the Ca 2+ -dependent inactivation is not linearly related to the Ca 2+ influx, and that Ca 2+ influx during pulses from +30 to +80 mV (with 10 mM external Ca 2+ ) causes considerable saturation of the Ca 2+ -dependent component of inactivation. The shape of this curve is in good agreement with the 20% reduction in peak Ca 2+ -dependent inactivation that is caused by a 50% reduction in Ca 2+ influx with 1 mM Co 2+ . The result that a fourfold reduction in Ca 2+ influx, caused by reducing extracellular Ca 2+ from 10 to 1 mM, only blocks half of the peak Ca 2+ -dependent inactivation also fits this same relationship . Several researchers have proposed that Ca 2+ may cause inactivation by binding to a site on the channel itself , or to an associated protein in the channel complex . However, we do not think that is the case in Lymnaea Ca 2+ channels, mainly because of two observations. The first is that 5 mM EGTA is able to suppress Ca 2+ -dependent inactivation completely. Since EGTA is a slow Ca 2+ buffer, 5 mM EGTA is fairly ineffective in reducing Ca 2+ concentration at the mouth of the channel and makes a significant contribution only at some distance from the channel. A mobile Ca 2+ buffer attenuates the intracellular Ca 2+ transient exponentially with distance from the channel, the space constant being ∼60 nm for 5 mM EGTA . At distances smaller than the space constant, the buffer has little effect on the Ca 2+ transient. Hence, we think the Ca 2+ -sensitive site mediating Ca 2+ -dependent inactivation of Lymnaea Ca 2+ channels must be at least 100 nm away from the channel itself. Secondly, if there were a Ca 2+ -binding site on the Ca 2+ -channel complex that causes the inactivation of the channel, then this site would have a low affinity for Ca 2+ (in the range of a few hundred micromolar, which is the concentration of Ca 2+ at the inner mouth of an open channel). However, Ca 2+ channels in Lymnaea neurons are sensitive to much lower levels of intracellular Ca 2+ . Perfusing these neurons with intracellular solutions buffered to 2 μM Ca 2+ causes a rapid loss of Ca 2+ current as soon as the intracellular saline diffuses within the cells . Also, a sudden increase in the Ca 2+ levels to a few hundred nanomolar by flash photolysis of Ca 2+ -loaded DM-nitrophen causes a reversible decrease in Ca 2+ current . We interpret these results to mean that the site mediating Ca 2+ -channel inactivation is sensitive to lower levels of intracellular Ca 2+ and, therefore, cannot be on the channel. Here we are assuming that the inactivation of Ca 2+ channels (as measured by 150-ms-long pulses), rundown (which develops with a much slower time course of minutes in whole-cell preparations and is largely irreversible) and block of Ca 2+ current by intracellular Ca 2+ (which has a time course of a few milliseconds) are all caused by similar mechanisms and, hence, have similar sensitivities to intracellular Ca 2+ . It is possible, however, that the mechanism of inactivation is independent of rundown and Ca 2+ block; in which case, we cannot use the above results to estimate the Ca 2+ sensitivity of the site mediating Ca 2+ -dependent inactivation. Studies on vertebrate L-type Ca 2+ channels have led several researchers to conclude that Ca 2+ -dependent inactivation in these channels is caused by Ca 2+ -ion binding directly to the channel or to a site closely associated with it . Giannattasio et al. 1991 showed that L-type Ca 2+ channels in a smooth muscle-derived cell line have Ca 2+ -dependent inactivation even in the presence of 10 mM intracellular BAPTA, suggesting that the Ca 2+ -binding site is very close to the Ca 2+ pore. This view has been supported by the identification of a putative Ca 2+ binding domain (an E-F hand) on the COOH terminal of the mammalian α 1C subunit of the Ca 2+ channel that controls the Ca 2+ -dependent inactivation of these channels , although some studies suggest that the binding of Ca 2+ ions to this site is unimportant for Ca 2+ -dependent inactivation . Recent studies on Ca 2+ channels containing the α 1C subunit suggest that the channel is stably complexed with calmodulin, and it is the binding of Ca 2+ ions to calmodulin that causes inactivation of the Ca 2+ channel . Considering that 5 mM intracellular EGTA completely eliminates Ca 2+ -dependent inactivation in Lymnaea neurons, it is likely that vertebrate L-type Ca 2+ channels have a different mechanism for Ca 2+ -dependent inactivation than do the Ca 2+ channels in Lymnaea neurons. Several studies have shown that voltage-gated Ca 2+ channels are sensitive to the state of the cytoskeleton . However, the role of the cytoskeleton in modulating Ca 2+ current is less clear. One study in chick ventricular myocytes shows that colchicine (a microtubule disrupter) and taxol (a microtubule stabilizer) strongly influence the inactivation kinetics of L-type Ca 2+ channels . This is in contrast with the present study, where we find that the inactivation of Lymnaea channels is not affected by 100 μM colchicine. In this study, we have shown that cytochalasin B, an actin microfilament disrupter, increases the inactivation of Ca 2+ channels; however, there is some suggestion in our results that actin filaments may not be involved in this effect of cytochalasin B on inactivation. First, the concentration of cytochalasin B that results in half-maximal increase in inactivation is ∼100 μM, which is very high for an effect on actin filaments. Second, cytochalasin D does not yield the same affect as cytochalasin B . Also, cytochalasin B applied intracellularly does not increase the amount of inactivation measured, and phalloidin applied intracellularly cannot block the effect of extracellularly applied cytochalasin B . (Although, in the last two instances, it is questionable whether these drugs can effectively diffuse through the cytoplasm to reach the cytoskeleton close to the membrane.) Additionally, the time course of the onset of the cytochalasin B effect is very fast, limited only by the delay in application, and the effect is readily and completely reversible, inactivation returning to its original levels within minutes of perfusion with the control solution. This suggests that cytochalasin B may have only an extracellular effect. The mechanism by which cytochalasin B increases inactivation currently remains unresolved. Cytochalasin B is known to inhibit the glucose transporter ; however, glucose is not present in the extracellular medium at the time of the experiments. Thus, the effect of cytochalasin B on the glucose transporter is unlikely to explain the increase in Ca 2+ channel inactivation. Our results are consistent with the model where cytochalasin B acts as a weak open channel blocker, entering the pore from the external side. The slow time constant of recovery from inactivation that we observe in cytochalasin B may be due to the slow rate of dissociation of cytochalasin B from the pore. More experiments are required to distinguish whether actin filaments play a role in inactivation, or if cytochalasin B acts like an open channel blocker. These studies leave unknown the mechanisms of Ca 2+ channel inactivation in Lymnaea neurons. While we conclude that serine/threonine phosphorylation does not play any role in Ca 2+ channel inactivation, it is possible that tyrosine phosphorylation may be involved. Indeed, tyrosine phosphorylation has been shown to modulate excision-activated Ca 2+ channels in Lymnaea . We cannot rule out a role of the actin cytoskeleton in Ca 2+ -dependent inactivation, although the large effect of cytochalasin B on inactivation is unlikely to involve the cytoskeleton. Recent studies suggest that calmodulin may cause Ca 2+ -dependent inactivation of mammalian L- and P/Q-type channels by interacting directly with the cytoplasmic domains of the α 1 subunits in a Ca 2+ -dependent manner . This remains a possibility for Lymnaea Ca 2+ channels also, although our results suggest that the Ca 2+ receptor in Lymnaea neurons is not stably associated with the Ca 2+ channel (as suggested by these studies for the L-type channels). Other intracellular proteins, such as G-proteins or phospholipid kinases, could also be involved in Ca 2+ -dependent inactivation of Ca 2+ channels. | Study | biomedical | en | 0.999995 |
10498673 | The high-resolution structure of the KcsA K + channel has invigorated current approaches to the molecular foundations of cellular electrical excitability . KcsA is a prokaryotic channel with little sequence similarity to eukaryotic K + channels except in the pore-forming region. However, its structure provides compelling explanations for ion permeation and gating phenomena observed over many years in a multitude of K + channels. Ironically, functional properties of KcsA have been described only in outline. Single-channel recording, flux measurements, and ligand-binding assays have shown KcsA to be a high-conductance, tetrameric, K + -selective channel with an externally located receptor site for charybdotoxin-family peptides . While its structure is largely in harmony with models of familiar K + channels, an unexpected characteristic of KcsA is its gating by protons . The channel reconstituted into planar bilayer membranes opens significantly only at pH values lower than ∼5. Despite the fact that most of the protein's water-exposed mass, with fully 85% of its dissociable protons, is located on the cytoplasmic side of the membrane, proteolysis protection studies have led to the contention that activating protons are sensed on the channel's external side . As a prelude to a full ion selectivity study of KcsA, we sought to establish a planar lipid bilayer system in which single purified KcsA channels may be recorded accurately and to survey several basic pore properties of the channel. Single KcsA channels can be observed at 5 kHz bandwidth in a low-noise planar bilayer system. We document functionally asymmetric characteristics of KcsA and use several of these to show that protons gate this channel from the cytoplasmic, not the external, side of the membrane. General chemicals were of reagent grade or higher. High-purity (>99.997%) KCl was obtained from Alfa Inorganics. [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET, Br salt) 1 and 2-(sulfonatoethyl)methanethiosulfonate (MTSES, Na salt) were obtained from Anatrace. Dodecylmaltoside was from Calbiochem Corp. and CHAPS from Pierce Chemical Co. Lipids (Avanti Polar Lipids) were 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and phosphatidylglycerol (POPG), stored in sealed ampules at −80°C. Charybdotoxin (CTX) was expressed in Escherichia coli and purified as described . Two slightly different constructs of KcsA were used. Most experiments employed a synthetic gene coding for the natural KcsA polypeptide sequence with a hexahistidine tag added to the NH 2 terminus. This was derived by truncating the previously described “SliK” construct after residue R160, the natural COOH terminus. For charybdotoxin blocking experiments, we used a wild-type KcsA construct kindly provided by Dr. R. MacKinnon (The Rockefeller University, New York) and “KcsA-Tx,” a triple-mutant of this (Q58A/T61S/R64D) that binds CTX . Solutions used for planar bilayer recording are coded according to the convention: nKm, where n and m are numbers denoting the concentration (mM) of K + ion, and the pH, respectively. The solutions also contained an appropriate anionic buffer. Thus, solution 200K7 consists of 195 mM KCl/5 mM KOH/10 mM HEPES, adjusted to pH 7.0 with HCl, and 20K4 consists of 15 mM KCl/5 mM KOH/10 mM succinic acid, adjusted to pH 4.0 with HCl. KcsA was expressed in E. coli and purified on Ni 2+ affinity columns as described . The purified channel was eluted in 400 mM imidazole at 1–5 mg/ml protein concentration quantified by the extinction coefficient at 280 nm . Immediately after purification, KcsA was reconstituted into liposomes at room temperature as follows. A micellar solution of phospholipids (7.5 mg/ml POPE, 2.5 mg/ml POPG) and 34 mM CHAPS in reconstitution buffer (450 mM KCl/10 mM HEPES/4 mM N -methylglucamine, pH 7.0) was prepared as described , and KcsA protein was added to final concentrations of 2.5–10 μg/ml, according to the number of channels per liposome desired. After 20–30 min incubation, 400 μl of the mixture was passed down a 20–ml Sephadex G-50 (fine) column equilibrated with reconstitution buffer. Liposomes eluted in the void volume with a dilution of approximately threefold and were stored in 75-μl aliquots at −80°C for up to 3 mo. Single-channel recordings of KcsA were performed in a horizontal planar lipid bilayer, with the following improvements over the system's previous description . Partitions used to hold the bilayers were cut from 80-μm overhead transparency film (Primo™ Partitions), and holes (50–90-μm diameter) were handcrafted by the melt-and-shave method . Electrodes were connected to the cis and trans chambers by salt bridges (2% agar, 200 mM KCl, 5 mM EGTA). Microphonic noise was greatly reduced by enclosing the bilayer chamber in a metal box soundproofed on all surfaces with “dB-Bloc” coating (Netwell). Electrophysiological data were collected with an Axopatch 200A amplifier and pCLAMP software (Axon Instruments). Data were sampled at 10–50 kHz and low-pass filtered at 2–5 kHz bandwidth (eight-pole Bessel filter). After sealing the partition between cis and trans chambers with a worm of Vaseline or silicone grease, the hole was pretreated with ∼0.5 μl of phospholipid solution (15 mg/ml POPE, 5 mg/ml POPG in n -decane) and was allowed to air dry for ∼30 min. The trans chamber was then filled with 20K4 solution and the cis with 200K7 or 100K7 solution. A bilayer was spread on the hole with a glass or plastic rod wetted with phospholipid solution kept at room temperature. Capacitances were typically 25–40 pF, and resistances were in the 1 TΩ range. For channel insertion, a day's supply of reconstituted vesicles was prepared by thawing an aliquot, transferring the suspension to a glass test tube, and sonicating in a cylindrical bath sonicator for ∼10 s. Vesicles were kept at room temperature throughout the day's use, and then discarded. A newly formed bilayer was ruptured by physical violence, ∼1 μl of reconstituted vesicles were added to the cis solution directly above the open hole and a new bilayer was immediately spread. Current was monitored at 100–200-mV holding voltages, and if channels failed to appear within 5 min, the bilayer was ruptured and the procedure was repeated. Typically, channels were observed in ∼50% of such attempts. After channel insertion, recording conditions were established by perfusion with desired solutions or by dilution of stock solutions into the bilayer chamber with mixing. In all experiments reported here, 100 mM K + was present on both sides of the membrane. All data reported and all standard errors displayed are based on three to seven independent experiments. An essential requirement for structure–function analysis of any ion channel is a firm knowledge of its orientation in the membrane under study. Most ion channels are studied in their native cellular membranes, where orientation is obvious. However, the KcsA channel is best investigated as a purified protein reconstituted in biochemically defined membranes. In such a system, transmembrane orientation of the channel is not assured and must be empirically established. This study proceeds towards a single goal: to assign specific functions to defined sides of the KcsA protein. We approach this goal in two steps. First, we show that several of the channel's fundamental properties of gating, permeation, and blockade are asymmetric with respect to the “cis” and “trans” sides of the reconstituted membrane. Then, the channel's absolute orientation is established by assigning specific residues in the KcsA structure to the corresponding sides of the bilayer. The planar bilayer system consists of two experimentally accessible aqueous phases: the cis solution to which KcsA-reconstituted liposomes are added, and the opposing trans solution. According to the electrical polarity convention used here, the cis chamber is the zero-voltage reference. Single KcsA channels were inserted into planar lipid bilayers under asymmetric salt conditions, and both sides of the bilayer were then flushed with the desired recording solutions. Like other two-transmembrane helix K + channels, KcsA lacks an S4 voltage sensor. However, its gating shows a definite, though weak, voltage dependence. Fig. 1 compares KcsA channel activity at opposite voltage polarities in a multiple-channel membrane. At high positive voltage (175 mV), channel activity is marked by frequent openings throughout the duration of the record, a pattern that changes dramatically when voltage is reversed in polarity. At high negative voltage (−175 mV), the channel open probability is much lower. Most channels (>80%) insert into the planar bilayer with this orientation; a minority show reversed orientation, with frequent openings at negative voltages. Thus, KcsA channels preferentially orient in the reconstituted membrane, but the results do not even hint at their absolute orientation; i.e., which side of the bilayer is equivalent to the cytoplasmic or external face of the channel protein. KcsA is a proton-activated channel. As described by Cuello et al. 1998 , the channel opens significantly in planar bilayers only at pH values below ∼5. Fig. 2 shows, as in the original observations, that proton binding is linked to gating in a strictly sided manner. KcsA activity is responsive only to the pH of the trans solution; cis pH has no effect. These pH-dependent changes in gating are immediate upon perfusion of the trans side of the bilayer and are fully reversible. The majority of channels incorporate into the membrane with sensitivity to trans pH, only a minority appearing with reversed sensitivity. (The channels sensitive to cis pH also show reversed voltage sensitivity.) We exploited this asymmetric pH sensitivity to ensure that all channels observed have a single orientation; all subsequent experiments were performed with trans pH 4 and cis pH 7, a maneuver that enforces a perfectly oriented set of active channels by silencing any channel inserting in the “minority” direction. In symmetric 100-mM K + solutions, KcsA shows open-channel rectification, as seen in the raw recordings and the open-channel current–voltage (I–V) curve of Fig. 3 . Channels are well defined in amplitude and do not display the substate behavior reported previously . Under these conditions, chord conductances are 56 and 31 pS at 200 and −200 mV, respectively, and zero-voltage slope conductance is 83 pS. The open channel is substantially noisier at negative potentials than at positive. At all potentials, the open state displays noise in excess of the inherent instrumental noise seen during closed intervals; this observation suggests that the open KcsA channel undergoes rapid, unresolved transitions. These transitions do not arise from endogenous blockers since the only cation present in this chemically defined system, aside from K + , is H + and since the excess noise is not noticeably altered by changes of cis pH (in the range 4–7), by cis addition of 1 mM EDTA, or by the use of “purissimum grade” KCl in the recording solutions (data not shown). Many eukaryotic K + channels are reversibly blocked by tetraethylammonium (TEA), which can bind to two distinct sites located near the two ends of the narrow selectivity filter . KcsA is also sensitive to TEA applied to either side of the membrane . TEA reduces the open-channel amplitude in a concentration-dependent fashion, an effect expected for a reversible, low-affinity blocker with kinetics too rapid to be resolved by the recording electronics . At 200 mV, inhibition constants of 3 and 23 mM are seen for TEA added to the cis and trans solutions, respectively. The difference in affinity from the two sides must arise from intrinsic chemical differences of the binding sites involved, since the voltage polarity promotes blocker binding from the trans side and hinders it from the cis. A biological imperative of K + channels is to prohibit permeation by Na + . However, far from being inert to K + channels, Na + is known to interact with them in a sided fashion, blocking K + currents in nerve and muscle membranes exclusively from the intracellular side . This Na + blocking asymmetry is satisfyingly rationalized in terms of the KcsA structure, which shows a widening of the pore in the center of the membrane . Small cations should be able to gain ready admittance to this region from the internal solution, but would be unable to pass further through the narrow K + selectivity filter. A Na + ion in this region would thus block outward, but not inward, K + current. Being rigorously excluded from the externally disposed selectivity filter, Na + would be a functionally inert cation from the extracellular side. This Na + blocking behavior seen in eukaryotic K + channels is echoed in KcsA . Addition of 30 mM Na + to the cis side has no effect on the open-channel I–V relation in symmetrical K + . In contrast, 10 mM Na + added to the trans side reduces the channel amplitude with voltage dependence strong enough to produce a negative conductance, as seen originally in squid axon K + channels . The sidedness of Na + action intimates, by precedent from excitable membrane channels, that the trans-facing side of KcsA is intracellular, and the cis side extracellular. This conclusion is uncertain, however, because of its reliance on an analogy to only a few carefully studied K + channels. We therefore scrutinized the absolute sidedness of KcsA by combining measurements of the functional influences of specific residues with the channel's known structure . It is well established in eukaryotic K + channels that the affinity of external TEA block is enhanced by an aromatic residue at the position equivalent to Y82 in KcsA . Accordingly, we examined the effects of substitutions at Y82 on TEA block . The affinity of TEA blockade from the cis side is responsive to substitution here, with tyrosine producing the strongest block ( K i = 3.2 mM) and the nonaromatic substitutions showing weaker blocking affinities (Y82C, K i = 23 mM; Y82T, K i = 143 mM). In contrast, TEA inhibition from the trans side is insensitive to these replacements. These results argue that position 82, known from the structure to be externally exposed, faces the cis aqueous solution. The introduction of cysteine at position 82 offers an independent means of assessing the channel's orientation. Devoid of cysteine, KcsA is an ideal target for analysis by site-specific modification. In eukaryotic K + channels, ion permeation is affected by substitutions at this position , as may be easily understood from the KcsA structure, which places Y82 close to the external opening of the narrow pore . We therefore sought functional evidence of chemical modification of Y82C by MTSET, a cationic, water-soluble sulfhydryl-modifying reagent. The Y82C substitution preserves the basic KcsA properties of trans pH sensitivity and voltage-dependent gating, but it alters the shape of the open-channel I–V curve, nearly eliminating the rectification normally observed . The I–V curve was unchanged immediately following addition of 70 μM MTSET to the cis compartment, but ∼3 min of exposure to the reagent led to a distinct asymmetry. This rectification resulted from a decrease in cis-to-trans current while leaving current in the reverse direction unchanged, as expected from an electrostatic influence of a positive charge near the channel's cis entryway. This effect persisted after removal of the MTSET by perfusion, and it was not reversed by several minutes of exposure to 5 mM dithiothreitol. Since MTSET is membrane impermeant , and since the acidic pH of the trans side greatly disfavors formation of the thiolate nucleophile, the reagent-induced alteration in I–V curve further supports a cis-facing location of residue 82. We have not attempted to determine the number of subunits modified by MTSET under these conditions. However, prolonged application of the reagent leads to the disappearance of the channel, and so we suspect that channels like the one shown in Fig. 8 have not been modified on all four subunits. In unsurprising control experiments without the cysteine substitution at Y82, MTSET had no effect (data not shown). The modification of Y82C provoked a supplementary experiment. We have already seen that mutations at Y82 affect external TEA block, and we anticipate from experiments on the equivalent position in Shaker channels that modification of this residue by MTSET would greatly reduce TEA affinity. Single Y82C channels were held at 200 mV (K + current towards the cis side), and upon cis addition of 10 mM TEA, open-channel current was reduced by ∼35% . MTSET was then added to the cis side. Initially, there was no effect of the reagent, but after ∼1 min, an increase of open-channel current occurred (concurrently with a shortening of open times). This increase reflects the expected decrease of TEA affinity arising from the introduction of a trimethylammonium group near the blocking site. This result corroborates the cis exposure of position 82. Scorpion venom peptides of the charybdotoxin family block eukaryotic K + channels by binding to a receptor site in the outer vestibule, thereby occluding the conduction pathway . Wild-type KcsA fails to bind CTX, but a triple mutant in the CTX-receptor region, KcsA-Tx, reveals a binding site for radiolabeled CTX , albeit with rather low affinity (0.1–1 μM). Fig. 10 demonstrates block of single KcsA-Tx channels by CTX added to the cis side. As expected , CTX is without effect on the wild-type channel and is profoundly inhibitory on KcsA-Tx. The toxin acts by shortening open times and by modestly lengthening closed times, as expected for a bimolecular process slow enough to resolve individual toxin-block events. CTX action is fully reversed upon perfusion with toxin-free solutions, as illustrated by the open-time histograms of Fig. 10 B. It is not our purpose to carry out a detailed analysis of toxin block, which would minimally require dissecting a four-state model . Qualitatively, however, the toxin dwell-time appears to be close to the normal closed times in the absence of toxin (10–100 ms). Furthermore, analysis of the shortening of open times leads to an estimated bimolecular association rate constant of ∼4 × 10 9 M −1 s −1 , 60-fold higher than that seen for Shaker channels at a similar ionic strength . In additional experiments, CTX-K27H, a toxin variant with greatly reduced affinity for Shaker K + channels , had a minimal effect on KcsA-Tx (data not shown). Thus, CTX block of KcsA mirrors its action in eukaryotic systems and buttresses the argument for fundamentally similar toxin receptor sites in eukaryotic and prokaryotic K + channels. The results dictate a cis-facing location for this externally exposed receptor site in KcsA. We did not perform experiments with trans addition of CTX, since the acid conditions on the trans side would make the expected negative results meaningless; the conclusion of a cis-facing CTX receptor stands without these controls. The above experiments all involved manipulations of residues exposed to the cis solution. For completeness, it was desirable to test a residue exposed on the opposite side of the membrane. We substituted cysteine at Q119 and monitored changes in channel conductance in response to covalent modification. While the experiment is identical in motivation to those with Y82C above, the low pH of the trans solution requires two special considerations. First, the reaction rate of cysteine with thiosulfonate reagents is negligible at pH 4, and so MTSES was applied in a pH 7 solution. Since under these conditions the channels were closed, after several minutes of exposure to the reagent, the trans chamber was returned to pH 4 to gauge the effect of the modification. Second, two criteria guided the selection of a target residue: (a) susceptibility to closed-state modification by MTSES, and (b) proximity to the conduction pathway, maximizing the likelihood of an electrostatic influence on conduction. On the basis of these requirements, we selected Q119 as the target residue. This residue is located in the COOH-terminal domain close to the cytoplasmic opening of the pore . The equivalent residue in the Shaker K + channel, H486, reacts with MTS reagents in both open and closed states . Fig. 11 shows that under control conditions, Q119C displays wild-type behavior, with normal rectification of the open-channel I–V curve. Several minutes of exposure to MTSES from the trans side led to an increase in single-channel current at positive voltages, with no discernible effect at negative voltages. This is as expected for the MTSES reaction, which places a negatively charged ethylsulfonate group near the channel's trans entryway. No effects were observed in control experiments in which the reagent was added to the cis side or to bilayers containing wild-type KcsA channels (data not shown). These experiments exploit a fully oriented reconstituted system in which single KcsA channels, purified after high-level expression in E. coli , can be studied electrophysiologically. We have taken pains to document functional asymmetries in KcsA to assign absolute sidedness to the system. Eight separate asymmetries were examined: voltage-dependent gating, proton activation, open-channel rectification, block by Na + , TEA, and CTX, and covalent modification of channels with cysteines substituted on either side of the membrane. Three independent lines of evidence establish the channel's orientation in the bilayer. The cis solution bathes the extracellular face of the channel protein containing the CTX receptor and the aromatic TEA blocking site. The trans solution bathes the intracellular face containing Q119. These assignments unambiguously demonstrate that the protonation sites linked to KcsA gating face the intracellular solution. This orientation makes sense from a statistical standpoint: of the 56 carboxylate groups in KcsA (16 asp, 36 glu, 4 COOH termini), 44 are located at intracellular positions; all 44 histidine residues in the His-tagged protein are also intracellular. Previously, Cuello et al. 1998 , arguing from protection of liposome-reconstituted KcsA against proteolysis, had concluded the reverse: that extracellular protons gate the channel. Our own results, in contrast, show robust proteolysis of KcsA under similar conditions (data not shown). Our results are perplexing from a biological perspective. Indeed, they suggest strongly that this channel is not gated by low pH in its native membrane, since Streptomyces , like most bacteria, tightly regulates cytoplasmic pH near neutrality. If the physiological role of KcsA is in fact to gate the K + conductance of the bacterial membrane, then it is likely that some factor other than pH, perhaps an as yet unrecognized partner protein, provides control of gating. Questions of this kind must remain unresolved until the physiological purposes of prokaryotic K + channels are clarified. In the course of assessing KcsA orientation, we have also shown that pore characteristics of this prokaryotic channel are remarkably similar to those of many well-studied eukaryotic K + channels. The functional familiarity of KcsA is not surprising, given how closely the structural features proposed for eukaryotic K + channels match those actually observed with KcsA . But it is nevertheless important to confirm this functional similarity by experiment, since KcsA provides the first opportunity for a direct structure–function assault on an ion-selective channel. | Study | biomedical | en | 0.999998 |
10498674 | The transport-related activity of the surface and glandular endometrial epithelium plays an important role in regulation of uterine lumen electrolyte composition, pH, and fluid volume, providing a suitable environment for fertilization and implantation of the developing embryo . Previous studies with native porcine endometrial epithelium demonstrated that amiloride-sensitive Na + absorption and K + secretion were modulated by PGF 2α , cAMP, and gastrin-releasing peptide . Other studies using primary cultures of rodent endometrial epithelial cells grown on permeable membrane filters demonstrated that anion secretion was stimulated by adrenergic agonists through β adrenergic receptors and by ATP through multiple subtypes of purinoceptors . In cultures of porcine glandular endometrial epithelial cells, it was reported that PGE 2 also stimulates anion secretion . In human endometrial epithelial cells, bradykinin, bombesin, and gastrin-releasing peptide have all been shown to affect transepithelial electrolyte transport . Thus, the results of these studies indicate that the absorptive and secretory activities of endometrial epithelial cells are modulated in response to a variety of signaling molecules. The first demonstration of an effect of insulin on transepithelial Na transport was reported by Herrera 1965 using isolated toad bladder epithelium. Since that time, insulin and insulin-like growth factor I (IGF-I) 1 have been shown to stimulate electrogenic sodium transport in a variety of cells . In many cases, this increase in Na + transport was a result of stimulation of the Na + -K + ATPase, either directly through changes in pump concentration or kinetic properties or indirectly through increases in intracellular [Na + ], which in turn leads to increased substrate availability and subsequent pump stimulation . In amphibian and rodent skeletal muscle cells, for example, stimulation by insulin increases Na + -K + ATPase transport activity by increasing pump translocation into the plasma membrane. This effect occurs within 15–30 min and is not blocked by inhibitors of protein synthesis. In rodent muscle, it appears that the α-2 isoform is selectively inserted into the plasma membrane . In another study, however, Clausen and Hansen 1977 demonstrated that the effect of insulin on [ 3 H] ouabain binding in intact skeletal muscle was an artifact arising from stimulation of the rate of binding. Steady state binding of [ 3 H] ouabain was unaffected, indicating that insulin does not induce translocation of the Na + -K + ATPase to the plasma membrane. These results were confirmed in a later study and demonstrated that IGF-I also had a stimulating effect on the Na + -K + ATPase in skeletal muscle . In rat adipocytes, insulin does not appear to alter pump concentration in the plasma membrane or to involve elevations in intracellular [Na + ] . In these cells, increased transport activity of the pump is associated with an increase in Na + affinity for the α-1 and α-2 isoforms and an increase in V max associated with the α-2 isoform of the pump. In hepatocytes, skeletal muscle cells, and 3T3-L1 adipocytes, increases in Na + -K + ATPase transport activity appear to be due to elevations in intracellular [Na + ] resulting from activation of either Na + -H + exchange or Na + -K + -2Cl − cotransport activity . In epithelial cells, insulin and IGF-I have also been demonstrated to stimulate Na + transport either by increasing the Na + affinity of the Na + -K + ATPase or by increasing the number of amiloride-sensitive Na + channels present within the apical membrane . Specific binding sites for insulin and IGF-I have been identified in normal endometrium and endometrial cancer cells . Thus, it was of interest to determine whether insulin and IGF-I play a role in regulation of Na + transport across the endometrial epithelium. In this study, we report the effects of insulin and IGF-I on transepithelial Na + transport function of cultured porcine endometrial epithelial cells under defined medium conditions. We show that insulin and IGF-I produce an acute increase in Na + transport that involves direct regulation of the Na + -K + ATPase and a basolateral K + channel. In addition, we demonstrate that long-term exposure to insulin (4 d) results in enhanced Na + absorption with a further increase in pump activity and an increase in apical membrane amiloride-sensitive Na + conductance. Insulin, IGF-I, ouabain, indomethacin, nonessential amino acids, and high purity grade salts were purchased from Sigma Chemical Co. Hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM) 3 ], wortmannin, okadaic acid, and PD-98059 (2′-amino-3′-methoxyflavone) were purchased from Biomol Research Laboratories. Amiloride, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and calyculin A were purchased from Research Biochemicals International, and benzamil from Molecular Probes. Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate buffer saline (DPBS), fetal bovine serum (FBS), collagenase (type 1), kanamycin, penicillin-streptomycin, and fungizone were purchased from GIBCO BRL. Porcine uterine tissues were collected from 4–5-mo-old Yorkshire-Pietrain cross pigs purchased from stock herds maintained by the University of Minnesota College of Agriculture. Uterine tissues from adult, precycling animals were used to minimize variability in electrolyte transport properties at different stages of the estrus cycle. Uteri were obtained from pigs that were killed at the University of Minnesota Meat Sciences Laboratory by the captive bolt euthanasia procedure approved by the University Animal Care Committee and supervised by a USDA certified veterinarian. Uterine tissue was placed in ice cold porcine Ringer solution containing (mM): 130 NaCl, 6 KCl, 3 CaCl 2 , 0.7 MgCl 2 , 20 NaHCO 3 , 0.3 NaH 2 PO 4 , 1.3 Na 2 HPO 4 , gassed with 95% O 2 /5% CO 2 , pH 7.4. After removal of the serosal muscle layer, the endometrial tissue was minced into small pieces (≈1 mm 3 ) and washed twice with Ca 2+ - and Mg 2+ -free DPBS. The tissue fragments were then subjected to collagenase digestion and the epithelial glands were isolated as described previously . The epithelial glands were suspended in DMEM supplemented with 3.7 g/liter sodium bicarbonate, 10% FBS, 5 μg/ml (850 nM) insulin, 1% nonessential amino acids, 5 μg/ml fungizone, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml kanamycin (standard media). They were then plated onto cell culture dishes and incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. Culture medium was changed after 24 h, and then every 2–3 d. The epithelial cells became confluent monolayers within 3–4 d. The remaining stromal cells were removed by adding 0.02% collagenase in serum-free medium for 24 h. The epithelial cells were then trypsinized and placed on 24-mm (4.5 cm 2 ) transparent permeable membrane filters (Corning Costar). To study the long-term effect of insulin and IGF-I, the epithelial cells were grown on permeable filters in DMEM supplemented with 10% FBS for 7 d, and then replaced with Phenol Red-free DMEM (serum-free media) supplemented with 850 nM insulin or 1.3 nM IGF-I for 4 d. Transepithelial resistance of the cell monolayers was measured using the EVOM epithelial voltohmmeter coupled to Ag/AgCl “chopstick” electrodes (World Precision Instruments). After 10 d, the confluent culture inserts were mounted in Ussing Chambers, bathed on both sides with standard porcine Ringer solution maintained at 37°C, and bubbled with 95% O 2 /5% CO 2 . Transepithelial potential difference, tissue conductance, and short circuit current (Isc) were measured with the use of voltage-clamp circuitry from JWT Engineering. The data from the voltage clamp experiments was digitized, stored, and analyzed using Workbench data acquisition software (Kent Scientific Corp.), and recorded with a Compaq pentium microcomputer. All cells were pretreated with indomethacin (10 μM) added to both apical and basolateral solutions at least 10 min before the beginning of the experiment. Pump current was measured using amphotericin B (10 μM) to permeabilize the apical membrane of monolayers mounted in Ussing chambers. To determine the [K + ] dependence of the pump, monolayers were bathed on both sides with NaMeSO 4 Ringer solution containing (mM): 120 NaMeSO 4 , 30 mannitol, 3 calcium gluconate, 1 MgSO 4 , 20 NaHCO 3 , 0.3 NaH 2 PO 4 , 1.3 Na 2 HPO 4 , gassed with 95% O 2 /5% CO 2 , pH 7.4. Increasing intracellular K + concentration was accomplished by replacement of NaMeSO 4 Ringer solution with an equivalent volume of KMeSO 4 Ringer solution. KMeSO 4 Ringer solution contained (mM): 120 KMeSO 4 , 30 mannitol, 5 NaCl, 3 calcium gluconate, 1 MgSO 4 , 20 KHCO 3 , 0.3 KH 2 PO 4 , 1.3 K 2 HPO 4 , gassed with 95% O 2 /5% CO 2 , pH 7.4. To determine the [Na + ] dependence of the pump, monolayers were bathed on both sides with N -methyl- d -glucamine (NMDG)–MeSO 4 Ringer solution containing (mM): 130 NMDGMeSO 4 , 30 mannitol, 3 calcium gluconate, 1 MgSO 4 , 10 KHCO 3 , 0.3 KH 2 PO 4 , 1.3 K 2 HPO 4 , gassed with 95% O 2 /5% CO 2 , pH 7.4, and a NaMeSO 4 Ringer solution containing different concentrations of Na + was used to replace NMDGMeSO 4 Ringer solution. The Na + and K + dependence of pump current was determined using the Hill equation: I p = I max [S] n /([S] n + K 0.5 ), and its linear expression: log ( I p / I max − I p ) = n log[S] − log K 0.5 , where I p is the pump current stimulated by an increase in intracellular [Na + ] or extracellular [K + ], I max is the maximal pump current, S is an intracellular [Na + ] or extracellular [K + ], K 0.5 is the apparent dissociation constant, and n is the Hill coefficient. The kinetic parameters were determined by nonlinear regression or by linear regression analysis (Prism™ 2.0; GraphPad Software). Current–voltage relationships were determined using amphotericin B–permeabilized monolayers mounted in Ussing chambers. The intracellular compartment was bathed with KMeSO 4 Ringer solution and amphotericin B (10 μM) was added to permeabilize the membrane. The extracellular compartment was bathed with standard porcine Ringer solution or NaMeSO 4 Ringer solution. An epithelial voltage clamp (World Precision Instruments) in combination with an LM-12 A-D interface (Dagan Corp.) were used to voltage clamp the monolayers and record the data. The voltage step commands and the resultant currents were generated using pCLAMP software (Axon Instruments). Current–voltage (I–V) relationships were obtained by a series of voltage step commands described in the figure legends. The compound-sensitive components were obtained by subtracting the currents before and after addition of the compound. The Na + :K + selectivity ratio ( P Na / P K ) was calculated from reversal potential ( E rev ) measurements using the Goldman-Hodgkin-Katz equation { E rev = RT / zF ln ( P Na [Na + ])/( P K [K + ])}. Epithelial cells (3 × 10 5 ) were subcultured onto 6.5-mm transparent permeable membranes containing 10% FBS in DMEM. After 3–4 d, the cells were placed in serum-free Phenol-Red–free DMEM for 48 h, followed by insulin treatment (850 nM) for 24 h. Specific [ 3 H] ouabain binding was performed at 37°C in humidified incubator of 5% CO 2 in air, using a procedure modified from Lobaugh and Lieberman 1987 . Cell monolayers were preincubated in Phenol-Red–free DMEM for 20 min, and then the media was replaced with loading solution containing [ 3 H] ouabain (Amersham Life Science) in DMEM. After incubation for 45 min, monolayers were rinsed with ice-cold DMEM for 10 s to remove unbound [ 3 H] ouabain. The filters were then cut from their supports and transferred to the scintillation fluid and assayed for radioactivity. Other filters were solubilized in lysis buffer and assayed for amount of protein. Nonspecific [ 3 H] ouabain binding was determined when an additional 500 μM ouabain was included in the loading solution. The magnitude of nonspecific ouabain binding did not exceed 30% of the total binding as determined at a concentration of 0.5 μM. Epithelial cells were allowed to grow on permeable membrane filters in DMEM supplemented with 10% FBS for 5–7 d, followed by serum-free Phenol Red–free DMEM for 2 d. Insulin (850 nM) was then added to the serum-free media for 2 d. The monolayers were then washed twice in DPBS, pH 7.4, permeabilized with 0.3% Triton X-100 in DPBS at room temperature for 10 min, and then fixed in methanol at −20°C for 10 min. After fixation, filters were cut from their supports. The cells were then washed with DPBS and incubated with DPBS containing 1% bovine serum albumin and 10% normal goat serum to block nonspecific binding at room temperature for 1 h. Then the cells were incubated overnight with isoform-specific mouse monoclonal antibody against the α-1 and polyclonal antibodies to α-2 and α-3 isoforms of the rat Na + -K + ATPase, 1:200 (Upstate Biotechnology Inc.) at 4°C. After washing, the cells were incubated with indocarbocyanine (cy3)-labeled goat anti–rabbit antibody, 1:400 (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After the final wash, the filters were placed on a glass slide. The cells and filters were embedded in fluoromount (Gallard Schlessinger) and examined by confocal microscopy, using a MRC1024 laser confocal microscope (Bio-Rad Laboratories) equipped with krypton-argon lasers. Cell monolayers, as prepared for immunocytochemistry, were solubilized with lysis buffer (50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 μg/ml aprotinin, and 1 mM NaF, pH 7.4) at 37°C for 30 min and homogenized. A protein assay was performed using a BCA Protein Assay Kit by Pierce. Proteins were separated by PAGE (8%). Electroblotting was done using Immobilon-P (Millipore Corp.). The electroblot assembly was placed into the electroblotting apparatus (Trans-Blot Cell; Bio-Rad Laboratories) and blotting was performed at 16 V overnight on ice. After the blots were removed, they were washed twice, and then blocked in freshly prepared 1× TBS-tween containing 3% nonfat dry milk (MLK) for 30 min at 20–25°C with constant agitation. After washing, blots were reacted overnight in primary antibody, 15 or 100 ml freshly prepared 1× TBS-tween containing 3% MLK with appropriate dilution of the primary antibody (anti–rat α-1 monoclonal antibody and rabbit-anti–rat α-2 polyclonal antibody from Upstate Biotechnology Inc.). The next day, blots were washed and reacted with secondary antibody, either goat anti–rabbit, alkaline phosphatase–labeled or goat anti–mouse, alkaline phosphatase–labeled. Secondary antibody was diluted 1:3,000 (33 μl into 100 ml) in 1× TBS-tween containing 3% MLK and reacted for ∼2 h. After washing, alkaline phosphatase color reagent was added to 100-ml 1× alkaline phosphatase color development buffer at room temperature. Blots were incubated in development buffer until bands were clearly developed. All values are presented as means ± SEM, n is the number of monolayers, and N is the number of animals in each experiment. The differences between control and treatment means were analyzed using a t test for paired or unpaired means, where appropriate. A value of P < 0.05 was considered statistically significant. The EC 50 values for insulin and IGF-I and the IC 50 values for benzamil and amiloride were determined using a four parameter logistic function to fit the data (Prism™ 2.0). The basal electrical properties of cultured epithelial cells used in this study have been previously described . Our data showed that endometrial epithelial cells cultured in standard serum-containing media were capable of both Na + absorption and Cl − secretion. To investigate acute and long-term effects of insulin and IGF-I on electrolyte transport in endometrial epithelial cells, we measured both NPPB-inhibitable Cl − secretion and benzamil-sensitive Na + absorption as shown in Fig. 1 . NPPB is an arylamino benzoate compound that has been previously shown to block apical membrane Cl − channels in cultured porcine endometrial epithelial cells . Amiloride and its more potent analogue benzamil, have been previously shown to block Na + channels in native porcine endometrial epithelium . For cell monolayers cultured in standard serum-containing media, 20% of the basal Isc was blocked by 10 μM benzamil and the remaining Isc was inhibited by 100 μM NPPB. In serum-free media, there was a significant decrease in both basal Isc from 34 ± 3 μA ( n = 12, N = 4) to 13 ± 2 μA ( n = 9, N = 4) and NPPB-sensitive Isc from 24 ± 3 to 4 ± 1 μA without a significant change in benzamil-sensitive Isc (from 7 ± 2 to 9 ± 2 μA) compared with serum-containing media. Treatment with 850 nM (5 μg/ml) insulin or 1.3 nM (10 ng/ml) IGF-I for 4 d significantly increased basal Isc to 48 ± 3 μA ( n = 23, N = 9) for insulin and 27 ± 5 μA ( n = 7, N = 4) for IGF-I, and increased benzamil-sensitive Isc to 41 ± 3 μA for insulin and 23 ± 5 μA for IGF-I. However, no change in benzamil-insensitive Isc was detected after treatment with either insulin or IGF-I. Addition of 850 nM insulin to the basolateral solution of monolayers produced an increase in Isc within 5 min that reached a maximal plateau response of 36 ± 2 μA ( n = 11, N = 5) in 30–45 min, as illustrated in Fig. 2 A. The maximal Isc response was sustained for as long as 3 h. Addition of 65 nM IGF-I to the basolateral solution produced the same response as insulin with a maximal plateau response of 36 ± 3 μA ( n = 6, N = 3). Subsequent addition of 10 μM benzamil to the apical solution inhibited both the basal and insulin-stimulated Isc. Concentration–response curves for benzamil and amiloride are shown in Fig. 2 B with IC 50 values of 25 nM for benzamil and 194 nM for amiloride. Pretreatment with 10 μM benzamil completely inhibited the insulin-stimulated Isc (data not shown). In addition, pretreatment with 50 μM HNMPA-(AM) 3 , a specific inhibitor of insulin receptor tyrosine kinase activity, for 30 min inhibited the insulin-stimulated increase in Isc (7 ± 2 μA, n = 6, N = 4), as illustrated in Fig. 3 A. The concentration–response relationships for insulin and IGF-I presented in Fig. 3 B demonstrated that threshold concentrations were ∼0.85 nM for insulin and 0.13 nM for IGF-I. The EC 50 values were 12 nM for insulin and 2.5 nM for IGF-I. To determine whether the increase in Isc produced by insulin was the result of stimulation of the Na + -K + ATPase, experiments were performed using amphotericin B–permeabilized monolayers mounted in Ussing chambers bathed on both sides with either NaMeSO 4 Ringer solution containing 5 mM BaCl 2 or NMDGMeSO 4 Ringer solution containing 10 mM KHCO 3 . BaCl 2 was used to inhibit basolateral K + channels and limit the contribution of K + recycling to pump activation. The increase in extracellular [K + ] was accomplished by replacement of NaMeSO 4 solution with an equal volume of KMeSO 4 Ringer solution containing different concentrations of K + . To determine the Na + dependence of the pump, NaMeSO 4 Ringer solution with 10 mM KHCO 3 and different concentrations of Na + was used to replace NMDGMeSO 4 Ringer solution. Fig. 4 A shows the increase in pump current ( I p ) produced by increasing K + concentrations in the basolateral solution of insulin-treated monolayers. The stimulated I p was completely inhibited by 10 μM ouabain added to the basolateral solution (data not shown). When I p was plotted as a function of basolateral [K + ], it revealed that insulin treatment increased I max from 20 ± 2 μA ( n = 11, N = 5) to 63 ± 6 μA ( n = 9, N = 5), with an increase in the K 0.5 value from 1.8 ± 0.2 to 2.9 ± 0.2 mM. Stimulation of pump current was also dependent on intracellular [Na + ], as shown in Fig. 5 A. The relationship of I p and [Na + ] revealed that insulin treatment increased I max from 18 ± 1 μA ( n = 8, N = 4) to 42 ± 5 μA ( n = 10, N = 4) with a significant decrease in the K 0.5 value from 39 ± 2 to 24 ± 2 mM. Hill coefficients were 2.1 ± 0.3 for K + and 1.2 ± 0.1 for Na + under insulin treatment, which was not significantly different from corresponding control values (1.9 ± 0.2 for K + and 1.3 ± 0.1 for Na + ), as illustrated in Fig. 4 B and 5 B. The acute (15–20 min) effects of insulin on the current–voltage relationship of the Na + -K + ATPase is shown in Fig. 6 . The experiment was performed using amphotericin B–permeabilized monolayers, as described in the previous section. The apical surface of the epithelium was bathed with KMeSO 4 Ringer solution supplemented with 5 mM NaCl, and amphotericin B was used to permeabilize the apical membrane. Standard Ringer solution was used to bathe the basolateral surface of the epithelium. The I–V relationship of the pump was obtained using a voltage step protocol ranging from −90 to +90 mV (15-mV steps) at a holding potential of 0 mV. The difference currents were obtained by subtracting the current before and 10 min after basolateral addition of 100 μM ouabain. The ouabain-sensitive current after pretreatment with 850 nM insulin for 15 min was approximately twofold greater in magnitude, with no change in conductance when compared with current responses under basal conditions. The effect of insulin on basolateral K + permeability is shown in Fig. 7 . The apical surface of the epithelium was permeabilized with amphotericin B and bathed with KMeSO 4 Ringer solution without NaCl, while the basolateral surface was bathed with standard Ringer solution. The insulin-sensitive current obtained after basolateral addition of 850 nM insulin for 15 min exhibited slight outward rectification with a mean reversal potential of −53 ± 3 mV ( n = 9, N = 4). Replacement of standard Ringer solution with KCl Ringer solution shifted the reversal potential toward zero (3 ± 2, n = 6, N = 4). Acute insulin (850 nM) treatment (15–30 min) had no effect on apical membrane conductance. To characterize the properties of the benzamil/amiloride-sensitive Na + channel located in the apical membrane, experiments were performed with amphotericin B–permeabilized monolayers, as previously mentioned. The monolayers were bathed on the apical side with NaMeSO 4 Ringer solution and on the basolateral side with KMeSO 4 Ringer solution containing amphotericin B. The benzamil-sensitive current was obtained before and 2 min after apical addition of 5 μM benzamil. The current–voltage relationship of benzamil-sensitive current is shown in Fig. 8 . Treatment with 850 nM insulin for 4 d showed a mean reversal potential of 65 ± 4 mV ( n = 7, N = 4), which was not significantly different from control cells in serum-free media (72 ± 4 mV, n = 5, N = 3). The basal benzamil-sensitive conductance was 0.17 ± 0.05 mS. Stimulation with insulin significantly increased the conductance to 0.37 ± 0.05 mS. Interestingly, a 30-min treatment with 850 nM insulin produced no significant change in reversal potential (69 ± 8 mV, n = 6, N = 3) or conductance (0.18 ± 0.05 mS) compared with control monolayers. The Na + to K + selectivity ratio of the benzamil-sensitive current in the presence of insulin was calculated to be 11.6:1. Previous studies using a rat skeletal muscle cell line demonstrated that the activation of Na + -K + ATPase by insulin may involve the phosphatidylinositol 3-kinase (PI-3 kinase) signaling pathway . Therefore, experiments were performed to determine the signaling cascade involved in insulin-stimulated Na + transport across endometrial epithelial cells. Incubation with 1 μM wortmannin, a PI-3 kinase inhibitor, markedly inhibited the insulin-induced increase in Isc from 47 ± 3 μA ( n = 12, N = 5) to 5 ± 3 μA ( n = 6, N = 4) compared with control, as shown in Fig. 9 A. Further experiments were conducted to determine the downstream components of the pathway using phosphatase inhibitors. It was demonstrated that pretreatment with 100 nM okadaic acid or 100 nM calyculin A, inhibitors of protein phosphatase PP-1 and PP2A, for 30 min also inhibited insulin-induced increases in Isc to 3 ± 3 μA ( n = 4, N = 3) and 5 ± 3 μA ( n = 4, N = 3), respectively. Previous studies of insulin signaling in adipocytes suggested the possibility that PI-3 kinase activation by the insulin receptor can stimulate mitogen-activated protein kinase (MAP kinase), so that insulin-dependent regulation of the Na + -K + ATPase in endometrial epithelial cells might involve components of the MAP kinase signaling pathway . To examine this possibility, we pretreated monolayers with a MAP kinase inhibitor and tested the effects of insulin. Although we observed a slight reduction in Isc stimulation produced by insulin (34 ± 4 μA, n = 5, N = 3), the results suggested that the MAP kinase signaling pathway did not play a major role in activation of the Na + -K + ATPase. Additional experiments were performed to determine the effect of phosphatase inhibitors on insulin-stimulated pump current, as illustrated in Fig. 10 . The Na-K ATPase was stimulated by basolateral addition of 5 mM KMeSO 4 Ringer solution before and after treatment with insulin for 30 min. Addition of 850 nM insulin to the basolateral solution had no effects on basal current (−5 ± 1 μA, n = 25, N = 9). A subsequent addition of 5 mM KMeSO 4 Ringer solution produced a rapid increase in current that was completely inhibited by 100 μM ouabain, as shown in Fig. 10 A. Compared with the control monolayers, insulin stimulated an increase in pump current from 18 ± 2 μA ( n = 12, N = 5) to 32 ± 1 μA ( n = 7, N = 4). Pretreatment with 100 nM okadaic acid for 30 min before addition of insulin abolished the insulin-stimulated pump current and decreased the basal pump current to 14 ± 2 μA ( n = 6, N = 4), as shown in Fig. 10 B. Fig. 11 compares the apical Na + conductance and the pump current stimulated by 30-min and 4-d treatments with insulin. Administration of insulin for 30 min significantly increased K + -stimulated pump current with no effect on Na + conductance. However, treatment with insulin for 4 d significantly increased both Na + conductance and K + -stimulated pump current, suggesting that long-term treatment with insulin produced an increase in Na + permeability of the apical membrane in addition to further activation of Na + -K + ATPase. The presence of α-1, α-2, and α-3 isoforms of the Na + -K + ATPase were determined by immunofluorescence and Western blotting. Fig. 12 shows the summation of immunofluorescence images (1–3) obtained at 3-μm steps as the microscope was focused from the filter towards the apical membrane of glandular epithelial cell monolayers. The cell monolayers were labeled using a monoclonal antibody to α-1 and polyclonal antibodies to α-2 and α-3 isoforms of fusion proteins of the rat Na + -K + ATPase. The cell monolayers were labeled with antibodies to the α-1 and α-2 isoforms, but not the α-3 isoform of the Na + -K + ATPase. The labeling pattern of both α-1 and α-2 isoforms was intensely localized to the basolateral membrane. The cells grown in serum-free media in the absence and presence of 850 nM insulin exhibited the same pattern of labeling for both α-1 and α-2 isoforms. The control monolayers incubated with preimmune serum gave images identical to that observed with the α-3 isoform antibody (data not shown). A representative Western blot is also presented in Fig. 12 and confirms the expression of α-1 and α-2 isoforms of the Na + -K + ATPase. The monoclonal anti–Na + -K + ATPase α-1 antibody labeled an ∼95-kD protein and the polyclonal anti–Na + -K + ATPase antibody labeled a protein of ∼100 kD, consistent with α-1 and α-2 isoforms of the Na + -K + ATPase. To determine whether insulin increased transport activity of the pump as a result of an increase in Na + -K + ATPase concentration in the basolateral membrane, specific [ 3 H] ouabain binding was performed with the cell monolayers cultured in serum-free media in the absence or presence of 850 nM insulin. Fig. 13 shows the specific binding of [ 3 H] ouabain to endometrial epithelial cells as a function of ouabain concentration. Analysis of [ 3 H] ouabain binding revealed a single class of binding sites with total receptor concentration ( B max ) of 13.9 ± 2.4 pmol/mg protein and K d of 252.8 ± 90.5 nM for insulin-treated cells ( n = 5). The B max and K d of insulin-treated cells did not significantly differ from those of control cells ( B max = 11.4 ± 3.9 pmol/mg protein and K d = 237.0 ± 173.4 nM). Early studies of toad urinary bladder demonstrated that insulin stimulates transepithelial Na + transport by activation of the Na + -K + ATPase without increasing apical membrane Na + permeability . Further studies of toad bladder and skeletal muscle showed that insulin-stimulated Na + transport resulted from activation of the Na + -K + ATPase. More recent studies of rat proximal tubule demonstrated that insulin increases the Na + affinity of the pump similar to that previously shown in adipocytes . These findings are consistent with the previously mentioned study on skeletal muscle and with a more recent study of adipocytes where insulin stimulation did not produce any change in Na + -K + ATPase concentration as measured using immunogold labeling . In contrast, studies of A 6 cells demonstrated that insulin-dependent stimulation of Na + transport was primarily due to an increase in Na + permeability of the apical membrane . Previous patch clamp experiments have suggested that insulin-dependent increases in Na + permeability resulted from increased open probability of apical Na + channels , whereas studies of intact A 6 monolayers using blocker-induced noise analysis indicated that insulin increased Na + channel density and apical membrane surface area . The idea that insulin functions to increase insertion of Na + channels into the apical membrane was further supported by recent experiments with brefeldin A, which demonstrated partial block of insulin-stimulated Na + absorption across A 6 cell monolayers . The effect of insulin on Na + transport in A 6 cells appears to be similar to the effects of insulin on glucose transport previously reported in adipocytes where insulin enhances glucose uptake by stimulating the insertion of GLUT 4 glucose carriers into the plasma membrane . Unlike the results obtained using A 6 cells, we find in the present study that the acute (within 30 min after stimulation) increase in Na + transport after treatment with insulin or IGF-I is not the result of an increase in apical Na + conductance. Insulin and IGF-I increase Na + absorption by stimulating Na + -K + ATPase activity and by increasing basolateral membrane K + permeability. Stimulation of Na + -K + ATPase activity involves increases in both V max and Na + affinity, and a small decrease in K + affinity. Previous studies of Na + -K + ATPase stimulation by insulin in skeletal muscle indicated that increases in V max may be the result of insertion of pumps into the membrane . This conclusion was supported by experiments with microtubule-disrupting agents such as colchicine, which blocked insulin-stimulated increases in V max of the Na + -K + ATPase in skeletal muscle myoballs . In the present study, insulin-stimulated increases in Isc and I P were not blocked by colchicine or brefeldin A (data not shown), suggesting that insulin-dependent increases in V max do not involve increases in the concentration of pumps present in the basolateral membrane. This interpretation is consistent with the results of ouabain binding experiments, which showed no significant increase in number of binding sites or K d after insulin stimulation. Immunocytochemical studies along with Western blot analysis of endometrial epithelial cells presented in this study demonstrate that α-1 and α-2 isoforms of the Na + -K + ATPase are present in the basolateral membrane. Previous studies with adipocytes showed that insulin stimulation produced decreases in K 0.5 for Na + of both the α-1 and α-2 isoforms of the pump . The insulin-dependent decrease in Na + K 0.5 (from 39 to 23 mM) for 86 Rb + uptake reported for the α-2 isoform was remarkably similar to pump current measurements reported in this study for endometrial epithelial cells (from 39 to 24 mM). Estimates of V max based on a theoretical fit of the 86 Rb + uptake data in adipocytes also suggested a twofold increase in V max , but this prediction was not experimentally confirmed. It was concluded from the studies of McGill and Guidotti 1991 that the fractional activity of the α-1 isoform was greater than that of the α-2 isoform in its contribution to basal pump activity in the absence of insulin. However, treatment with insulin produced selective activation of the α-2 isoform so that α-2 fractional activity was dominant under insulin-stimulated conditions. Stimulation of the α-2 isoform by insulin has also been reported in brain . A similar mechanism of insulin action on activity of the α-2 isoform could account for both the increase in Na + affinity and V max observed after insulin stimulation reported in this study for endometrial epithelial cells. However, it is worth noting that in renal epithelia, where only the α-1 isoform is present, insulin also produces a marked stimulation of the Na + -K + ATPase. Although insulin did not produce an acute increase in apical Na + conductance, we found that longer-term treatment (4 d) with insulin resulted in a greater than twofold increase in apical Na + conductance and an additional increase in pump current. One possible explanation for the enhanced Na + absorption following longer term exposure to insulin may be related to the growth-stimulating effects of insulin on cultured cells. Although we did not examine this possibility directly, it is worth noting that no significant change in total protein content was observed in monolayers treated with insulin for 4 d compared with control monolayers under serum-free conditions. In addition, primary endometrial epithelial cells exhibit density-dependent arrest, making it less likely that the cell population would increase twofold after achieving confluence. Another possible explanation could involve regulation of Na + channel expression at the transcriptional level that could result in an increase in apical membrane Na + conductance. Regulation at this level would likely follow a time course on the order of hours or perhaps days that could be consistent with the longer-term actions of insulin on Na + conductance. At present, evidence in support of this idea is not available, but insulin is known to stimulate DNA replication and transcription of other proteins involved in cell cycle regulation. An interesting observation from this study, relating to the Na + dependence of the Na + -K + ATPase, is that the degree of cooperativity for Na + ion binding is less than that previously reported for the Na + -K + ATPase in adipocytes , proximal tubule epithelial cells , and colonocytes . For most cells where the Na + dependence of the pump has been determined, the Hill coefficient has a value near 2, whereas in endometrial epithelial cells, a value of 1.3 was obtained. This difference in cooperativity leads to a higher level of pump activity at lower Na + concentrations in endometrial epithelial cells, where cooperativity is low. Reduced cooperativity combined with the increase in Na + affinity of the pump after stimulation by insulin may be important in lowering intracellular Na + activity within the cell. This would enhance the driving force for Na + entry across the apical membrane through amiloride-sensitive Na + channels. The electrogenic character of the Na + -K + ATPase would also contribute to the driving force for Na + entry by producing an increase in cell hyperpolarization after stimulation with insulin. Thus, we suggest that stimulation of the pump by insulin increases both the electrical and chemical driving forces for Na + uptake across the apical membrane. Experiments using amphotericin B to permeabilize the apical membrane demonstrated the presence of an insulin-activated, outwardly rectifying conductance in the basolateral membrane. Replacement of standard porcine Ringer solution with high KCl Ringer solution shifted the reversal potential to near 0 mV, suggesting K + as the current-carrying ion. Insulin-dependent increases in basolateral K + conductance presumably contributes to the electrical driving force for apical Na + uptake and could serve to offset decreases in K + recycling that would occur in the face of sustained hyperpolarization of the basolateral membrane. It is generally accepted that the first step in the insulin/IGF-I signaling cascade involves receptor binding followed by stimulation of receptor-mediated tyrosine kinase activity. Previous studies in A 6 cells demonstrated that insulin-stimulated Na + transport was inhibited by tyrosine kinase inhibitors . In the present study, we also demonstrated that a specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM) 3 , blocked insulin-stimulated Na + transport. This result supports the conclusion that an initial enzymatic step in insulin-dependent regulation of Na + transport in endometrial cells involves receptor autophosphorylation. A model summarizing our hypothesis regarding post-receptor signaling events responsible for regulation of Na + -K + ATPase transport activity in endometrial epithelial cells is presented in Fig. 14 . We suggest that phosphorylation of PI-3 kinase either directly by the insulin/IGF-I receptor or by IRS-1 constitutes one of the early postreceptor phosphorylation steps that leads to pump activation. This suggestion is based on the observation that wortmannin, a selective inhibitor of PI-3 kinase activity, can completely abolish the stimulatory effects of insulin and IGF-I on Na + -K + ATPase activity. We further speculate that PI-3 kinase, presumably acting through protein kinase B/AKT protein or through protein kinase C activates a protein phosphatase (presumably PP-1 or PP-2A), which in turn dephosphorylates the Na + -K + ATPase, resulting in an increase in transport activity. Although we have no direct evidence to identify either PKB/AKT protein or PKC in this pathway, it is known in other systems that these serine-threonine kinases can be activated by PI-3 kinase and can modulate the activity of protein phosphatases, including PP-1 and PP-2A. Evidence supporting the involvement of a protein phosphatase in this signaling cascade comes from experiments showing that okadaic acid and calyculin A act to inhibit insulin/IGF-I–dependent activation of the pump at concentrations that block protein phosphatase activity. Previous studies using cultured L 6 rat skeletal muscle cells demonstrated that pretreatment with okadaic acid and calyculin A blocked the effects of insulin on Na + -K + ATPase activity and insulin-dependent dephosphorylation of the Na + -K + ATPase. In addition, the presence of wortmannin also blocked insulin-stimulated PP-1 activation as well as dephosphorylation and activation of the pump . Thus, insulin appears to regulate Na + -K + ATPase activity in L 6 cells by promoting dephosphorylation of the α subunit through activation of PP-1, and PI-3 kinase appears to be involved in an earlier step in the signaling cascade. In addition, recent studies in A 6 cells demonstrated that PI-3 kinase inhibitors blocked insulin-stimulated Na + transport and insulin-stimulated PI-3 kinase activity . The results of the present study demonstrate that Na + -K + ATPase transport activity in primary cultures of endometrial epithelial cells is subject to regulation by insulin and IGF-I through a postreceptor signaling pathway that ultimately leads to dephosphorylation of resident pumps in the basolateral membrane. The signaling pathway responsible for insulin/IGF-I activation of the Na + -K + ATPase in endometrial epithelial cells appears to follow a similar pattern previously proposed for L 6 rat skeletal muscle cells and A 6 epithelial cells. Insulin and IGF-I have been previously shown to stimulate endometrial epithelial cell proliferation . Moreover, it was proposed that the growth-promoting actions of estrogen are mediated, in part, by release of IGF-I from endometrial cells . In human endometrium, IGF-I mRNA is primarily localized within stromal cells, and mRNA levels are most abundant during the proliferative phase of the cycle. IGF-I receptors are present in both stromal and epithelial cells, but are relatively more abundant in the epithelium . The levels of IGF-I receptor do not appear to change during the cycle. These results suggest that the endometrial epithelium can respond to locally released IGF-I from stromal cells and that IGF-I regulation of epithelial cell growth is maximal during the proliferative phase of the cycle. In porcine uterus, IGF-I activity changes with the early development of the placenta . Increases in IGF-I mRNA levels within the endometrium peak at a time when conceptus estrogen secretion reaches its maximum, and decreases significantly after implantation. The role of IGF-I and insulin in endometrial epithelial cell transport function has not been previously explored. We speculate that increases in Na + absorption in the presence of IGF-I may have some role in regulating the volume of fluid present within the uterus at the time of implantation. In porcine uterus, uterine fluid secretion is significantly stimulated just before ovulation and remains high for the next 2–3 d . The increase in fluid secretion is believed to be critical in providing a fluid environment for conceptus migration and positioning along the uterine horns before implantation. Increases in IGF-I secretion before implantation may provide a means of reducing uterine fluid volume once migration is complete. Stimulation of amiloride-sensitive Na + absorption may also account for the lower concentrations of Na + previously measured in mammalian uterine fluid . At this time, the effects of uterine fluid ionic composition on implantation are not understood, but the observation that Na + transport can be stimulated by IGF-I suggests the possibility that regulation of uterine fluid volume and ionic composition may play some role in the process of implantation. | Study | biomedical | en | 0.999997 |
10498675 | Ca 2+ plays a crucial role in a variety of cellular biochemical and physiological processes. The application of Ca 2+ -sensitive fluorescent indicators and the development of imaging technology have greatly advanced our ability to understand the mechanisms involved in global and, more recently, localized Ca 2+ handling. Localized Ca 2+ signaling is of particular importance because not only can the generation of a highly localized intracellular Ca 2+ pulse regulate such physiological events as exocytosis and ion channel activation, but it can also affect resting Ca 2+ levels and contribute to global elevations of Ca 2+ , and consequently affect distant cellular events . The localized Ca 2+ transients investigated so far include such “elementary events” as “Ca 2+ sparks” and “Ca 2+ puffs/blips” arising from the opening of ryanodine receptors or IP 3 receptors, respectively. Although the fluorescence changes associated with these events are well characterized and the studies of such elementary events have greatly enhanced the understanding of local and global Ca 2+ signaling, the precise relationship between these fluorescence changes and the channel openings that underlie these events (number of receptors/channels involved, channel kinetics, channel unitary current, etc.) is not clear . To determine this relationship, it would be useful to record the fluorescence transient due to Ca 2+ influx through a verifiable single channel opening—the “fundamental event” . Since the amount of Ca 2+ entry can be determined from the channel unitary current and open time, a Ca 2+ -permeable plasma membrane ion channel would be a good candidate for obtaining such a fundamental event, provided the fluorescence transient can be detected while recording the unitary single channel current. Another advantage of using a plasma membrane ion channel is that the rate of Ca 2+ entry can be adjusted by altering the driving force for Ca 2+ via changes in the membrane potential and extracellular Ca 2+ concentration. Such a channel has been identified and the fluorescence transients associated with single openings of this channel are described in this study. Previously, a plasma membrane, Ca 2+ permeable, nonselective cation channel was characterized in toad stomach smooth muscle cells . This channel appears to be directly activated by caffeine; activation does not require changes in membrane potential, intracellular Ca 2+ concentration, or cyclic AMP levels. In the solutions used for the studies by Guerrero et al. 1994a , Guerrero et al. 1994b , the caffeine-activated channel conductance is 80 pS and the zero-current potential is near 0 mV. The open times of the channel can be long (longer than 500 ms). Since, under whole-cell current recording conditions, at most ∼10–15 channels are open at the same time, the caffeine-activated channel apparently has either a low plasma membrane density or a low probability of being open. These characteristics make it possible to resolve single channel openings in the whole-cell current recording. Using a dual wavelength microfluorimeter to obtain measurements of global Ca 2+ with fura-2 as the Ca 2+ indicator, Guerrero et al. 1994b determined that ∼20% of the channel current is carried by Ca 2+ at −80 mV. Since, at physiological concentrations of extracellular Ca 2+ , there would be >1 pA Ca 2+ current (out of >5 pA total current) passing through this channel, we thought that it might be possible to image the fluorescence increase associated with the Ca 2+ transient that occurs when this channel opens and simultaneously record the (large and long duration) unitary currents. To this end, a wide-field digital imaging microscope was used with fluo-3 as the fluorescent Ca 2+ indicator to image the distribution of Ca 2+ in the cell while recording the unitary currents. Thapsigargin and/or ryanodine were used in the experiments to eliminate possible effects of intracellular Ca 2+ stores. The results presented here demonstrate the detection of the localized discrete fluorescence transient due to Ca 2+ entry through a single opening of this caffeine-activated plasma membrane cation channel while simultaneously recording the unitary current. Moreover, the location of the plasma membrane channels can be obtained from the images of the transients. Computer simulations of the events associated with a channel opening were used as an aid to interpret the time course of the observed transients. Smooth muscle cells were enzymatically dispersed from the stomach of the toad, Bufo marinus , as previously described and used on the same day. All experiments were carried out at room temperature. Whole-cell currents were recorded with an Axopatch-1D amplifier (Axon Instruments) using standard patch-clamp techniques, and caffeine-activated channel unitary currents were usually recorded with the membrane potential held at −80 mV. After rupturing the patch membrane, 5–10 min elapsed before collecting data to allow the fluo-3 concentration inside the cell to reach a steady level. The membrane potential was sampled at 1 kHz, as was the whole-cell current after the latter was low-pass filtered at 200 Hz. Two-dimensional fluorescent Ca 2+ images were obtained using the Ca 2+ indicator fluo-3 (50 μM) loaded into the cells through the patch pipette. Fluo-3 was used because of its low background fluorescence and its ∼200× fluorescence increase upon binding Ca 2+ . The 488-nm line of an argon-ion visible laser was used as the source of the excitation light. For these studies, fluorescence images were acquired using a custom built high-speed digital imaging microscope . Images were acquired at 15-, 30-, or 50-ms intervals, depending on the purpose of the experiment, with a 6-ms exposure time. A maximum of 200 images could be acquired during any recording sequence at the repetition rates used. In an attempt to catch the entire fluorescence transient associated with the channel opening, a circular-buffer protocol (software provided by Dr. Karl D. Bellvé, Biomedical Imaging Group) was used in some experiments to control image sampling. For this protocol, a circular buffer of 100 images was continuously refreshed once sampling started and the whole-cell current was monitored on an oscilloscope. When a channel opening was observed after caffeine application, manual triggering caused the previous 100 and next 100 images to be saved, so that the whole image set was composed of 200 images. Several image sets were generally obtained from the same cell. The laser shutter control voltage (sampled at 1 kHz) was simultaneously recorded with the current to facilitate the alignment of the fluorescence trace with the corresponding current trace. Images were processed using custom designed software. Each image is composed of 128 × 128 pixels either 500- or 333-nm square. At each pixel, the fluorescence in the absence of transients ( F 0 ) and during a transient ( F ) was used to construct the ratio images [ F / F 0 or Δ F / F 0 = ( F − F 0 )/ F 0 ], which were then smoothed (with a 3 × 3 kernel approximating a Gaussian with σ = 1 pixel) before analysis and display. The resting fluorescence, F 0 , was obtained by averaging the fluorescence intensity of 10 consecutive images when there was no fluorescence transient. Each of the fluorescence traces in the figures was obtained from the pixel where the greatest fluorescence change was observed during a localized Ca 2+ transient. The outline of the cell in the fluorescence ratio images was usually determined by applying a fluorescence intensity threshold. For some of the images, the background was masked using an outline of the cell obtained by manually tracing the fluorescence image. When necessary, to correct for the photo bleaching of fluo-3, the cell fluorescence was normalized to the fluorescence of that part of the cell that did not have any transient activity. A pseudocolor intensity ratio scale was generated for each set of images and is displayed in each of the figures. Generally, the focus was adjusted to what appeared to be the center of the cell. Therefore, if the cell was cylindrical in shape and was lying on the bottom of the chamber, then a transient at the edge of the two-dimensional image would be more likely to be in focus. Moreover, the farther the transient was from the edge and the closer it was towards the middle of the cell image (on the top or on the bottom half of the cell), the more likely it would be out of focus . If the diameter of the cell changed with length, or the focal plane was not in the center of the cell, then an in-focus transient might be observed in the middle of the two-dimensional image of the cell. A transient occurring near the middle of the cell image, whether in or out of focus, would be expected to have a smaller amplitude ( F / F 0 ) than one in the same focal plane near the edge of the cell. This occurs mainly because the background resting fluorescence, F 0 , would be higher due to the greater thickness at the center of the cell. Therefore, for the same Δ F , F / F 0 is necessarily smaller. To understand the processes underlying the fluorescence images when the channel opens, we simulated the distribution in time and space of free Ca 2+ , bound and free fluo-3, and bound and free stationary endogenous Ca 2+ buffers. To do this, finite difference approximations were used to solve a set of partial differential equations for the reaction–diffusion kinetics in a cylindrical coordinate system. This representation, an enhanced version of the simulation by Kargacin and Fay 1991 , assumed that the cell was cylindrically symmetric so that the simulation of three-dimensional kinetics required only two coordinates, radius and length, thus significantly reducing the numerical calculations. Two models of the smooth muscle cell were used. One was a longer cylinder (length = 30 μm, radius = 6 μm) with a channel in the center . The other was a shorter cylinder (length = 12 μm, radius = 6 μm) with the channel centered at one end. These simulations were “oriented” relative to an optical axis, and then numerically blurred (convolved) with a point spread function of a microscope (see below). We examined the longer cylinder with its axis perpendicular to the optical axis, and the shorter cylinder with its axis either perpendicular to or aligned with the optical axis. These orientations of the shorter cylinder provided, respectively, a view of the transient with the channel on the side or on the top/bottom of the cell. All three configurations (i.e., the longer cylinder or the shorter cylinder in two orientations) have their advantages and disadvantages. For example, the shorter cylinder with the channel at the end more closely approximates the transient near the channel; i.e., for hemispherical diffusion. On the other hand, the longer cylinder better resembles the shape and basal fluorescence spatial profile of a real smooth muscle cell. However, all three configurations produced qualitatively similar results. Ignoring blurring, the results presented for a channel in the center of the cylinder with a given current are the same as those for a channel at the end of the cylinder with one half that current. For Fig. 4 and Fig. 6 , the results are shown using the longer cylinder. For the simulations, the following parameters were used: channel opening of 600 ms with a 1.2-pA Ca 2+ current; initial free intracellular Ca 2+ concentration of 100 nM; total endogenous stationary buffer concentration of 230 μM and total fluo-3 concentration of 50 μM; an on-rate of 80 μM/s for fluo-3 and 100 μM/s for the stationary buffer; an off-rate of 90 μM/s for fluo-3 and 100 μM/s for the stationary buffer (yielding a K d of 1.13 μM for fluo-3 and 1 μM for the stationary buffer); a diffusion constant of 2.5 × 10 −6 cm 2 /s for Ca 2+ and 2.2 × 10 −7 cm 2 /s for free and Ca 2+ -bound fluo-3. The effects of plasma membrane pumps and leaks were negligible and no other intracellular activity (e.g., Ca 2+ -induced Ca 2+ release, Ca 2+ uptake) was included. Diffusion, rate and dissociation constants, and the concentration of stationary buffer are from Smith et al. 1998 or Kargacin and Fay 1991 . Images of the intracellular concentrations of the free Ca 2+ , fluo-3, and Ca 2+ -bound fluo-3 were typically generated every millisecond. The time intervals used in the simulation were actually much finer to ensure stability of the explicit method used in the finite difference approximation to the partial differential equations. After simulation in a two-dimensional coordinate system at 100-nm spatial resolution, images were converted to three dimensions at the appropriate pixel size (e.g., 333 nm). The Ca 2+ -bound fluo-3 concentration image was then convolved with the theoretically derived point spread function (oil lens with a NA of 1.3, 530 nm emission) of wide-field and confocal (assuming an infinitely small pinhole) microscopes to mimic the optically blurred observation through the camera. The point spread functions were sharper than those expected from real microscopes. The fluorescence from free fluo-3 was ignored since it is only ∼1/200 the brightness of the Ca 2+ -bound fluo-3. Fluorescence changes over time ( F / F 0 or Δ F / F 0 ) were then extracted from various planes through the cell to determine what would be observed for both in- and out-of-focus transients. For example, the fluorescence change 2 μm above the channel was extracted and plotted together with the in-focus fluorescence change . The sensitivity of the results (e.g., predicted F / F 0 , estimated Ca 2+ concentration, etc.) to a variety of parameters was examined by carrying out simulations with various values for cell size, diffusion constants, concentrations of fluo-3 and stationary endogenous buffers, and their on and off rates for Ca 2+ binding, channel location, and current amplitude. The final values used here were adopted from the literature to illustrate the basic findings from these simulations and were not meant to be predictive of the values underlying the measured results. A difference between the simulations carried out here and those generally found in the literature dealing with single Ca 2+ channels is that, because of the long channel openings and the small cell radius, volume limitations were taken into account. Other simulations without these limitations generally follow the diffusion process into an infinite volume . In addition, theoretical point spread functions were also used here to take into account the optical blurring of the microscope, especially because of the large depth of field of wide-field optics . In most experiments, 40 μl of cells in dissociation solution were added to a 1.5-ml chamber with a standard bathing solution containing (mM): 127 NaCl, 3 KCl, 1.9 CaCl 2 , 1 MgCl 2 , 10 HEPES, pH 7.4. The standard whole-cell patch pipette solution contained (mM): 130 KCl, 1 MgCl 2 , 20 HEPES, 3 Na 2 ATP, 1 Na 3 GTP, 0.05 fluo-3 (pentapotassium salt; from Molecular Probes, Inc.), pH 7.2. These solutions differed somewhat from those used by Guerrero et al. 1994a , Guerrero et al. 1994b , resulting in a slightly larger unitary current at −80 mV. Modifications of the solutions for specific experiments are indicated in the text and figure legends. The free Ca 2+ concentrations in solutions containing DiBrBAPTA (Molecular Probes, Inc.) were calculated using MAXChelator, a computer program based on the work of Bers et al. 1994 . To remove possible effects of intracellular Ca 2+ stores, for most experiments, thapsigargin (1 or 3 μM; Sigma Chemical Co.) was added to the bathing solution and/or ryanodine (100 μM; Sigma Chemical Co.) was added to the pipette solution. Usually both were present. The stock solutions for thapsigargin (10 mM) and ryanodine (10 or 100 mM) were in DMSO and stored at −20°C. The occurrence of caffeine-activated channel openings and associated fluorescence transients does not require the presence of thapsigargin and/or ryanodine since they could be recorded in the absence of these agents (not shown). Moreover, spatially averaged transients correlated with the opening of the caffeine-activated channels without these agents have been described previously . Caffeine (usually 20 mM dissolved in the bathing solution) was applied to the cells by pressure ejection from a micropipette (puffer pipette) using a picospritzer (General Valve Corp.). The ejection pressure, caffeine application time, and the distance between the puffer pipette and the cell were usually adjusted to optimize the recording of single channel openings such that only one or two channels were open at the same time. In general, brief (subsecond) caffeine applications to the cells were repeated before data acquisition to help empty intracellular Ca 2+ stores and test for the presence of the caffeine-activated channels. Caffeine was applied continuously for 3 s before sealing onto some of the cells, bathed in a thapsigargin-containing solution for at least 15 min, to maximally release Ca 2+ from intracellular stores . There was no noticeable difference between the results obtained from these cells and from cells that were not exposed to caffeine for such an extended period of time. In some experiments, two picospritzers were used so that caffeine in different solutions could be applied to the same cell . The puffer control monitor was sampled at 1 kHz. Application of caffeine to a smooth muscle cell with the membrane potential held at −80 mV causes inward unitary currents . When fluorescence images were acquired while simultaneously recording the unitary currents, discrete, localized, transient fluorescence increases appeared at various times and at various locations throughout the cell. For the cell in Fig. 1 , repeated image sets revealed localized transients in at least eleven different locations . For the current recording shown , with the images acquired every 50 ms, the discrete fluorescence increases were found at eight different locations . When the fluorescence traces from these locations were aligned in time with the whole-cell current trace , every one of these fluorescence transients was found to correspond to a single channel opening in the current trace. However, the reverse was not the case since the transients corresponding to some of the channel openings either occurred out of the image field or, less likely, were possibly masked by a nearby transient. To examine the temporal relationship between the fluorescence transient and the channel opening in more detail, the transient at one location (d) and the corresponding unitary current trace are plotted on an expanded time scale . The 10 sequential images comprising this transient, starting with the one immediately preceding the channel opening, are also shown . When the channel opened, the fluorescence increased from the resting level and continued to rise during the ∼200 ms the channel was open. The fluorescence decreased when the channel closed and stayed closed. In the images , the punctate appearance of the fluorescence increase appeared as soon as the channel opened and the fluorescence intensity and spatial spread kept increasing during the time the channel was open. When the channel closed, and therefore the Ca 2+ source was removed, the local fluorescence declined and became more diffuse with time. These observations suggest that the fluorescence transient was following the Ca 2+ influx through a single opening of the caffeine-activated plasma membrane cation channel located in a particular area of the cell membrane. Because of its punctate appearance and steep spatial gradient, this transient was most likely more in focus than some of the other transients, which had a more diffuse appearance . Since each and every observed transient corresponded to a single channel opening, the location of the transient revealed the location of the channel on the plasma membrane (projected onto these two-dimensional images). Although not rigorously studied, it appears that caffeine-activated channels are not localized in any one particular region of the cell, but rather seem randomly distributed over the cell surface. It is unlikely that intracellular Ca 2+ stores contributed to these transients since the bathing and pipette solutions contained thapsigargin and ryanodine, respectively. If the fluorescence transients described above were indeed due to Ca 2+ entry through the caffeine-activated channels, then they would be expected to be abolished when the channels open in a bathing solution where the Ca 2+ concentration is sufficiently reduced. To see if this would be the case, cells were bathed in a solution in which the Ca 2+ concentration was lowered to ∼14 μM, which should substantially decrease the fraction of the unitary current carried by Ca 2+ . Application of caffeine dissolved in this bathing solution activated the channels; however, no clear fluorescence transients were detected (in any of the 38 image sets from five cells, with at least 5 image sets per cell). When caffeine dissolved in a solution containing 2 mM Ca 2+ was briefly applied to the same cells, discrete fluorescence transients were observed (in 26 of 27 image sets with at least three image sets from each cell showing a transient). Similar results were also obtained when Ca 2+ in the bathing solution was buffered to 3.6 μM (2 mM Ca 2+ and 4 mM DiBrBAPTA) or when the bathing solution contained 200 μM BAPTA and no added Ca 2+ (“zero Ca 2+ bathing solution”). No fluorescence increases were observed when the channels were opened by applications of caffeine dissolved in either bathing solution (in all six image sets from two cells in the 3.6 μM Ca 2+ bathing solution with at least two image sets from each cell; and in all of the 29 image sets from five cells in the zero Ca 2+ bathing solution with at least four image sets from each cell). However, discrete localized fluorescence transients were evident in the same cells when the channels were opened by applications of caffeine dissolved in a solution containing 2 mM Ca 2+ (in five of nine image sets in the 3.6 μM Ca 2+ bathing solution with at least two image sets from each cell showing a transient, and in all 19 image sets in the zero Ca 2+ bathing solution with at least three image sets from each cell). Therefore, sufficiently reducing the driving force for Ca 2+ entry by decreasing the Ca 2+ gradient across the cell membrane abolishes the fluorescence transients. In the zero Ca 2+ bathing solution, applications of caffeine caused mainly shorter duration channel openings; i.e., longer openings were much less frequent than was the case in the 14 μM Ca 2+ bathing solution. Chiefly because of the shorter openings in the zero Ca 2+ bathing solution, the 14 μM Ca 2+ bathing solution was a better control for demonstrating the requirement for extracellular Ca 2+ . Further evidence that these fluorescence transients are due to Ca 2+ entry was obtained by changing the membrane potential to alter the driving force for Ca 2+ entry. Fluorescence transients were recorded from the same cell in the standard bathing solution with the cell membrane potential alternately held at −50 and −100 mV. If the fluorescence transients were due to Ca 2+ entry, they should have a faster initial rate of rise at −100 than at −50 mV because the initial rate of rise (measured as the slope of the initial linear part of the fluorescence change) represents a measurement of the underlying current that is carried by Ca 2+ . This current should be larger at −100 than at −50 mV. There are three factors that complicate these measurements. First, often there were brief closures of the channel that might interrupt the increase of the fluorescence transient. Hence, the measurement for the initial rate of rise could only be obtained up to the time when the first brief closure occurred. Second, for these studies, the images were acquired every 15 ms and the channel opening might occur anytime during this 15-ms period. Therefore, counting only the first data point showing a fluorescence increase after the channel opened could affect the apparent initial rate of rise of the transient. Because of these two factors, the initial rate of rise of a transient was usually measured as the slope of the linear part of the fluorescence trace: between the point immediately preceding channel opening and the fourth point into the transient in the absence of closures, or between the point immediately preceding channel opening and the point immediately preceding the closure if it occurred earlier than the fourth sampling point. Third, the initial rate of rise of each recorded transient depended on how well it was in focus (see below). Therefore, paired comparisons were made between the fluorescence transients at the two membrane potentials at the same location in the cell. The mean ratio (±SEM) of the initial rate of rise at −100 mV to that at −50 mV was 2.1 ± 0.5, which is significantly greater than 1 (14 different locations in five cells, P < 0.01, two-tailed t test based on the difference between the natural logs of the rates of rise when compared with zero), indicating that fluorescence transients at −100 mV had a greater average initial rate of rise than those from the same location at −50 mV. The temporal order of the holding potential did not appear to affect the measurements. This result is consistent with the generation of a larger Ca 2+ influx at more negative holding potentials. In summary, all of these results obtained by altering the driving force on Ca 2+ , either by changing the extracellular Ca 2+ concentration or by changing the membrane potential, suggest that the observed localized fluorescence transients are indeed due to Ca 2+ influx through single openings of a plasma membrane ion channel. These fluorescence transients are associated with increases in the underlying intracellular free Ca 2+ concentration or single channel Ca 2+ transients (SCCaTs) and, therefore, can be designated as single channel Ca 2+ fluorescence transients (SCCaFTs). 1 These terms are analogous to the terms “Ca 2+ sparks” and “sparks” or “fluorescence sparks” used by Cheng et al. 1993 or Blatter et al. 1997 . However, the term “Ca 2+ sparks” has been used to convey both meanings. To examine the time course of the rising phase of the SCCaFT in more detail, SCCaFTs were monitored at a higher temporal resolution by acquiring images every 15 ms. For the channel located at the center of the image , a very long opening without discernible closures occurred after a briefer opening of the same channel. When a long channel opening occurs, especially in the near absence of brief closures with the channel in or nearly in focus, it becomes quite clear that while the channel is open, the SCCaFT is composed of a rapid initial rise followed by a slower rise. Moreover, when the channel closes, the fluorescence decrease is composed of a fast initial phase followed by a slower phase as the fluorescence returns towards its resting level. This time course seems to be characteristic of the SCCaFT as imaged with wide-field optics. Consistent with two phases for the rise and fall of the transients, neither the rise nor the fall for the long transient in Fig. 3 could be well fit with a single exponential. Instead, the sum of two exponentials was required for a reasonably good fit . The rise and fall of other transients also appeared to be fit by the sum of two exponentials, but it was difficult to make comparisons because both the degree of focus and the presence of brief closures affect the results. To understand the time course of the fluorescence transient and the underlying events when a channel opening occurred, we simulated a caffeine-activated channel opening in a smooth muscle cell using a modification of the simulation carried out by Kargacin and Fay 1991 . For the simulation described here , the model cell was cylindrically symmetric, 30-μm long and 12 μm in diameter, with a 1.2-pA unitary Ca 2+ current (approximately the portion of the current passing through the channel that is carried by Ca 2+ ). Cell dimensions were chosen so that the cell was long enough to eliminate the end-effects that would also be absent with a real cell, while the diameter, close to that of the cells used, would provide the volume effects of the real cell. The channel was located in the center of the cell to simplify calculations. See methods for further details and for the rationale. From the simulation, it is clear that when the channel opens, Ca 2+ -bound fluo-3 as well as free Ca 2+ increase abruptly in the immediate vicinity (<100 nm) of the channel , and each establishes a large spatial gradient. Ca 2+ (either free or bound to fluo-3) then quickly diffuses away from the channel so that near steady state levels (with a slow rise) are maintained for both Ca 2+ and Ca 2+ -bound fluo-3 in the vicinity of the channel. After the channel closes, the three-dimensional diffusion of Ca 2+ and fluo-3 rapidly abolishes the spatial gradients to establish a homogeneous equilibrium in the cell determined by (in the absence of Ca 2+ removal mechanisms) the amount of Ca 2+ entry and the cell volume . To obtain from the simulated data what would be observed with the camera after blurring by the microscope optics, the simulated Ca 2+ -bound fluo-3 image was convolved with the theoretical point spread function of a wide-field microscope. Similar to what was observed experimentally , for the pixel overlaying the channel, the fluorescence transient in these blurred images also reveals two distinct phases when the channel opens or when it closes . When the channel opens, the initial rapid rise corresponds to Ca 2+ entry and the quick establishment of the local near steady state. The slower rise corresponds to the increasing fluorescence farther from the channel as Ca 2+ and Ca 2+ -bound fluo-3 are distributed throughout the cell . The optical blurring averages the near-channel fluorescence with the fluorescence from the accumulation of Ca 2+ -bound fluo-3 occurring within a larger cell volume. When the channel closes, the sudden removal of the Ca 2+ source causes the rapid decrease in fluorescence near the channel, while the slower phase represents the processes whereby Ca 2+ and Ca 2+ -bound fluo-3 diffuse away from the channel. The in-focus image better reflects the kinetics and amplitude of the rapid phases than the out-of-focus images. As can be seen in the 2-μm out-of-focus fluorescence change in Fig. 4 C, the kinetics are slowed and the amplitude is decreased with defocus. These effects are due to the fact that less of the near-channel fluorescence is being collected in the same size pixel in the out-of-focus image. For comparison, the simulated Ca 2+ -bound fluo-3 image was also convolved with a theoretical point spread function of a confocal microscope. The blurred line scan image and the fluorescence change from the pixel overlying the channel are displayed in Fig. 4A and Fig. C , respectively. Characteristics of the in- and out-of-focus fluorescence traces obtained from the simulation are clearly present in the recorded images of relatively in- and out-of-focus SCCaFTs shown in Fig. 5 . As the respective channels opened and closed, the in-focus SCCaFT clearly showed two phases and a larger amplitude, while the out-of-focus SCCaFT was slower in its time course and smaller in amplitude. Moreover, with the out-of-focus SCCaFT, the fast fluorescence changes due to brief closures of the channel became smaller and tended to disappear compared with the large decreases (or “valleys”) in fluorescence with the in-focus SCCaFT. Thus, the fluorescence changes of the in-focus SCCaFT have a much stronger correlation with channel openings and closings, indicating that they better represent the Ca 2+ (or Ca 2+ -bound fluo-3) changes in the vicinity of the channel. The fast phases are revealed only when imaging with high temporal resolution, even if the transient is relatively in focus . Most recorded SCCaFTs fall between the examples shown in Fig. 5 . In summary, compared with out-of-focus SCCaFTs, in-focus SCCaFTs have a larger fluorescence change and a much more rapid initial rise and fall when the channel opens and closes. Furthermore, the fluorescence of an in-focus SCCaFT has a more limited or punctate-appearing spatial spread when the channel initially opens, and a steeper spatial gradient throughout the opening . This study, to our knowledge, is the first to report the imaging of the localized fluorescence transient due to Ca 2+ entry through a verifiable single opening of an ion channel. Using fluo-3 as a fluorescent Ca 2+ indicator and a plasma membrane Ca 2+ -permeable cation channel, it is possible to simultaneously record this transient (SCCaFT) and the unitary current at physiological extracellular Ca 2+ concentrations. Because the wide-field microscope collects fluorescence from outside the focal plane of the camera in addition to the in-focus fluorescence, it is possible to detect the fluorescence transients anywhere in any plane within the image field. This is helpful for these studies because the location of the channels is not known before recording begins, and they do not appear to have a defined distribution pattern, as is the case for the sites of Ca 2+ sparks in cardiac cells . Moreover, imaging Ca 2+ entry through openings of Ca 2+ -permeable ion channels with a wide-field microscope provides a method for the determination of channel location in a manner similar to that used to locate mechanically gated channels in hair cells, though with different optical methods . For the caffeine-activated channels in toad stomach smooth muscle cells, the channel appears to be randomly located in the plasma membrane . In addition, multiple SCCaFTs often appear at the same location in one image set, and multiple image sets reveal a sparse SCCaFT distribution. These observations, plus the observation that only ∼10–15 channels are open at the same time during maximal activation with caffeine , suggest that rather than having a low probability of being open these channels have a low density on the plasma membrane. However, the possibility of closely clustered channels with (or without) some sort of local coordination of channel gating cannot be eliminated. SCCaFTs reported here should not be confused with sparks or puffs/blips, which are the fluorescence transients reflecting localized increases in intracellular Ca 2+ concentration due to Ca 2+ release from intracellular Ca 2+ stores through ryanodine or IP 3 receptors, respectively. These Ca 2+ release events have usually been recorded with confocal line-scan imaging. Sparks have also been recorded in toad stomach smooth muscle cells with the same wide-field imaging system used here . However, the sparks observed in these smooth muscle cells were much shorter in duration (rise time ∼20 ms) and smaller in amplitude than SCCaFTs (presumably due to briefer underlying channel openings and possibly a smaller current) and, unlike SCCaFTs, could be detected in the absence of extracellular Ca 2+ and were abolished when Ca 2+ uptake into intracellular stores was blocked by the presence of thapsigargin . Moreover, SCCaFTs were always associated with the openings of a plasma membrane channel. Brief SCCaFTs, due to short openings of plasma membrane cation channels, having mainly the fast phases of the fluorescence change , resemble sparks. This suggests that the major components of the spark (the rising phase and the first part of the falling phase) reflect the fast phases of fluorescence change. The slow rising phase is not usually observed when recording sparks because they are normally brief events and there is little time for Ca 2+ and Ca 2+ -bound fluo-3 to diffuse further away before the channels close. A slow falling phase is also seen in some sparks. However, it is difficult to make exact comparisons because it is unclear how many ryanodine receptors are involved in spark formation and how well their openings and closings are synchronized, if indeed multiple channel openings are involved . The SCCaFTs associated with long openings of the caffeine-activated channel in toad cells show some similarity to the prolonged sparks sometimes recorded when there were “modal changes in spark activity” or when the underlying ryanodine receptors were presumably in a long duration conductance state in the presence of ryanodine , imperatoxin A , or FK506 . Arguments similar to the above can also be made for puffs and blips that occur with the opening of IP 3 receptors. The variation in the rising and falling phases of puffs , assuming they were all in focus, indicates asynchronous openings and closings of IP 3 receptors, in agreement with puffs being due to the opening of a cluster of IP 3 receptors. Moreover, the time course of the fluorescence change for certain prolonged blips resembles that of SCCaFTs . The parameters used in the computer simulation of events underlying SCCaFTs were adopted from the literature. Changes in these parameters did not qualitatively affect the fluorescence transient obtained from the simulation. However, there were quantitative effects. For example, decreasing the concentration of the stationary buffer or increasing the unitary current increased the initial rate of rise and the amplitude of the SCCaFT; increasing the resting Ca 2+ concentration or cell diameter (resulting in an apparent increase in F 0 ) decreased the amplitude of the SCCaFT; decreasing the on and off rates of the stationary buffer (keeping K d constant) increased the initial rate of rise and fall of the simulated SCCaFT. In general, these changes did not affect the overall shape of the fluorescence traces, all showed a rapid initial rise followed by a slower rise during channel opening and a rapid fall followed by a slower fall after the channel closed. The results of the simulation were also in agreement with other aspects of the observed fluorescence trace, such as a slower initial rate of rise and a smaller amplitude when the SCCaFT was out of focus. Fluorescence transients have been used by others to obtain estimates of the free Ca 2+ concentration change (or Ca 2+ transient) and the underlying Ca 2+ influx (or Ca 2+ current). Shown in Fig. 6 A are the relationships between the measured fluorescence change and the free Ca 2+ concentration change based on either the assumption that fluo-3 is in equilibrium with Ca 2+ or the simulation used for Fig. 4 . The simulation is not constrained by the equilibrium assumption, which is invalid within nanometers of the open channel . From the simulation, given the same underlying Ca 2+ transient (or current), an in-focus fluorescence transient observed with a confocal microscope is always larger than that observed with a wide-field microscope, and both are smaller than would be observed without blurring . Thus, the greater the optical blurring the smaller the recorded fluorescence transient. Moreover, the free Ca 2+ concentration (i.e., Ca 2+ transient) at any Δ F / F 0 is grossly underestimated by assuming equilibrium conditions. Therefore, the relationship between fluorescence transients and Ca 2+ transients is complex and cannot be uniquely determined without a set of underlying assumptions about Ca 2+ handling and the point spread function of the microscope . Extrapolation from the time course of fluorescence change to the underlying Ca 2+ current is even more complex. When trying to calculate the Ca 2+ current simply from the peak amplitude of the fluorescence transient (for example, calculating the current passing through the ryanodine receptor from the amplitude of an observed spark), the result can vary significantly depending on how long after the onset of the transient the peak occurs . For this reason, the initial rate of rise of the fluorescence trace is a better measure of the Ca 2+ influx , but this measurement should be made with the fluorescence transient in focus because fluorescence changes at different rates in different focal planes . To avoid the requirement of imaging the fluorescence transients in focus as stated in the previous paragraph, the fluorescence change at any time on the rising phase can be spatially integrated over the image. As shown in Fig. 6 C, when the area of integration is large enough, the total fluorescence change is essentially the same whether the channel is in focus or 2 μm out of focus. The Ca 2+ current can be determined from the rate of rise of the total fluorescence . This is similar to the insightful approach used by Sun et al. 1998 , who estimated the total Ca 2+ release underlying Ca 2+ puffs and blips by integrating the one-dimensional confocal linescan profile over three dimensions (the “signal mass”). In our case, the integration can be obtained directly from the two-dimensional wide-field images. While this seems to be an appealing way for calculating Ca 2+ current from the fluorescence measurement, it still has its limitations. Ca 2+ removal and transport systems, as well as the concentrations of free Ca 2+ and Ca 2+ bound to buffers other than the fluorescent indicator, still need to be taken into account (as for any of these methods). In the absence of a detailed description of Ca 2+ handling, these experiments would best be done with higher concentrations of Ca 2+ indicator to capture more of the Ca 2+ influx and/or by recording SCCaFTs and fluorescence events in the same cell or cell type. Then, the known current underlying the SCCaFT can be used to estimate the unknown currents giving rise to the other fluorescence events. The estimates will be more accurate if SCCaFTs and the fluorescence events are recorded at high imaging frequency, occur near the same location, and have close to the same rate of rise of fluorescence. The last can be adjusted by changing the underlying SCCaFT Ca 2+ current by altering the Ca 2+ driving force. From the above discussion, as an example, an estimate of the Ca 2+ current underlying Ca 2+ sparks in toad stomach smooth muscle cells can be obtained using the fluorescence measurement from SCCaFTs. The initial rate of rise of fluorescence for the in-focus SCCaFTs from the cell in Fig. 5 is ∼1.7%/ms (average of five transients), corresponding to a 1.4-pA Ca 2+ current, assuming that 20% of the current was carried by Ca 2+ . The initial rate of rise of a spark is ∼1%/ms for a spark rise time of 20-ms with a 20% fluorescence increase . Therefore, based on the near-linear relationship between the initial rate of rise of fluorescence and the Ca 2+ current , and neglecting the effect of any Ca 2+ removal process, the Ca 2+ current underlying the spark should be ∼0.8 pA (≈1.4 pA × 1%/1.7%). If the ryanodine receptor in toad stomach smooth muscle cells has similar properties as those described by Mejía-Alvarez et al. 1999 , this result would suggest that approximately three ryanodine receptors underlie the spark. A more precise estimate requires a more complete data set. Moreover, systematic studies should be carried out using the “signal mass” or spatial integration . The applicability of these methods is dependent upon the assumption that the fluorescence events (for example, sparks) are caused either by a single channel opening or by well synchronized openings of a few tightly clustered channels. If this does not occur or if there are brief channel closures, then the current determined in this way would be the average current. | Review | biomedical | en | 0.999995 |
10499913 | B10.D2nSn/J and two Ig transgenic (Tg) mouse models were used in the experiments: the 3-83 μδ Ig Tg mice express IgM and IgD specific for H-2K k 1 , and Ig Tg anti–hen egg lysozyme (α-HEL) mice express IgM and IgD specific for HEL 2 . Bone marrow (BM) was obtained from 3–4-mo-old mice; a single-cell suspension was prepared by flushing femurs with IMDM to dislodge cells, followed by gentle deaggregation using a 5-ml syringe. BM was depleted of erythrocytes using Gey's solution, washed twice in IMDM, and cultured at 5 × 10 5 cells/ml per 10-cm petri dish (7% CO 2 , 37°C) in IMDM containing 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin, 2 mM l -glutamine, 1 mM sodium pyruvate, 50 μM 2-ME, 10% FCS (Hyclone), and 50–100 U IL-7 34 (derived from culture supernatant of J558L cells transfected with mIL-7 cDNA, a gift from Dr. A. Rolink, Basel Institute for Immunology, Switzerland). Typically, cells were harvested after 6–7 d in culture, washed twice with IMDM, and used in subsequent experiments. Resting splenic B cells (ρ > 1.066) were obtained from 3–4-mo-old adult mice and prepared as previously described 35 . Cell viability was assessed using trypan blue dye exclusion. Antigens: an H-2K k mimetic peptide (CSGFGGFQHLCCGAAGA) that binds specifically to the 3-83 receptor 35 36 was synthesized and multimerized by coupling to N -ethylmaleamide–activated dextran (a gift from CorTech, Inc.) at a 100:1 peptide/dextran molar ratio; HEL was purchased from Sigma Chemical Co. Antibodies directed against the following molecules were used: MHC class II (Ia b/d ; D3.137); CD69 (H1.2F3, PharMingen); CD86 (GL-1, PharMingen); CD21/35 (CR2/CR1; 7G6, PharMingen); CD19 (1D3, PharMingen); IgM (b-7-6); IgD (JA12.5); CD22 (CY34.1.2); CD23 (IgE Fc receptor; B3B4, PharMingen); CD45 (I3/12.5); and CD45R (anti-B220; RA3-3A1 and RA3-6B2). Ionomycin was purchased from Calbiochem Corp.; propidium iodide was from Sigma Chemical Co. Cells were washed once, resuspended in PBS containing 1% BSA and 0.1% sodium azide, and incubated with an optimal amount of biotinylated or directly fluorescinated antibody. Cells were incubated for 30 min at 4°C and washed twice in PBS/BSA/azide. In the case of biotinylated antibodies, cells were incubated as before with avidin–FITC or avidin–PE (Becton Dickinson). After washing, cells were analyzed on a flow cytometer. Histograms were constructed based on analysis of 10,000 cells. For measurements of [Ca 2+ ] i , cells were loaded with Indo-1AM (Molecular Probes, Inc.), suspended at 10 6 cells/ml in IMDM, and stimulated with either antigen or anti-IgM antibody (b-7-6). Mean [Ca 2+ ] i was evaluated over time using a flow cytometer (Model 50H; Ortho Diagnostic Systems Inc.) with appended data acquisition system and MultiTime software (Phoenix Flow Systems) as previously described 35 . Levels of recombinase activator gene (RAG)-2 and CD19 mRNA were determined by RT-PCR assay as described in detail elsewhere 34 . To obtain a semiquantitative estimate of gene expression, the signal for RAG-2 was normalized to the CD19 signal. Analysis of molecular mechanisms underlying the responses of immature and mature B cells to antigen required generation of homogeneous populations of antigen-specific cells. To generate antigen-specific immature B cells, cells were isolated from BM of 3-83 μδ (specific for H-2K k ) Ig Tg 1 or α-HEL Ig Tg 2 mice and cultured for 6–7 d in IL-7–containing medium. Over this period, the total number of cells per dish increased from 5 ×10 6 to 30–40 × 10 6 (data not shown). The percentage of B cells (CD45R + ) increased from 12–18% at the onset of the culture to >95% after 5 d, demonstrating the selective outgrowth of IL-7–responsive B cell precursors 34 37 . Phenotypic analysis further indicated that few cells from non-Tg BM grown in IL-7 progressed to the IgM + IgD − immature B cell stage compared with the Ig Tg animals . Cells from Tg BM grown under these conditions were >90% IgM low IgD − ; upon removal of IL-7, cells progressed through IgM high IgD − , the immature stage, to IgM high IgD low , the transitional stage, and finally to mature B cells (data not shown and reference 34). Relative expression of mIgM and mIgD transgenes as well as other markers was consistent with development of a homogeneous population of immature cells in these cultures : cells were CD45 low (also CD45R low , not shown), CD22 low , and CD19 low (all compared with mature B cells), whereas they were negative for CD21/35 and CD23, which are only expressed on mature B cells 38 39 40 . Equivalent phenotypes were seen when BM cells from α-HEL Tg animals were grown in IL-7 (data not shown). These data confirm a previous report 34 that the presence of mIgM transgenes is sufficient to accelerate B cell maturation through the pro- and pre-B cell stages to an immature phenotype. Although it is difficult to exclude the possibility that the cells grown in this system are developmentally pre-B cells that simply express a Tg BCR on their surfaces, these cells express all known markers associated with the immature B cell stage 41 42 . We then compared early biologic responses of the respective B cell populations to antigen, analyzing induction of rag gene expression, indicative of clonal elimination by receptor editing 3 , and CD69, CD86, and MHC class II expression, indicative of BCR signal transduction leading to initiation of an immune response. Immature B cells from BM cultures and mature splenic B cells from 3-83 μδ or α-HEL Tg animals were incubated with antigen, and the expression of activation markers was assessed . Mature B cells upregulated CD69, CD86, and MHC class II molecules within 12 h of stimulation, as previously shown 43 44 45 . In immature 3-83 μδ Tg cells, however, only slight upregulation of CD69 and CD86 was detected, and this was seen only at 12 h after stimulation. HEL stimulation of immature, α-HEL Tg cells also led to upregulation of these markers at 12 h. The responses were more pronounced than those of immature 3-83 μδ cells but much less than those of mature α-HEL Tg B cells . No antigen induction of MHC class II was observed in immature cells. The difference in the degree of upregulation between the 3-83 μδ and α-HEL Tg cells is probably due to the affinity of the transgene-encoded receptor for its ligand (∼2 × 10 −5 M for 3-83 μδ [reference 46 ] and 2 × 10 −9 M for α-HEL [reference 2]). These results demonstrate that immature B cells are unable to effectively upregulate molecules essential for T–B cell collaboration; this probably prevents their participation in T cell–dependent immune responses. Recent studies demonstrate that binding of antigen to mIgM on immature B cells induces recombinase gene expression and L chain receptor rearrangements 16 17 , a process that results in alteration of receptor specificity, termed receptor editing 3 . This process leads to clonal elimination of immature cells but may also contribute to increasing diversity in a germinal center reaction 47 . To compare the ability of immature and mature B cells to undergo a receptor editing response to antigen, we tested for antigen-induced reactivation of rag genes using RT-PCR. These analyses demonstrated a greater than twofold (normalized to CD19 mRNA levels) increase in RAG-2 expression upon antigen stimulation in immature cells , resembling responses observed in vivo 3 . No RAG-2 mRNA was detectable in mature B cells before or after 18-h antigen stimulation. Finally, no antigen-induced cell death was observed over the 18-h period in mature or immature cells (data not shown). Thus, as previously shown 16 , antigen-induced cross-linking of the BCR in immature cells results in the induction of receptor editing, and this response is not seen in mature, naive B cells. Together, the data demonstrate two mutually exclusive response patterns in mature and immature cells. These distinct patterns may ensure that autoreactive, immature B cells do not mature into autoreactive, mature B cells and that only B cells that have been properly vetted to eliminate those that are autoreactive can effectively upregulate ligands for CD28 (CD86) and T cell antigen receptor (MHC class II) and thus participate in a productive immune response. The observed differences in the biologic responses of mature and immature cells may result from differences in BCR coupling to proximal signal transduction pathways. Alternatively and/or additionally, these B cells may be programmed to respond differently to the same constellation of “second” messengers. Calcium mobilization plays an important role in triggering a wide array of cellular processes 48 , including RAG induction 49 , and could drive the BCR-mediated expression for CD69, MHC class II, and CD86. To determine if calcium mobilization is sufficient to initiate the biologic responses of immature and mature B cells, ionomycin was used to induce graded rises in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ). Ionomycin induced comparable increases in [Ca 2+ ] i in immature and mature B cells at each concentration used . Analysis of marker induction after 18 h of ionomycin stimulation demonstrated profound upregulation of CD86 in mature B cells but only at doses of ionomycin that induced increases of >600 nM [Ca 2+ ] i ; small increases were observed at doses that raised [Ca 2+ ] i by 80–120 nM, and no upregulation was seen when [Ca 2+ ] i was raised by 50 nM . Consistent with the effects of antigen , ionomycin did not induce CD86 in immature B cells. Similar observations were made for MHC class II and CD69 (data not shown). This demonstrates the sufficiency of calcium mobilization in antigen activation of CD86 and MHC class II expression in mature B cells. As previously reported 49 , elevation of [Ca 2+ ] i induced RAG-2 expression in immature B cells. However, as shown in Fig. 3 , this response followed much smaller increases in [Ca 2+ ] i than required to induce CD86 in mature cells: RAG-2 expression was increased 1.7–2.3-fold after 80–120-nM rises in [Ca 2+ ] i and 3.1–5-fold after a 50-nM rise in [Ca 2+ ] i . High doses of ionomycin (inducing >600 nM increase in [Ca 2+ ] i ) only raised RAG-2 marginally and were accompanied by an increased apoptotic response (two- to threefold increase over unstimulated control), whereas little to no apoptosis was induced at 100 and 10 nM ionomycin (data not shown and reference 49). This suggests that extreme increases in [Ca 2+ ] i in immature B cells (possibly reflecting a signal through a high-avidity interaction with self-antigen) leads to an apoptotic rather than a receptor editing response. No RAG-2 induction was detected in mature B cells. These data demonstrate that increases in [Ca 2+ ] i can mediate the unique, antigen-induced changes in CD86 and RAG-2 seen in mature and immature B cells, respectively. These distinct biologic responses could be generated via two mechanisms. First, BCR stimulation could lead to activation of different transcription factors that are developmental stage specific in their expression. Second, different genetic loci could be more accessible in immature and mature B cells by regulated demethylation and chromatin remodeling; it has been demonstrated that these processes are involved in differential induction of egr-1 in immature versus mature B cells 9 , allelic exclusion during B cell development 10 , and regulation of cytokine gene expression in Th1 and Th2 cells 50 . According to this model, BCR stimulation would activate calcium-dependent transcription factors (e.g., NFAT [nuclear factor of activated T lymphocytes]), leading to rapid transcription of the accessible loci in each cell type; these would include rag in immature cells and CD86 and MHC class II in mature cells. The ability of HEL to induce transient upregulation of CD86 in immature cells appears inconsistent with both of these hypotheses. The high frequency of cells displaying this response excludes the possibility that it is a consequence of contaminating mature cells. It is possible that the accessibility of the loci is not absolute. Perhaps a sufficiently strong antigen receptor signal can lead to some expression of a relatively inaccessible locus. More studies are required to resolve this question. The observations presented here are consistent with the behavior of the 3-83 μδ Ig Tg model of tolerance induction wherein immature B cells encounter autoantigen in the BM and are induced to edit their receptors 1 3 . However, they appear inconsistent with the HEL/α-HEL model. Specifically, findings that HEL induces RAG in immature B cells from α-HEL mice predict that B cells in HEL/α-HEL double-Tg mice should undergo editing in vivo. Evidence for receptor editing is observed when HEL is expressed in a membrane-associated form 51 but is less obvious when HEL is present as a secreted protein 2 . In the latter double-Tg animals, however, a decline in the number and frequency of HEL-specific B cells is observed, suggesting that a portion of B cells may have been deleted (e.g., the IgM high cells) 2 . It is possible that soluble HEL may not reach sufficient concentrations in the BM milieu to generate a strong enough signal to induce RAG in most developing B cells. Upon exiting the BM, B cells would encounter concentrations of soluble HEL in the periphery sufficient to render them anergic. Considering that the soluble HEL Tg is under the control of a metallothionein promoter, highest secretion of soluble HEL would be expected in the liver, kidney, or pancreas 52 53 . Thus, it is conceivable that immature B cells would only encounter low concentrations of soluble HEL in the BM. In addition, it could be hypothesized that only a limited number of B cells (e.g., only IgM high B cells) receive a signal strong enough to induce receptor editing; these cells are then either deleted or successfully edited away from their specificity for HEL but are not seen in vivo because of the size of the anergic B cell population. Mice expressing Ig Tg receptors for another soluble but multivalent ligand, DNA, demonstrate that B cells are rendered anergic when recognizing single-stranded DNA (low affinity), whereas B cells binding double-stranded DNA (high affinity) are deleted 4 54 . This supports the hypothesis that antigen valency in combination with receptor affinity determines the mechanism of tolerance induction in immature B cells. Resolution of these and other alternatives awaits further study. The data presented also demonstrate a clear difference in sensitivity of the responses of mature and immature B cell to rises in intracellular calcium. Differences in these responses may be linked to the observed difference in tolerance sensitivity 5 6 7 , where low signals in immature B cells (induced by a low antigen concentration) result in clonal elimination, presumably by receptor editing. Similarly, the higher signal threshold for activation of mature B cells in the periphery protects against autoimmunity by preventing activation of mature B cells with low affinity for self. The question remains whether BCR-proximal signal transduction events differ between immature and mature B cells. It will be especially important to assess differences pertaining to the generation of Ca 2+ mobilization, as this leads to the activation of specific genes. The increased sensitivity to antigen imposed by these mechanisms in immature B cells is likely to play a very important role in repertoire development, purging the repertoire of cells with even low affinities for self-antigens. | Study | biomedical | en | 0.999997 |
10499914 | C57BL/6 (B6) mice and their Ly-5.2 congenic variant (B6.Ly-5.2) were purchased from the National Cancer Institute animal facility (Frederick, MD). Class II–deficient (CII − ) mice and class I/II–deficient mice on a B6 background (12th backcross, CI/II − ) were purchased from Taconic Farms. The B6 Thy-1 congenic variant strain, B6.PL–thy-1 a Cy, as well as the mice genetically deficient in the CD4 gene 18 , which were used as class II + recipients, and the natural H-2K b and H-2 I-A b coisogenic variants of B6 mice, B6.C–H-2 bm1 (bm1) and B6.C–H-2 bm12 (bm12), respectively, which were used as stimulators in the MLR, were purchased from The Jackson Laboratory. Female mice, between 7 and 10 wk of age, were used in all experiments. The allele-specific anti-Ly5.1 (104-2.1) and anti-Ly5.2 (A20-7.1) mAbs ( 19 ; obtained from Dr. U. Hammerling, Memorial Sloan-Kettering Cancer Center) were produced as ascitic fluid, purified, and conjugated to FITC or biotin in our laboratory. All other antibodies were purchased from PharMingen. Anti-CD4–conjugated microbeads, the VS+ and RS+ separation columns, and the SuperMacs ® separation device were purchased from Miltenyi Biotec and were used per the manufacturer's instructions. All other antibodies and reagents, as well as the cytotoxic elimination of CD8 hi/med cells by mAb + C′ and separation of the resulting population into CD8 lo and CD8 − by panning, were described previously 9 . CD4 hi populations from each subset were then purified to >99% purity, either by flow cytometry (FCM) using a Mo-Flo sorter (Cytomation), or by two sequential immunomagnetic bead sorting steps using the VS+ separation columns and anti-CD4–conjugated microbeads (Miltenyi Biotec), as indicated. For the cell sorting, cells were stained with mAbs against CD4, CD11c + , and MHC class II, and were sorted to obtain a CD4 hi class II − CD11c − fraction. Upon reanalysis, these cells contained <0.1% cells positive for class II, CD11b, or CD11c, and were used for Exp. 2 in Table Table Table . CD8 − CD4 hi cells were used in the intravenous transfer experiment to confirm the stringency of cell purification, and in the intrathymic “spiking” experiment aimed to test the role of contaminating cells; the CD8 lo CD4 hi cells were used for intravenous and intrathymic transfers. B6 mice were depleted of CD4 cells using two intraperitoneal injections of 100 μl (∼500 μg) mAb GK1.5 in the form of ascites, 2 d apart. Antibody-injected mice were then rested for 4 wk before they entered the experiment to allow elimination of the antibody. They were subsequently used as recipients for intrathymic transfers or were also thymectomized and used as recipients for the intravenous transfer. Genetically deficient mice (CD4 − , CII − , and CI/II − ) used as recipients were not pretreated before intrathymic transfer. Intrathymic injections were performed as described 20 21 , with 0.85–2 × 10 6 thymocyte subsets of the B6.Ly-5.2 and/or B6.PL origin injected into each thymic lobe. The thymectomies were performed using the same surgical approach as for the intrathymic injections, except that vacuum suction was applied to remove the organ. Killed mice were dissected to verify the removal of the thymus. 4–5 mo after intravenous or intrathymic transfer, spleens and mesenteric lymph nodes (mLNs) from the recipient mice were used to analyze the phenotype and function of CD4 T cells. Phenotypic analysis was performed by FCM, using anti-CD4 and donor- and recipient-specific Ly-5 mAbs, using a FACScan™ instrument (Becton Dickinson) equipped with the Lysys II ® software, as described previously 9 . For functional analysis, donor-derived CD4 + cells were magnetically purified from the pooled spleens of the recipients, using FITC-labeled donor allele–specific mAbs, followed by the anti-FITC–conjugated paramagnetic microbeads and the RS+ separation columns (Miltenyi Biotec). 10 5 cells/well were seeded in a flat-bottomed 96-well plate, alone or with 2 × 10 5 irradiated (30 Gy) nucleated spleen cells of bm1, bm12, or B6 origin. The assays were done in quadruplicate. Culture supernatants were collected 72 h later and frozen for subsequent IL-2 quantification by using the IL-2–dependent CTLL indicator line. 5 × 10 3 CTLL cells were incubated in a 1:1 dilution of the supernatants for 18 h, the last 6 h in the presence of 1 μCi of 3 H-TdR (New England Nuclear). The cells were then harvested, and the degree of incorporation of radioactive thymidine was determined in a beta-counter (Amersham Pharmacia Biotech). To investigate the role of MHC class II molecules in the final maturation of incompletely mature CD8 lo CD4 hi thymocytes, these cells were intrathymically transferred into recipients genetically deficient in MHC class II molecules. To that effect, CD8 lo CD4 hi and CD8 − CD4 hi thymocytes were purified (>99%) from B6.Ly-5.2 mice, as described in Materials and Methods. The key separation step was achieved by panning, and, in our hands, this was found to be the most reliable method of separating these two functionally nonoverlapping subsets. By FCM, the phenotype of these two subsets was characteristic of that described previously : both subsets were CD8 − CD4 hi TCR-αβ hi by FCM, but the CD8 lo CD4 hi cells contained more CD69 + cells and expressed higher levels of CD24 and somewhat lower levels of CD44 than CD8 − CD4 hi thymocytes (not shown). However, as shown previously 9 , most of these molecules are expressed by both subsets in at least partly overlapping fashion. We observed that the most consistent difference in the phenotype of these two subsets was in the level of expression of CD45RB: the levels observed on CD8 − CD4 hi cells were more than twice higher than those on CD8 lo CD4 hi thymocytes , similar to the findings of Penit's group 15 . This feature correlates very well with the difference in functional responsiveness of the two subsets and the function of CD45: the former subset is functionally mature, whereas the latter cannot respond with the full spectrum of functional activities 12 . To further confirm the identity of the above purified cell populations, both subsets were transferred intravenously into thymectomized and CD4-depleted syngeneic recipients. Under such circumstances, only the CD8 − CD4 hi thymocytes can give long-term (>1 mo) repopulation of the peripheral lymphoid organs, whereas the CD8 lo CD4 hi cells disappear 3–4 wk after transfer 9 . Results shown in Table confirmed this finding, and demonstrated that the CD8 lo CD4 hi thymocyte subset isolated in this study was phenotypically and functionally indistinguishable from the previously described cells 9 12 . Moreover, results of this experiment demonstrate that the cross-contamination and outgrowth of the contaminating cells between the two subsets isolated as described in Materials and Methods was negligible or nonexistent, as the putative progeny of the cross-contaminating CD8 − CD4 hi cells was not detectable 4–5 mo after injection of purified CD8 lo CD4 hi cells. CD8 lo CD4 hi thymocytes from the same cellular preparation were also injected into the thymi of control class II + CD4 −/− recipients, which express wild-type levels of the MHC class II molecules (but lack CD4 + T cells , thus facilitating the detection of CD4 + progeny of the injected cells), or of the CII − recipients, which genetically lacked these molecules (and consequently only had very low CD4 + numbers, owing to a severe defect in CD4 T cell positive selection ). In both types of recipient, injected immature CD8 lo CD4 hi thymocytes yielded long-lived progeny that populated the peripheral lymphoid organs ( Table , Exp. 1). Therefore, class II molecules appeared not to be necessary either for the terminal differentiation of CD4 hi thymocytes or for their survival in the periphery. Again, a caveat to this conclusion was the long time course of our experiment, which was conducive for the outgrowth of a minor population of contaminating CD8 − CD4 hi thymocytes. Although the results shown in Table strongly argue against this possibility, we performed another control experiment in which we deliberately spiked the transferred CD8 lo CD4 hi cells with Thy-1 congenic (Thy-1.1) CD8 − CD4 hi thymocytes. When we added 1% CD8 − CD4 hi thymocytes to the sorted >99% pure CD8 lo CD4 hi thymocytes, we could not detect the progeny of the admixed Thy-1.1 cells ( Table ). The progeny of 10% contaminating cells was detectable, but these cells showed no selective proliferative advantage: their representation remained at or below the 10% level among the transferred thymocytes even after 5 mo in vivo ( Table ). Therefore, the above results cannot be accounted for by the contaminating mature thymocytes. Another possibility was that we transferred a substantial number of class II + cells with our thymocytes. However, cells transferred in Exp. 2 ( Table ) were sorted to exclude class II + and CD11c + cells, and, after sorting, contained no cells positive for class II, for the dendritic cell (DC) marker CD11c, or the macrophage (Mφ) marker CD11b (<0.1% in Exp. 2, Table [see legend]). Coupled with the fact that the turnover of class II + cells (DCs, B cells, and Mφ) is relatively rapid and certainly complete within 1 mo, it is extremely unlikely that those cells could have promoted the final maturation of CD4 thymocytes in the thymus and/or the periphery. A possibility also existed that MHC class I molecules may provide critical signals to keep CD4 + T cells alive in the peripheral organs. To test this possibility, we used CI/II − mice as recipients for thymocytes. Interestingly, upon such transfer, we recovered more CD4 T cells than when recipients only lacked class II molecules ( Table ), similar to the results of a recently published study 22 . It is possible that the complete lack of T cells, observed in CI/II − mice 17 , may be conducive to an even more extensive expansion than the one occurring in CII − mice. Indeed, from the spleen only we recovered amounts of CD4 cells that were tenfold higher than those injected intrathymically. However, one caveat of this experiment is that the injected CD4 cells themselves expressed class I, as they were derived from the class I + mice (this was the only way to unambiguously ascertain the donor origin of these cells); therefore, their contact with each other could have provided the survival signal. However, at a minimum, we can still reliably conclude that the presence of the MHC molecules on stromal cells or on APCs is not necessary for either the terminal CD4 thymocyte differentiation or the survival of at least some CD4 T cells in the periphery. To test whether the peripheral CD4 progeny of the CD8 lo CD4 hi thymocytes was functional in the absence of MHC molecules, we isolated >99% pure CD4 + Ly-5.2 + donor-derived cells from the recipient mice, stimulated them with allogeneic cells in vitro, and measured the IL-2 release in response to such stimulation ( Table ). CD4 cells of donor origin produced IL-2 at levels comparable to those produced by unmanipulated peripheral CD4 cells, establishing that these cells were functional and immunocompetent. The fact that donor-derived CD4 + Ly5-2 + cells responded vigorously to MHC class II–disparate I-A bm12 stimulators, but not to the class I–disparate K bm1 stimulators ( Table , Exp. 2) clearly demonstrated that their precursors must have been appropriately positively selected on MHC class II molecules before the point of transfer. Similar results were obtained with the donor-derived CD4 T cells recovered from the CI/II − recipients (data not shown). The primary objective of this study was to investigate the dependence of the final phase of intrathymic maturation on MHC class II molecules. Several reports indicated that the TCR–MHC contact must be maintained throughout several discrete stages in T cell development, but none of these has addressed the importance of this contact for terminal maturation of SP cells. As pointed out in our previous publications 9 12 , the CD8 lo CD4 hi cells in our studies differ significantly from the cells called CD8 lo CD4 + 23 24 25 : theirs correspond to the CD8 int CD4 hi cells, which bear levels of CD8 lower than those on double-positive (DP) thymocytes but clearly detectable by FCM. Many of these latter cells actually belong to the CD8 lineage. The CD8 lo CD4 hi cells studied here belong to the CD4 lineage and score within the CD4 SP population by FCM: their CD8 levels are undetectable by FCM (for a full phenotypic difference between our CD8 lo CD4 hi cells and the CD8 lo CD4 + cells of the other authors, see reference 9). Our data resolve the class II dependence of the last phase of intrathymic maturation of SP CD4 thymocytes unambiguously: no MHC class II–TCR contact is required for this late differentiation. This conclusion is further supported by the recent data of Hare et al. in the reaggregation organ culture system 26 , showing that thymocytes at or past the CD69 + DP stage no longer require contact with class II molecules to become functional CD4 SP thymocytes. At present, we can only speculate on the nature of the final maturation signal. Among the obvious contenders are the cytokines, the costimulatory molecules, and/or the matrix–integrin interactions, some of which are currently being investigated. Survival and peripheral homeostasis of T cells were not the key subjects of this study. However, our results beg a discussion of these issues. Perhaps surprisingly, the peripheral progeny of intrathymically transferred CD8 lo CD4 hi cells was able to survive for >5 mo in the absence of MHC class II molecules (albeit the numbers were ∼50–60% of those in class II + mice 5 mo after the transfer). This survival was not due to the outgrowth of contaminating mature CD8 − CD4 hi thymocytes ( Table ) nor to the contaminating class II + cells (which were undetectable in the inoculum). We conclude that MHC class II molecules are not necessary for the long-term survival of at least some CD4 T cells, but are required for their optimal expansion and homeostasis, since both the percentages and the absolute numbers of CD4 + T cells were about twofold higher in the presence of class II molecules. The issue of whether MHC molecules are required and necessary for the survival of peripheral T cells has recently gained much attention. Although it appears that MHC class I molecules are necessary for the survival of CD8 + cells 27 28 29 30 , the consensus is more tenuous in the case of CD4 + cells 22 31 32 33 . Takeda et al. 31 using the transfer of fetal thymic lobes into CII − mice, concluded that class II molecules are not essential for survival but affect the half-life of CD4 cells. Rooke et al. 22 reported on a model of retroviral reconstitution of class II expression in CII − mice, and found that the half-life in reconstituted mice that expressed no class II in the periphery was similar to that of Takeda et al. 31 . These authors concluded that the lack of class II molecules curtailed (but did not abolish) the survival of CD4 + cells, and showed that the lack of class I molecules did not further pronounce this curtailment. In these experiments, the uneven expression of intrathymically injected and retrovirus-driven class II molecules could have caused a situation in which class II molecules were not expressed in all the intrathymic compartments relevant to the T cell longevity. This could have resulted in a paucity of intrathymic signals that regulate the half-life of T cells, thus reducing the numbers of long-lived cells or their half-life. By contrast, the study of Brocker 32 argued for an absolutely essential role of class II on peripheral APCs in the survival of CD4 T cells, based on the results of transplantation of the APC-depleted class II + thymi into CII − mice bearing no transgenes or expressing an MHC class II transgene on DCs alone. In these experiments, good repopulation and intrathymic differentiation of CD4 T cells were observed, but no extrathymic CD4 T cells were detected unless the DCs expressed the class II transgene. Brocker postulated that class II molecules are essential for survival of CD4 T cells. Kirberg et al. 33 showed that CD4 T cells bearing a TCR transgene required self class II molecules for long-term survival in the periphery. Finally, Viret et al. showed that the CD4 T cell repertoire is incompletely maintained when a single peptide is present on the majority of MHC class II molecules 34 . Our data certainly agree with all of the above studies in that class II molecules are required for optimal CD4 + expansion and the filling up of the peripheral compartment. But our results, together with those of Takeda et al. 31 , also clearly show that a substantial proportion of peripheral CD4 T cells do not require MHC class II molecules. Results of Viret et al. 34 are also compatible with this notion, since these authors saw a reduction, but not disappearance, of CD4 T cells in mice unable to exchange the class II–associated invariant chain peptides for other peptides, owing to a lack of H-2Mα. However, the period of observation in these experiments was rather short. In distinction to the studies of Kirberg et al. 33 , we used polyclonal T cells, and it is quite possible that only some cells from the original inoculum have survived, reflecting the requirement of some, but not all, cells for the particular MHC class II ligand. It is possible that in our system, we have selected for only those cells that do not require class II for peripheral survival, and we are testing this possibility at present. We are also investigating whether these cells express a diverse repertoire, as suggested by their vigorous response to alloantigens ( Table ). At face value, our data appear contrary to those of Brocker 33 . However, these differences can be reconciled if class II molecules, expressed on hematopoietic cells rather than being essential for CD4 T cell survival, are necessary for the export of selected CD4 cells from the thymus. If this is the case, both studies would have to be reinterpreted slightly. In our study, then, the transferred CD8 lo CD4 hi cells either would already have received class II–mediated signals before transfer, or would have been contaminated with sufficient numbers of class II + cells to provide this signal after transfer. We believe that the latter possibility is highly unlikely, for two reasons. First, the magnetic bead separation retains most DCs and Mφ on the columns nonspecifically, and since the largest theoretical contamination of the inoculum with CD4 − cells was in the range of 600–800 cells , even assuming all of these cells were MHC class II + , this number appears too small to permit substantial proliferation and export of maturing thymocytes. Second, the presence of class II + , CD11c + , or CD11b + cells was undetectable among FCM-sorted inoculated thymocytes (see legend to Table ). In the experiments of Brocker 32 , the class II + DCs would provide their essential export signal intrathymically (indeed, DCs are well known to reside in the thymus ), and would also help the CD4 expansion in the periphery. The first function would be absolutely critical, and its lack would mean that no CD4 T cells could be detected in CII − mice grafted with class II + epithelium. Finally, with regard to CD4 T cell homeostasis, our study demonstrated that the proliferation of the injected thymocytes can occur in the absence of class II molecules, as well as in the absence of class I + APCs. Interestingly, this proliferation was particularly pronounced in CI/II − mice, paralleling the findings of Rooke et al. 22 . At present, it is unclear whether the SP thymocytes in our study proliferated intrathymically 10 11 or whether the proliferation occurred in the periphery, and additional studies will be needed to address this issue. | Study | biomedical | en | 0.999998 |
10499915 | A KLH-specific and I-A k –restricted murine Th1-type T cell clone, 23-1-8 19 20 , was maintained in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME, and 5 ng/ml murine IL-2. These T cells were stimulated with KLH (100 μg/ml; Calbiochem Corp.) and irradiated C3H/HeJ spleen cells every 3–4 wk. Retrovirus packaging cell line BOSC23 was provided by Dr. T. Kitamura (The University of Tokyo, Tokyo, Japan) and cultured in DMEM supplemented with 10% FCS, 100 μg/ml l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 μg/ml xanthine, 15 μg/ml hypoxanthine, 6 μg/ml mycophenolic acid, and 50 μM 2-ME. Anti–CTLA-4 mAb (UC10.4F10.11) and anti-CD3∈ mAb (145-2C 11 ) were provided by Dr. J. Bluestone (University of Chicago, Chicago, IL). Anti-CD3ζ mAb (H146-968) was provided by Dr. R. Kubo (Cytel Corp., San Diego, CA). Stimulatory anti-CD28 mAb (PV-1) was previously described 21 . Retrovirus vector pMXneo 22 was provided by Dr. T. Kitamura and used for stable transfection of WT CTLA-4 and its mutants into T cell clone 23-1-8. To construct the retrovirus vector carrying cDNA for WT CTLA-4, the full length cDNA (a gift from Dr. P. Golstein, INSERM-CNRS, Marseille, France) was subcloned into the EcoRI site of pMXneo. DNA fragments carrying point mutations and deletions of the cytoplasmic region of CTLA-4, Y165G ( 165 Tyr-Gly), Y182G ( 182 Tyr-Gly), Y165/182G ( 165 Tyr-Gly and 182 Tyr-Gly), and ΔCP7 (deletion of the cytoplasmic region except for the membrane-proximal 7 amino acids) were generated by PCR and also introduced into the same vector. These retroviral constructs were transiently transfected into the packaging cell line BOSC23 by lipofection using LipofectAMINE (GIBCO BRL). 24 h later, the T cells prestimulated with KLH and irradiated C3H/HeJ spleen cells were added to the culture and cocultivated for 24 h with the virus-producing BOSC23 cells. Thereafter, T cells were harvested and subjected to G418 (Geneticin; GIBCO BRL) selection at 1 mg/ml. T cells were incubated with biotinylated anti–CTLA-4 mAb for 30 min at 4°C, followed by PE–streptavidin. Flow cytometry was performed using a FACScan™ flow cytometer (Becton Dickinson), and 10 4 cells were analyzed using the CELLQuest™ program (Becton Dickinson). T cells (10 7 ) were precultured in a methionine-free medium for 30 min, metabolically labeled with 18.5 mBq/ml [ 35 S]methionine (Tran 35 S-Label™, ICN Pharmaceuticals, Inc.) for 3 h, harvested, washed three times in PBS, and then lysed in 1% NP-40 lysis buffer (1% NP-40, 50 mM Tris, 150 mM NaCl, 5 mM EDTA, 10 μg/ml aprotinin, 12.5 μg/ml antipain, 12.5 μg/ml chymostatin, 50 μg/ml leupeptin, 25 μg/ml pepstatin A, 1 mM PMSF, and 2 mM Na 3 VO 4 ). The precleared lysates were immunoprecipitated with either anti–CTLA-4 or anti-CD3∈ mAb as control. Where indicated, immunoprecipitates were treated with N -glycosidase F to remove N-linked oligosaccharide chains. Immunoprecipitated samples were resuspended with 20 μl of 0.1 M 2-ME/0.5% SDS, denatured by boiling, resuspended in 60 μl of a buffer containing 1.5 mU N -glycosidase F (Calbiochem Corp.), 167 mM Tris-Cl, pH 8.0, 13 mM 1,10-phenanthrorine, and 1.3% NP-40, incubated overnight at 37°C, and analyzed on 13% SDS-PAGE. The dried gels were analyzed with a BAS-2000 image analyzer (Fuji Photo Film Co.). Cell surface biotinylation was performed as previously described 23 . Cells were solubilized in 0.5% NP-40 lysis buffer. Immunoprecipitates were treated with N -glycosidase F as described above and then analyzed on 14% SDS-PAGE. For antigen stimulation, 10 5 23-1-8 T cells were cultured with 5 × 10 5 irradiated C3H/HeJ spleen cells and 10 μg/ml KLH in a 96-well flat-bottom plate for 48 h at 37°C in 5% CO 2 . For stimulation by FcR-dependent cross-linking with anti-CD3∈ and anti-CD28 mAbs, 2 × 10 5 T cells were cultured with 10 5 irradiated C3H/HeJ spleen cells in the presence of 0.1 μg/ml anti-CD3∈ alone or in combination with 1 μg/ml anti-CD28 mAb. In both stimulations, anti–CTLA-4 mAb (or control anti-CD3ζ mAb) was added at 50 μg/ml. For the last 8 h of incubation, 37 kBq [ 3 H]TdR (Amersham Life Sciences) was pulsed and the uptake was measured with a liquid scintillation counter (MicroBeta™; Pharmacia). Flat-bottom 96-well plates were sequentially coated with 0.1 μg/ml of anti-CD3∈ mAb and then with anti–CTLA-4 mAb and/or control anti-CD3ζ mAb to keep a constant sum concentration of mAbs of 10 μg/ml. T cells were added at 10 5 /well in 200 μl of complete RPMI 1640 in the presence or absence of 1 μg/ml anti-CD28 mAb. Supernatants from triplicate cultures were collected, and the titer of IL-2 was determined by ELISA with rIL-2 as a standard as described previously 24 . The Ab to detect IL-2 was purchased from PharMingen. Total RNA was extracted from stimulated or unstimulated T cells by the AGPC (acid guanidium-phenol-chloroform) method. Reverse transcriptase (RT) reaction was carried out using random hexamer primers (Takara Biomedicals) and Superscript II (GIBCO BRL). Twofold dilutions of the RT product were amplified with the specific primers for IL-2, IFN-γ, Bcl-X L , and β-actin to ensure that the PCR reactions were performed in a linear range. T cells were stimulated with 0.5 μg/ml immobilized anti-CD3∈ mAb and 10 μg/ml soluble anti-CD28 mAb and lysed for 30 min at 4°C with a lysis buffer (20 mM Hepes, pH 7.4, 300 mM NaCl, 2 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, 20 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 1 mM PMSF, 10 μg/ml aprotinin, 12.5 μg/ml antipain, 12.5 μg/ml chymostatin, 50 μg/ml leupeptin, and 25 μg/ml pepstatin A). Protein concentration in the lysate was determined using the Bradford method (Bio-Rad Labs.), and 50 μg of total protein was subjected to immunoprecipitation with polyclonal rabbit anti–extracellular signal-regulated kinase 2 (ERK2) Ab (Santa Cruz Biotechnology, Inc.). After the immunoprecipitates were washed with a kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl 2 , and 1 mM dithiothreitol), kinase reactions were carried out at 30°C in 30 μl of the kinase buffer containing 20 μM ATP, 5 μCi of γ-[ 32 P]ATP, and 3 μg of myelin basic protein (MBP; Sigma Chemical Co.) as a substrate and stopped after 20 min. Samples were resolved on 15% SDS-PAGE, and phosphorylation of MBP was visualized and the radioactivity of each band was quantified using a BAS-2000 image analyzer (Fuji Photo Film Co.). ERK2 immunoblots were performed as previously described 25 . 40 μg of whole cell extracts was analyzed on 10% SDS-PAGE and transferred onto a polyvinyl difluoride membrane (Immobilon-P; Millipore Corp.). The membranes were blotted with murine monoclonal anti-ERK2 (Santa Cruz Biotechnology, Inc.), followed by a peroxidase-conjugated anti–murine IgG. Blots were developed using the enhanced chemiluminescence system (ECL kit; Amersham Life Sciences). Although it has been shown that Y-165 of CTLA-4 is crucial for association with AP-2 complex and endocytosis from the cell surface in a model system using nonlymphoid cells 12 13 14 15 , the regulation of the cell surface expression of CTLA-4 in normal T cells has not been analyzed. Therefore, we examined which of the two tyrosines in the cytoplasmic tail of murine CTLA-4 (Y-165, Y-182, or both) are important for the regulation of the cell surface expression and inhibitory signals of CTLA-4 in normal T cells. For this purpose, we generated various forms of mutant CTLA-4 with a single tyrosine→glycine mutation (Y165G and Y182G), double mutations (Y165/182G), and a deletion of most of the cytoplasmic tail except for the membrane-proximal 7 amino acids (ΔCP7) . These constructs were transfected into a murine T cell clone, 23-1-8, by retrovirus-mediated gene transfer, and the G418-resistant bulk population of transfected T cells was analyzed for the surface expression of CTLA-4 . CTLA-4 was hardly detected on the surfaces of resting T cells transfected with the vector alone (CT T cells). T cells transfected with the WT CTLA-4 (WT T cells) showed only a dull expression of CTLA-4 on the cell surface, as did T cells transfected with the Y182G mutant CTLA-4 (Y182G T cells). In contrast, the mutation of 165 Tyr→Gly (Y165G), which blocks the association with AP-2, induced constitutively high expression of CTLA-4 on the surfaces of resting T cells. Interestingly, Y165/182G mutants exhibited a higher expression of surface CTLA-4 than Y165G mutants, and ΔCP7 mutants showed even greater expressions than Y165/182G mutants. To test whether the significant differences in the surface expression of CTLA-4 can be attributed to its transcriptional level in these T cells, we performed Northern blot analysis using a CTLA-4 cDNA probe. The expression level of CTLA-4 mRNA of these WT and mutants of CTLA-4 T cells at resting stage varied within a twofold difference . We also examined the protein level of CTLA-4 in T cells expressing these mutants and WT CTLA-4. T cells were metabolically labeled with [ 35 S]methionine, and the cell lysates were immunoprecipitated with either anti–CTLA-4 mAb or anti-CD3∈ mAb as control. As CTLA-4 is heavily glycosylated, simple immunoprecipitation of the labeled CTLA-4 showed very broad bands. Therefore, all precipitates were treated with N -glycosidase F to remove all N-linked oligosaccharide chains and then the core proteins were analyzed. As shown in Fig. 1 D, CTLA-4 protein was not precipitated in control cells. WT and all mutant CTLA-4 cells (Y165G, Y182G, Y165/182G, and ΔCP7) showed fairly similar amounts of CTLA-4 proteins. These data demonstrate that the difference in the surface expression of CTLA-4 was not due to the different expression level of mRNA nor protein of CTLA-4 but rather due to translocation of CTLA-4 protein from the cytoplasm to plasma membrane by the mutation or deletion of the tyrosine motif in the cytoplasmic tail. Preliminary analysis with confocal microscopy disclosed that CTLA-4 protein was located predominantly in the cytoplasmic granules in resting WT T cells, whereas the major portion was localized on the cell surface in Y165G or ΔCP7 T cells (Iida, T., C. Nakaseko, H. Ohno, and T. Saito, manuscript in preparation). The finding that Y165/182G T cells and ΔCP7 T cells exhibited higher expressions of surface CTLA-4 than Y165G T cells indicates that Y-165 is basically the tyrosine required for regulation of the surface expression of CTLA-4 through endocytosis mediated by the clathrin–AP-2 complex, but Y-182 or other regions of the cytoplasmic tail of CTLA-4 are also involved in the regulation of the surface expression of the molecule. Previous reports demonstrated that both intact and monovalent fragments of CTLA-4 augmented T cell proliferation in allogeneic MLR, whereas the same Ab inhibited proliferation under the condition where FcR cross-linking was provided 6 7 . To investigate the region in the cytoplasmic tail of CTLA-4 responsible for delivering negative signals, we performed functional analyses using these T cells transfected with various mutant forms of CTLA-4. First, we examined the effect of CTLA-4 mutants in terms of Ag-specific proliferative response. Because 23-1-8 is a KLH-specific T cell clone, transfectants expressing various CTLA-4 mutants were stimulated with KLH and APCs in the presence or absence of anti–CTLA-4 mAb. As shown in Fig. 2 A, the proliferative response of CT T cells was not altered by the addition of anti–CTLA-4 mAb. However, proliferation of WT T cells was suppressed in comparison with CT T cells. The addition of anti–CTLA-4 mAb restored the response to a level almost equal to that in control cells. In T cells with high expression of surface CTLA-4, such as Y165G, Y165/182G, and ΔCP7 T cells, the suppression was more significant than in WT T cells, in accordance with their surface expression. For these cells, the addition of anti–CTLA-4 mAb induced dramatic restoration of the Ag-specific response. These results suggest that mutant forms of CTLA-4, with either the tyrosine mutation or deletion of the cytoplasmic region, could deliver a negative signal for Ag-specific proliferation. Alternatively, these molecules might inhibit the proliferation by merely competing for ligand binding with CD28. To examine the effect of cross-linking various forms of CTLA-4 with anti–CTLA-4 mAb, T cells were stimulated with a suboptimal dose of anti-CD3∈ mAb and optimal costimulation with anti-CD28 mAb by cross-linking on FcR-bearing syngeneic APCs. In this system, suboptimal stimulation with anti-CD3∈ mAb resulted in a minimum costimulation effect by CD80/CD86 on APCs, and the addition of stimulatory anti-CD28 mAb greatly augmented T cell proliferation . Under these conditions, the cross-linking of CTLA-4 mutants with Y165G, Y165/182G, and ΔCP7 by anti–CTLA-4 mAb resulted in significant inhibition of CD3/CD28-mediated proliferation. Together with the results in Fig. 2 A, these data strongly suggest that these mutant forms of CTLA-4 as well as WT CTLA-4 could transduce negative signals for proliferative response upon engagement of TCR and CD28. For detailed analysis of the suppressive mechanism via CTLA-4, we used another system in which T cells were stimulated by cross-linking of CD3, CD28, and CTLA-4 separately with the respective specific Abs in the absence of APCs and examined the early response by measuring IL-2 secretion. This system made it possible to avoid the effect of interaction between CTLA-4 and/or CD28 and CD80 and/or CD86 on APCs and to analyze the molecular events of CTLA-4 cross-linking both in the early and late phases after T cell activation. To this end, T cells expressing WT CTLA-4, Y165G, ΔCP7, and control T cells were stimulated by cross-linking with a suboptimal dose of immobilized anti-CD3∈ mAb in the presence or absence of soluble stimulatory anti-CD28 mAb, and the negative effect via CTLA-4 in IL-2 production was examined by cross-linking with immobilized anti–CTLA-4 mAb. Under this condition, the costimulatory effect of anti-CD28 mAb was optimal for IL-2 production . In CT T cells, IL-2 secretion was inhibited by endogenous CTLA-4 by at most 60–70% of the response upon CTLA-4 cross-linking . In WT T cells, CTLA-4 cross-linking resulted in significant inhibition of IL-2 production in a dose-dependent manner . In ΔCP7 as well as Y165G T cells, consistent with the results of the proliferative response shown in Fig. 2 , cross-linking of these CTLA-4 mutants induced stronger inhibition of IL-2 production than was seen in WT T cells . As ΔCP7 T cells express extremely high levels of mutant CTLA-4 on their cell surfaces (severalfold higher than those expressed by Y165G T cells), we intended to analyze the function of ΔCP7 T cells with relatively low expression of the mutant CTLA-4 on the cell surface. For this purpose, we isolated low-expressing cells (ΔCP7-low) by cell sorting. The cell surface expression of ΔCP7 on these cells was much lower than that of Y165G T cells, though it was still higher than that of WT T cells. However, when mRNA and protein of the mutant CTLA-4 in ΔCP7-low T cells was analyzed, the expression of both mRNA and protein was found to be extremely low . Analysis of the function of such ΔCP7-low T cells revealed that these cells with much lower expression level of ΔCP7 strongly inhibited IL-2 production upon CTLA-4 cross-linking . These results demonstrated that the engagement of not only tyrosine mutants but also the cytoplasmic tail deletion mutant of CTLA-4 could inhibit IL-2 secretion as an early T cell response as well as proliferation as a late response. Specific suppression of IL-2 secretion was demonstrated by analyzing mRNA expression using a semiquantitative RT-PCR analysis . mRNAs were isolated from various T cells after stimulation by cross-linking of CD3 and CD28 for 4 h. Similar to IL-2 secretion, ligation with anti–CTLA-4 mAb induced strong inhibition of IL-2 mRNA expression in WT, Y165G, and ΔCP7 T cells but not in CT T cells. This inhibitory effect was not observed for the expression of IFN-γ or Bcl-X L . The expression of the latter is known to be induced upon TCR stimulation in the presence of CD28 costimulation 26 27 28 . These data support the notion that the high expression of cell surface CTLA-4 did not physically block TCR and CD28 stimulation and that the engagement of these mutant CTLA-4, which induced high expression on the cell surface, actively inhibited IL-2 mRNA production as early as 4 h after T cell activation. These results also demonstrate that CTLA-4 does not merely inhibit the signal through CD28, because the induction of CD28-dependent molecules such as Bcl-X L were not affected by CTLA-4 cross-linking. As ERKs are involved in the induction of IL-2 gene transcription and it has been shown that CTLA-4 engagement downregulates ERK activity in preactivated T cells 25 , we investigated whether mutant CTLA-4 such as ΔCP7 CTLA-4 has an inhibitory effect on ERK2 upon activation of resting T cells by immune complex kinase assay. Under the stimulation condition with suboptimal anti-CD3 and optimal anti-CD28 mAbs as described above, the ERK2 activity was so weakly upregulated that the effect of CTLA-4 cross-linking was hardly observed. Therefore, T cells were stimulated by optimal CD3/CD28 stimulation, and the effect of CTLA-4 cross-linking was examined. As shown in Fig. 5 A, ERK2 activity was significantly upregulated by the optimal CD3/CD28 costimulation within 20 min. The cross-linking of CTLA-4 significantly inhibited ERK2 activity to about half the level of the control in WT, Y165G, and ΔCP7 T cells but not in CT T cells. These data demonstrated that WT and tailless CTLA-4 might inhibit ERK2 activity during early activation of resting T cells when expressed on the cell surface. The inhibition of IL-2 production in the transfectants expressing mutant CTLA-4, such as Y165G or ΔCP7, is likely to be mediated through the expressed mutant CTLA-4. However, the possibility still remains that this suppression could be mediated through the endogenous CTLA-4 accumulated on the cell surface by forming heterodimers with the mutant CTLA-4. To examine this possibility, T cells were activated with immobilized anti-CD3 and anti-CD28 mAbs for 48 h, surface-biotinylated, and immunoprecipitated with anti–CTLA-4 mAb. We analyzed the surface expression of CTLA-4 on CT, WT, Y165G, and ΔCP7 T cells before and after T cell activation . Whereas CT T cells did not induce the surface expression of CTLA-4, WT T cells slightly enhanced the expression upon activation. The enhancement of the surface expression of CTLA-4 upon stimulation was also observed in Y165G and ΔCP7 mutant CTLA-4 transfectants. Because CTLA-4 is heavily glycosylated , it migrates broadly on SDS-PAGE, and we can hardly distinguish WT from mutant CTLA-4. Therefore, we treated the immunoprecipitates similarly to those in Fig. 1 D. In CT T cells, whereas CTLA-4 on the cell surface was hardly detected without N -glycosidase F treatment, a small amount of endogenous CTLA-4 of 18 kD was observed by immunoprecipitation with anti–CTLA-4 mAb after the treatment . In WT T cells, WT CTLA-4 was clearly detected by the treatment, and the amount of WT CTLA-4 was almost 10-fold greater than that on CT T cells . In ΔCP7 T cells, although the mutant CTLA-4 on the cell surface was detected as a strong band of 15 kD, the endogenous CTLA-4 of 18 kD was hardly detected . These data demonstrate that the enhancement of the surface expression of CTLA-4 on ΔCP7 T cells was not caused by accumulation of the endogenous CTLA-4 but rather by the upregulation of the ΔCP7 mutant upon activation. Considering that proliferation and IL-2 production were strongly inhibited in ΔCP7 but not in WT T cells, these data demonstrate that CTLA-4 with tyrosine mutation or tailless mutant did transduce negative signals through the mutant CTLA-4 molecules themselves rather than by forming heterodimers with endogenous CTLA-4. Recently, we and other groups demonstrated that endocytosis of CTLA-4 is induced by association of its cytoplasmic tail with the μ2 subunit of clathrin-associated adaptor complex AP-2 12 13 14 15 . The tyrosine-based motif containing 165 YVKM within the cytoplasmic tail of CTLA-4 is responsible for the binding to μ2. However, there has been no data so far that demonstrated the same regulation in normal T cells. In this study, we demonstrated that the surface expression of CTLA-4 on WT CTLA-4–transfected T cells was dull at the resting stage and that CTLA-4 was predominantly localized in the cytoplasm rather than on the cell surface. Whereas the expression of CTLA-4 on T cells transfected with the Y182G mutant was similar to WT CTLA-4 T cells, Y165G mutants exhibited a constitutively high expression of the cell surface CTLA-4, suggesting that Y-165 is critical for the regulation of surface expression of CTLA-4 in normal T cells. In addition, we found that the surface expression of Y165/182G and ΔCP7 mutant CTLA-4 at the resting stage was higher than that of the Y165G mutant. Furthermore, the surface expression of these mutants as well as WT CTLA-4 was enhanced upon activation. These data strongly suggest that Y-182 and/or other regions of the cytoplasmic tail are also involved in the regulation of the surface expression of CTLA-4. The mechanism of transport of CTLA-4 from intracellular endosomal pool to the plasma membrane might contribute to this regulation along with clathrin-associated endocytic machinery. Although functional analyses of CTLA-4 engagement have been extensively performed in vivo and in vitro, very little is known about the signaling events resulting from ligation of CTLA-4. PI3 kinase has been shown to associate with the cytoplasmic tail of CTLA-4 upon receptor ligation 17 . The same kinase also binds to the cytoplasmic tail of CD28 29 30 , and then the suppressive effect of CTLA-4 can only be explained by sequestering PI3 kinase by the high expression of CTLA-4. However, the fact that the expression level of CD28 is much higher than that of CTLA-4 suggests that this possibility is unlikely. It has also been suggested that the role of PI3 kinase binding to CTLA-4 may regulate the transport of molecules from intracellular vesicles to the cell surface 4 5 29 . Other investigators have reported that phosphatase SHP-2 associates with the same tyrosine of CTLA-4 upon activation and can be coimmunoprecipitated with phosphorylated CTLA-4 peptide 15 16 . Such CTLA-4 immunoprecipitates could dephosphorylate p52 Shc in vitro. Furthermore, it has been shown that the kinases Fyn, Lck, and ZAP-70 were activated and other proteins, including CD3ζ and p52 Shc, were constitutively hyperphosphorylated in T cells from CTLA-4–deficient mice. However, these findings might be attributed to the hyperactive status of T cells in CTLA-4–deficient mice rather than being a direct consequence of the lack of CTLA-4 9 10 . Frearson et al. reported that SHP-2 plays a critical role in connecting TCR to the Ras/MAPK (mitogen-activated protein kinase) pathway in TCR stimulation of Jurkat T cells 31 , in which the expression of mutant SHP-2 significantly inhibited TCR-induced activation of ERK2 but had no effect on CD3ζ tyrosine phosphorylation or TCR-elicited Ca 2+ mobilization. Taken together with the observation in signaling through several receptor tyrosine kinases that SHP-2 has been demonstrated to stimulate rather than inhibit growth factor–induced Ras/MAPK activation 32 33 , these reports suggest that SHP-2 plays a positive role in TCR stimulation rather than serving as a negative regulator of CTLA-4 engagement. In this study, we identified a new mechanism of negative signal transduction by CTLA-4. We analyzed CTLA-4–transfected T cells by cross-linking CD3, CD28, and CTLA-4 separately with specific Abs to avoid the complexity derived from the T cell–APC interaction. One of the mechanisms of suppression by CTLA-4 has been thought to be the competition of the ligand CD80/86 with CD28 34 35 . In our system, we proved that ligand-independent cross-linking of CTLA-4 induces suppression. Engagement of mutant forms of CTLA-4 with Y-165 substitution or complete deletion resulted in strong inhibition of IL-2 production upon CD3/CD28 costimulation. Under these conditions, IL-2 mRNA expression was significantly suppressed by CTLA-4 cross-linking just 4 h after stimulation. If these mutants were functionally inactive and served as dominant negative mutants of CTLA-4, their engagement would augment proliferation or IL-2 secretion. However, these mutants lacking the tyrosine motif did not serve as dominant negative forms for endogenous CTLA-4. Another argument for the strong inhibition of T cell activation by CTLA-4 mutants might be explained if the cross-linking of highly expressed CTLA-4 could physically interfere with the CD3/CD28 stimulation on the cell surface. However, the observation that the induction of IFN-γ or Bcl-X L mRNA expression was not inhibited by CTLA-4 cross-linking in the same T cells ruled out this possibility. Thus, we demonstrated that CTLA-4 cross-linking inhibits IL-2 production and proliferation induced by CD3/CD28 stimulation even in the absence of its tyrosine motif within the cytoplasmic tail. Therefore, in contrast to the current model for an inhibitory mechanism through SHP-2 activation, our results clearly show that the association of CTLA-4 with PI3 kinase and SHP-2 is not required for CTLA-4–mediated suppression of T cell activation, although it is still possible that the tyrosine motif may also partly contribute to CTLA-4–induced inhibition. Recently, CTLA-4 was found to associate with the CD3ζ chain in primary T cells 36 . It has been suggested that the interaction of CTLA-4 and CD3ζ recruits SHP-2 into the complex that induces CD3ζ dephosphorylation. However, these analyses showed only the effect on ζ phosphorylation; the functional consequence has not yet been determined. Considering that this analysis demonstrated that the association depends on the cytoplasmic tail of CTLA-4 and the tailless CTLA-4 did not associate with CD3ζ 36 , the inhibition through ΔCP7 CTLA-4 in our study could be induced by a different mechanism than CTLA-4–CD3ζ interaction. Upon CD3/CD28 stimulation, the surface CTLA-4 expression on Y165G or ΔCP7 mutant T cells was significantly increased. Analysis of CTLA-4 species on the surfaces of ΔCP7 T cells demonstrated that the mutant CTLA-4 did not form heterodimers with endogenous CTLA-4 on the cell surface, ruling out the possibility that endogenous CTLA-4 accumulated on the cell surface upon activation and delivered a negative signal. Inhibition of both proliferation and IL-2 production in WT T cells was much less than that in ΔCP7 mutant T cells. These results prove that cross-linking of the ΔCP7 mutant did deliver an inhibitory signal. Heterodimers between endogenous CTLA-4 and the mutant were not detected; if there are any, they are probably endocytosed by internalization machinery. The target molecule of inhibition by CTLA-4 has not yet been identified. As CTLA-4 inhibits CD28-dependent TCR activation, the target molecules may be related to CD28 signals. Both ERKs and Jun NH 2 -terminal kinases (JNKs) have been reported to be involved in the induction of IL-2 gene transcription, and it has been reported that CTLA-4 engagement downregulates these MAPKs 25 . We investigated whether Y165G or ΔCP7 mutant CTLA-4 has an inhibitory effect on ERK2 upon activation of resting T cells. However, under the condition of suboptimal CD3/optimal CD28 stimulation, where IL-2 secretion was maximally inhibited by CTLA-4, the ERK2 activity was only weakly upregulated and the effect of CTLA-4 cross-linking was not clearly observed. When we examined ERK2 activity upon optimal CD3/CD28 stimulation, it was upregulated in T cells and was reduced to an approximately half level by CTLA-4 cross-linking, even in Y165G or ΔCP7 mutant CTLA-4 as well as WT CTLA-4–transfected cells. In addition, the suppression of JNK was not clearly observed in our system (data not shown). Taken together with the results of Frearson et al. 31 , the inhibition of ERK2 upon CTLA-4 engagement is not mediated by SHP-2. Although CTLA-4 may suppress more efficiently upon stimulation with suboptimal CD3/optimal CD28 stimulation under physiological conditions, it is difficult to conclude from these results that the suppression of the early event of ERK2 upregulation upon TCR/CD3 stimulation is the primary cause of CTLA-4–mediated inhibition of IL-2 production. Further analysis will be required. Collectively, these data suggest that negative signals by CTLA-4 for IL-2 production and proliferation could be mediated through the membrane-proximal region of CTLA-4 rather than the 165 YVKM motif. The suppression is probably mediated through the association with as yet unidentified transmembrane or intracellular molecules, or, alternatively, is due to the proper positioning of the extracellular region upon T cell activation. The recent finding that CD28 mediates reorganization of TCR signaling machinery to rafts 37 may suggest that CTLA-4 inhibits such CD28 function. The tyrosine motif plays a role in regulating the surface expression of CTLA-4 through clathrin-mediated endocytosis and, in addition, might have other unknown signaling functions. | Study | biomedical | en | 0.999997 |
10499916 | Blood and LNs were obtained from patients with advanced stage malignant melanoma selected on the basis of HLA-A2 antigen expression. PBLs were separated from heparinized blood by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech), washed three times, and cryopreserved in RPMI 1640, 40% FCS, and 10% DMSO. Vials containing 5–10 × 10 6 cells were stored in liquid nitrogen. LNs collected by surgical dissection were dissociated to single cell suspensions in sterile RPMI 1640 supplemented with 10% FCS, washed, and cryopreserved as indicated above for PBLs. Aliquots were placed in 24-well tissue culture plates (Costar Corp.) in 2 ml of IMDM (Life Technologies) supplemented with 0.55 mM Arg, 0.24 mM Asn, 1.5 mM Gln, 10% pooled human A + serum, recombinant human (rh)IL-2 (100 U/ml), and rhIL-7 (10 ng/ml). The melanoma cell line Me 290 and the A2/Melan-A–specific CD8 + T cell clone 17 were established from surgically excised melanoma metastases from patient LAU 203 as described 26 . The cells were maintained in DME supplemented with 0.55 mM Arg, 0.24 mM Asn, 1.5 mM Gln, and 10% FCS. PBLs were typed for HLA-A2 by flow cytometry using the allele-specific mAb BB7.2 27 . Complete HLA-A and -B typing was performed by serology, and HLA-A2 allele typing and HLA-C typing were done by PCR using sequence-specific oligonucleotides 28 . Since all patients were HLA-A*0201 positive, they all expressed HLA signal peptides able to bind to HLA-E and to serve as ligands for CD94/NKG2A 29 30 . All six patients shown in Fig. 1 and Fig. 2 had at least one of the known p58.2 binding ligands Cw1, Cw3, Cw7, and Cw8. In some patients (LAU 181 and LAU 155) both alleles were p58.2 ligands, while in the other patients there was only one p58.2 ligand. The Me 290 melanoma cell line and the Melan-A–specific CD8 + T cell clone 17 were A1, A*0201, B7, B8, Cw07, Cwx. Antibodies specific for the NK receptors p58.2/CD158b (GL183), CD94 (XA185 and Y9), and the heterodimer CD94/NKG2A (ZIN199) were provided by A. Moretta, Università di Genova, Genova, Italy 31 . The ILT2-specific antibody HP3F1 14 was obtained from M. López-Botet, University Hospital Princesa, Madrid, Spain. Nonlabeled IgG1 and IgG2a antibodies, and PE-labeled antibodies specific for p58.2 and CD94 were purchased from Immunotech. mAbs specific for human CD3, CD8, CD14, CD16, CD28, CD45RA, and TCR-α/β were obtained from Becton Dickinson. All flow cytometry stainings for CD94 were done with the mAb XA185, while Y9 was used in the cytotoxicity assays. In this study, the term “NKT cell” was used for all NK receptor–positive cells expressing CD3 and/or TCR-α/β. Complexes were synthesized as described 24 25 . In brief, purified HLA heavy chain and β2-microglobulin were synthesized by means of a prokaryotic expression system (pET; R&D Systems, Inc.). The heavy chain was modified by deletion of the transmembrane cytosolic tail and COOH-terminal addition of a sequence containing the BirA enzymatic biotinylation site. Heavy chain, β2-microglobulin, and peptide were refolded by dilution. The 45-kD refolded product was isolated by fast protein liquid chromatography and then biotinylated by BirA (Avidity) in the presence of biotin, adenosine 5′-triphosphate, and Mg 2+ (all from Sigma Chemical Co.). Streptavidin–PE conjugate (Sigma Chemical Co.) was added in a 1:4 molar ratio, and the tetrameric product was concentrated to 1 mg/ml. As the antigenic peptide, the Melan-A 26–35 A27L analogue (ELAGIGILTV) was used, which has a higher binding stability to HLA-A*0201 and a higher T cell antigenicity and immunogenicity than the natural Melan-A decapeptide EAAGIGILTV or the nonapeptide AAGIGILTV 26 . In this paper, the abbreviation “tetramer” is used for the HLA-A*0201/Melan-A 26–35 A27L tetramers produced for this study. LN cells were thawed and cultured for 16–20 h in IMDM supplemented with 0.55 mM Arg, 0.24 mM Asn, 1.5 mM Gln, 10% pooled human A + serum, rhIL-2 (100 U/ml), and rhIL-7 (10 ng/ml). PBLs were thawed and stained after washing. Cells (0.5–1 × 10 6 ) were stained with tetramers and FITC-, peridinin chlorophyll protein (PerCP™), and allophycocyanin-labeled mAb conjugates in 50 μl of PBS, 2% BSA, and 0.2% azide for 40 min at 4°C. For indirect fluorescence labeling, cells were incubated (a) with tetramers, (b) with the primary (NK receptor–specific) antibody and washed, (c) with sheep anti–mouse FITC-labeled antibody and washed twice, (d) with IgG1 and IgG2a antibodies, and (e) with PerCP™- and allophycocyanin-labeled antibodies. Cells were washed once in the same buffer and analyzed immediately in a FACSCalibur™ machine (Becton Dickinson). With this method, the percentages of tetramer-positive cells were slightly lower compared with methods without multiple wash steps after tetramer incubation (not shown). Data acquisition and analysis were performed using CELLQuest™ software. Only cells falling in the “lymphocyte gate” were analyzed; this gate was defined by forward/side scatter settings corresponding to a cell population expressing >98% CD45 and <1% CD14 (as determined by control CD45/CD14 stainings). PBLs were thawed, stained with HLA-A2/Melan-A tetramers, and sorted using a FACStar™ machine (Becton Dickinson). The cells were then cultured in IMDM supplemented with 0.55 mM Arg, 0.24 mM Asn, 1.5 mM Gln, 10% pooled human A + serum, rhIL-2 (100 U/ml), and rhIL-15 (10 ng/ml) starting at day 2 to upregulate CD94/NKG2A as described 32 . 1 wk later, the cells were again FACS ® sorted after staining with CD94/NKG2A-specific antibodies and with HLA-A2/Melan-A tetramers. Cytolytic activity was tested in 51 Cr-release assays against the HLA-A*0201–expressing melanoma target cell lines Na8 and Me 290 26 . Target cells were radiolabeled with Na 51 CrO 4 for 1 h at 37°C, 5% CO 2 , then washed and coincubated in V-bottomed microwells at the indicated E/T ratio (10 3 target cells per well) in the presence of the IgM mAbs specific for CD94 (or control CD16). After 4 h at 37°C, supernatants were collected and counted in a Top-count™ (Canberra Packard) gamma counter. Percent specific lysis was calculated as (experimental release − spontaneous release) × 100/(total release − spontaneous release). In PBLs, the frequency of CTLs specific for single MHC/peptide epitopes is usually very low (range 1 in 10 4 –10 6 CD8 + T cells). Their frequency during acute infections may be much higher (up to 1 in ∼10 3 ), but such high frequencies are not observed for tumor-specific CD8 + T cells because immune responses to tumors involve much lower antigen-specific T cell numbers compared with acute microbial infections. The frequencies of tumor-specific CD8 + T cells may be significantly higher in tumor-infiltrated lymph nodes (TILNs) than in PBLs, since TILNs may contain up to 3% HLA-A2/Melan-A tetramer–positive cells among CD8 + cells when analyzed ex vivo 25 . To study the phenotype of tumor-specific CTLs in more detail, we cultured the TILNs for 2 wk in the presence of IL-2 and IL-7. This procedure induced expansion of the tumor antigen–specific T cells, allowing us to perform multiparameter FACS ® analysis. Fig. 1 shows the results of TILNs stained with tetramers and antibodies specific for CD8 and one of four different NK receptors. The TILNs from the three patients contained high percentages of CD8 + tetramer-positive cells (1.3–5.1%). In patient LAU 181, these cells were largely negative for the NK receptors p58.2 and ILT2, but they expressed CD94 and CD94/NKG2A at high percentages. The Melan-A–specific lymphocytes from patient LAU 203 expressed some ILT2 but only low levels of the other three receptors. Finally, ILT2, CD94, and CD94/NKG2A were also expressed by some Melan-A–specific T cells from patient LAU 267. The Melan-A–specific TILNs from four further patients (data not shown) expressed no or only low levels (<0.5%) of NK receptors. It has been demonstrated that melanoma patients develop vitiligo more frequently than individuals without melanoma, and that vitiligo is associated with an ongoing immune response directed against melanoma cells 33 34 . We have recently reported that patients with vitiligo have high frequencies of Melan-A–specific circulating CTLs which were detectable ex vivo using HLA-A2/Melan-A tetramers 24 34a . The relatively high percentages of tetramer-positive circulating CTLs provided us the unique opportunity of investigating NK receptor expression by specific CTLs in vivo. We obtained PBLs from three HLA-A2–positive melanoma patients with vitiligo, and found 0.10–0.17% CD8 + tetramer-positive cells . These cells expressed the following NK receptors: in patient LAU 155, practically all tetramer-positive cells were negative for each of the four NK receptors analyzed (p58.2, ILT2, CD94, and CD94/NKG2A). In contrast, in patient LAU 156, most of the tumor antigen–specific CTLs expressed CD94 and CD94/NKG2A, and about half of them expressed ILT2. Finally, in the third patient (LAU 269), there were low percentages of NK receptor–positive, tetramer-positive T cells. In conclusion, some tumor antigen–specific T cells may express NK receptors in vivo, and there are situations where this is the case for a large fraction of a given CTL population. The relatively high frequency of tumor-specific CTLs in the patients with vitiligo allowed us to investigate the in vivo phenotype of these cells in more detail. We investigated the expression of CD45RA, a marker for naive T cells 35 36 . In PBLs of patients LAU 155 and LAU 269, the tetramer-positive cells expressed high levels of CD45RA . In contrast, a reduced level of CD45RA was found in patient LAU 156, i.e., in the cells with high levels of NK receptor expression. We also analyzed the costimulatory molecule CD28 and the “adhesion” molecule CD57, since downregulation of CD28 and upregulation of CD57 have been shown to occur in activated “effector” CTLs 37 38 , and we have recently demonstrated that NK receptor expression by T cells is primarily confined to the CD28 − population 39 . Interestingly, the tetramer-positive CD8 + cells of the patient with high percentages of NK receptor–expressing CTLs (patient LAU 156) were predominantly CD28 − and CD57 + . In contrast, the tumor-specific CTLs of the other two patients were mostly CD28 + and CD57 − . It has been shown that antigen-specific T cell cytotoxicity can indeed be inhibited through triggering of CD94/NKG2A 20 or p58.2 16 18 19 . However, these results were obtained with selected T cell lines or clones, leaving the question open whether NK receptor inhibition is functional in polyclonal T cell responses. Therefore, we investigated the cytotoxicity by tetramer-sorted PBLs. Several of our attempts failed, since we did not obtain enough tetramer-positive, NK receptor–positive cells through FACS ® sorting. However, one of our patients (LAU 156) had exceptionally high percentages of A2/Melan-A tetramer–positive, CD94/NKG2A-positive cells in the peripheral blood . After FACS ® sorting, the tetramer-positive cells were cultured for 1 wk and sorted again to obtain pure A2/Melan-A tetramer CD94/NKG2A double positive CTLs. These cells were tested in cytotoxicity assays against the melanoma cell line Me 290, previously stimulated with IFN-γ for 48 h to increase MHC expression and inhibitory receptor triggering 40 . Indeed, the killing was enhanced in the presence of blocking anti-CD94 antibody Y9 compared with the killing in the presence of isotype-matched control anti-CD16 antibody (dashed line). The target cells were efficiently killed by the NK receptor–negative CTLs of the patient, and by the HLA-A*0201/Melan-A–specific CTL clone 17, both to a similar extent in the presence of anti-CD94 or control antibody. The killing of the Me 290 melanoma cell line was antigen specific, since all of the CTLs did not lyse the Melan-A–negative melanoma cells Na8 unless synthetic Melan-A peptide was added (data not shown). In summary, the data demonstrate that the lysis of melanoma cells was inhibited due to the inhibitory receptor CD94/NKG2A expressed by the Melan-A–specific effector CTLs. TILNs of seven melanoma patients were investigated. In one patient, the Melan-A–specific CD8 + T cells were mostly CD94/NKG2A positive. In other patients, they were partly ILT2 positive. Similar NK receptor–positive tumor antigen–specific T cells were also found in peripheral blood from at least one melanoma patient with vitiligo. Furthermore, NK receptor triggering inhibited the cytolytic activity of T cells from one patient. Together, these results show that CD8 + T cells may express NK receptors in vivo and that this may lead to inhibition of melanoma cell lysis. The early observation that NK cells preferentially lyse target cells that lack MHC class I molecules led to the formulation of the “missing self” hypothesis 41 . This surmised that NK cells were endowed with the ability to recognize and destroy cells lacking expression of MHC class I, whereas normal cells positive for MHC class I would be protected from NK cell lysis. Only after the identification and cloning of several NK receptors could this hypothesis be confirmed 13 14 15 . It is now established that the binding of inhibitory NK receptors to MHC class I molecules on target cells can lead to the delivery of signals that inhibit the cytolytic function of NK cells. As a consequence, abnormal (infected or malignant) cells lacking MHC class I may be destroyed preferentially. NK receptors were also found to be expressed by T cells. However, although NK cells frequently express these receptors, only small percentages of NK receptor–positive T cells (NKT cells) were identified 16 . In addition, some NKT cell populations were found to be mono- or oligoclonal, as indicated by very limited diversity of T cell receptor rearrangements 42 . Based on these findings, it was postulated that NKT cells are a small subpopulation and may represent particular lymphocyte lineages with special antigen specificity and/or special function. Recently, Ikeda et al. described a melanoma-specific CD8 + T cell clone that was inhibited by p58.2 recognizing HLA-Cw7 on autologous melanoma cells 18 . Noppen et al. characterized sister T cell clones bearing the same T cell receptor specific for a melanoma-associated antigen 20 . Some clones were NK receptor negative, but others expressed the inhibitory receptor CD94/NKG2A and showed reduced cytotoxicity to melanoma cells. These results demonstrated that cloned NKT cells may bear functional T cell receptors specific for classical peptide antigens presented by MHC class I. The above-mentioned studies were done with selected T cell clones that may not be representative, since the majority of T cell lines and clones are NK receptor negative (data not shown). To investigate whether NKT cells are rare or frequent in vivo, we applied the recently developed tetramer technology which allowed us to visualize and phenotype peptide antigen–specific, yet polyclonal CD8 + T cells without in vitro cultivation. Our data show that tumor-specific T cells expressing NK receptors can indeed be found at high percentages in some patients. Thus, NKT cells may not necessarily represent separate cell lineages, but can belong to the large pool of T cells with classical peptide/MHC class I specificity. Why should the CTL activity be modulated through this pathway? Current evidence suggests that CTLs which are activated over a prolonged period may need and have inhibitory mechanisms, and that human NKT cells may fall in this category of activated CTLs. Some NK receptor–expressing T cells have been described to be CD28 negative and express activation markers such as CD56 43 , CD18, and CD45RO 42 . Using a large panel of different NK receptor–specific mAbs, we have recently demonstrated that the majority of NKT cells in the circulation are TCR-α/β 1 , CD8 + , and CD28 − . Furthermore, these cells account for a large fraction of CD28 − T cells, which make up 10–80% of circulating CD8 + T cells in melanoma patients 39 . CD28 − T cells have also been shown to be strongly cytotoxic and to proliferate poorly in vitro 37 . Thus, T cells may downregulate CD28 and upregulate NK receptors in association with prolonged activation for cytolytic effector function. It is likely that NK receptors are involved in peripheral regulatory mechanisms avoiding overwhelming immune responses and immunopathology, particularly in situations of strong and/or long-lasting immune activation. The increased numbers of CD28 − T cells in prolonged infections such HIV 44 or CMV 45 may support this notion. The function of NK receptors as inhibitory pathways has the advantage that abnormal infected or tumor cells lacking MHC class I may still be efficiently eliminated by the CTLs. In summary, we found tumor-specific TCR-α/β 1 CD8 + T cells expressing NK receptors that could inhibit the lysis of melanoma cells. These data strongly suggest that NK receptors may modulate T cell activities in vivo. NK receptor expression by T cells varies depending on the cellular activation status and cytokines such as IL-12, IL-15, and TGF-β 31 32 46 . Possibly, new therapeutic strategies modifying NK receptor expression or function may help improve the efficacy of immunotherapy in infectious disease, cancer, autoimmunity, and transplantation. | Study | biomedical | en | 0.999997 |
10499917 | Vα14 NKT-deficient (NKT-KO) mice were established by specific deletion of the Jα281 gene segment with homologous recombination and aggregation chimera techniques 12 . In these mice, only Vα14 NKT cells are missing and other lymphoid populations, such as T, B, and NK cells remain intact. The Vα14 NKT-KO mice were backcrossed 7 times with C57BL/6 (B6) mice. Vα14 NKT (RAG −/− Vα14Tg Vβ8.2Tg) mice with a B6 background were established by mating RAG −/− Vβ8.2Tg mice and RAG −/− Vα14Tg mice as previously described 12 13 . In the Vα14 NKT mice, because they lacked gene rearrangement of endogenous TCR-α/β genes, only transgenic TCR-α/β (Vα14Tg and Vβ8.2Tg) are expressed, and resulted in preferential development of Vα14 NKT cells with no detectable number of conventional T cells. IFN-γ–deficient mice were provided by Y. Iwakura (Institute of Medical Science, the University of Tokyo, Tokyo, Japan) 54 . Pathogen-free B6, (B6 × BALB/c)F1 mice were purchased from Japan SLC Inc. All mice used in this study were maintained in specific pathogen-free conditions and used at 8–12 wk of age. Freshly prepared splenocytes were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide. In general, 10 6 cells were preincubated with 2.4G2 (PharMingen) to prevent nonspecific binding of mAbs via FcR interactions, and then cells were incubated on ice for 30 min with FITC-conjugated anti–TCR-α/β (H57-597-FITC) and PE-conjugated anti-NK1.1 (PK136-PE) as previously described 12 . Both reagents were purchased from PharMingen. Flow cytometry analysis was performed on Epics-Elite (Coulter Electronics). Wild-type and NKT-KO mice were injected intravenously with 1.5 μg of anti-CD3 mAb (PharMingen, 145-2C11) in 200 μl PBS. 90 min after the anti-CD3 treatment, splenocytes were separated and cultured (5 × 10 6 cells/ml) in 6-well culture plates for 1 h at 37°C, and then the supernatants were collected and subjected to ELISA for IL-4. For activation of Vα14 NKT cells with α-GalCer, mice were intraperitoneally injected with α-GalCer (100 μg/kg) or control vehicle as previously described 13 . α-GalCer was provided by Kirin Brewery Co. The α-GalCer stock solution did not contain detectable endotoxins, as determined by Limulus amebocyte assay (sensitivity limit 0.1 ng/ml) as previously described 55 . The stock solution (220 μg/ml) was diluted in control vehicle, and a mouse received 2 μg of α-GalCer. Whole spleen cells were prepared 0.5, 1, 2, and 24 h after the injection and washed extensively with ice-cold PBS, and the amounts of IFN-γ were determined by RT-PCR. In some experiments, sera were taken 2 and 24 h after the α-GalCer injection and subjected to ELISA for IFN-γ. IFN-γ and IL-4 concentrations in sera or the culture supernatants were measured by ELISA as previously described 13 . The amounts of IFN-γ transcript were determined with reverse transcriptase (RT)-PCR. Total cellular RNA from splenocytes was prepared using TRIZOL according to a manufacturer's protocol. 10 μg of RNA were reverse transcribed in 20 μl of mixture by using oligo dT primers, and 1 μl of reaction mixture was subjected to PCR as previously described 10 . Mice were subcutaneously infected with 750 third stage larvae of Nb 56 . 3 and 6 wk after infection, the mice were immunized with 10 μg DNP-conjugated Nippostrongylus adult antigen (DNP-Nb) mixed with 2 mg of alum [Al(OH) 3 ; Wako Chemical. Co.] as an adjuvant. For OVA immunization, 10 μg of OVA or DNP-OVA were mixed with 5 mg of alum. The immunized mice were treated intraperitoneally with α-GalCer (100 μg/kg) or control vehicle on days 1, 5, and 9. 4 wk later, mice were challenged with 10 μg of DNP-OVA in alum. Serum was collected 2 and 3 wk after primary OVA immunization, and 1 wk after the secondary challenge. IgE production was determined by passive cutaneous anaphylaxis (PCA) and ELISA, and the production of IgG1 and IgG2a was determined by ELISA. The total serum IgE level was determined by ELISA as previously described 57 . In brief, 96-well plates (Dynatech) were coated with an anti–mouse IgE mAb (6HD5), then plates were blocked with 1% BSA. After application of serum samples and standards (anti-DNP IgE mAb, SPE-7; Seikagaku Kogyo), biotinylated anti–mouse IgE mAb (HMK-12) was added for 30 min, followed by addition of avidin-peroxidase. The plates were washed with PBS containing 0.5% Tween 20. The substrate solution (ABST and H 2 O 2 ) was added, and the reaction was stopped with citrate and read at 450 nm with an ELISA reader (Bio-Rad 550). For detecting anti-DNP IgG1 or IgG2a antibody, 96-well plates were coated with DNP-BSA. After blocking, standard anti-DNP IgG1 mAb (NKIgG1), standard anti-DNP anti-IgG2a mAb (DO5-1C4), and samples were added. For second antibodies, an affinity-purified rabbit anti–mouse IgG1-peroxidase (Zymed) or a rabbit anti–mouse IgG2a-peroxidase (Zymed) was used, respectively. Anti-DNP-specific and anti-OVA-specific IgE antibody was detected by PCA reaction in rats as previously described 57 . Serial dilution of serum was injected intradermally into normal Wistar rats. After a 24-h sensitization period, OVA or DNP-BSA was intravenously injected with Evans Blue. The reaction was examined at 30 min after the challenge. Titers were expressed as the highest dilution eliciting a reaction. Naive CD44 low CD4 + T cells from (B6 × BALB/c)F1 or Ly5.1 B6 spleen were prepared as follows: splenocytes were incubated with a mixture of anti-CD8 (53-6.72), anti-NK1.1 (PK136), and anti-CD44 (IM7) mAbs (PharMingen). The treated cells were washed, then incubated on plastic dishes coated with goat anti–mouse IgGs (which cross-react with rat IgG, including 53-6.72, PK136, and IM7). The nonadherent cells were used as naive CD44 low CD4 + T cell population. Contaminations of CD44 high cells, NK1.1 + cells, or CD8 T cells were <3% in either marker. The CD44 low CD4 + T cells (1.5 × 10 6 ) were stimulated with immobilized anti–TCR-α/β mAb in the presence of IL-2 (30 U/ml) and graded doses of IL-4 to induce Th1 and Th2 cells in vitro as previously described 58 59 . After stimulation for 2 d, the cells were harvested and cultured for an additional 3 d without anti-TCR stimulation in the presence of IL-2 (30 U/ml). Intracellular staining of IL-4 and IFN-γ was performed as previously described 58 59 . Biotinylated anti-K d or anti-Ly5.1 mAbs (PharMingen) and allophycocyanin-conjugated avidin (The Jackson Laboratory) were used for identifying responding naive T cells from (B6 × BALB/c)F1 and Ly5.1 B6 mice, respectively. Vα14NKT cells from α-GalCer–injected Vα14 NKT mice with a normal Ly5.2 background were added to the induction culture. The cell number of Vα14NKT cells added was adjusted by prestainings of an aliquot of NKT spleen cells with anti–TCR-β and anti-NK1.1 mAbs. Where indicated, anti–IFN-γ mAb (5 μg/ml; PharMingen) was added in the Th1/Th2 cell differentiation culture. The goal of this study was to clarify the effector mechanisms of Vα14 NKT cell regulation of Th2 cell differentiation and the subsequent induction of IgE responses. To address this question, we used NKT-KO mice in which the development of Vα14 NKT cells was dramatically inhibited , and which produced essentially no primary IL-4 upon anti-CD3 stimulation or α-GalCer-treatment (data not shown). NKT-KO mice with B6 background were infected with Nb, and 3 wk later the mice were immunized with DNP-conjugated Nb in alum for the induction of DNP-specific IgE production. To our surprise, the levels of total IgE and DNP-specific IgE detected in wild-type mice were almost identical to those observed in NKT-KO mice . In addition, DNP-specific IgG1 and IgG2a levels in NKT-KO mice were comparable to those observed in the wild-type mice . We also examined the involvement of Vα14 NKT cells in the regulation of IgE response induced by OVA, a conventional protein antigen. NKT-KO mice were immunized with OVA in alum, and OVA-specific IgE and IgG1 productions were measured. Primary and secondary responses showed no significant differences in the serum antibody levels detected in wild-type and NKT-KO mice . These results indicated that Vα14 NKT cells were not required for antigen-specific IgE responses induced by either Nb infection or OVA immunization. It is well documented that IgE and IgG1 responses are mediated by antigen-specific Th2 cells, and that IgG2a responses depend on Th1 cells 60 . Consequently, we induced a specific activation of Vα14 NKT cells in vivo by using α-GalCer, and the antigen-specific IgE, IgG1, or IgG2a production was assessed. As shown in Fig. 3 A, anti-DNP IgE response induced by DNP-OVA immunization was dramatically reduced in wild-type mice after α-GalCer injection, whereas no inhibition was observed in NKT-KO mice. Some inhibitory effect was also observed in DNP-specific IgG1 response in wild-type mice . In contrast, anti-DNP-IgG2a responses were not reduced, but rather slightly enhanced in wild-type mice . These results suggested that the stimulation of Vα14 NKT cells with α-GalCer resulted in the suppression of OVA-specific Th2 responses with a subsequent decrease in IgE and IgG1 production while maintaining an intact or enhanced Th1-dependent IgG2a production. Since IFN-γ has a potent inhibitory effect on Th2 responses 37 , we next assessed serum levels of IFN-γ in B6 mice after in vivo treatment with α-GalCer, which activates Vα14 NKT cells. Spleen cells were prepared 0.5, 1, 2, or 24 h after intravenous administration of α-GalCer, and IFN-γ transcripts were detected by RT-PCR . Serum levels of IFN-γ were also assayed by ELISA . IFN-γ transcripts were detected within 2 h after α-GalCer injection in wild-type mice, whereas no transcript was detected in NKT-KO mice. Essentially similar results were obtained by the assessment of serum IFN-γ . These results strongly suggested that a large amount of IFN-γ was produced by Vα14NKT cells after α-GalCer treatment in vivo. Next, we used IFN-γ–deficient mice and examined whether the α-GalCer–induced IgE suppression was detected or not. IFN-γ–deficient mice were immunized with DNP-OVA in alum, and primary IgE and IgG1 responses and secondary IgE responses were assessed . As we expected, no suppression in the production of IgE was observed in either primary or secondary responses in IFN-γ–deficient mice. In addition, IgG1 response was not impaired. Vα14 NKT cells in IFN-γ–deficient mice produced an equivalent level of IL-4 upon stimulation with α-GalCer (data not shown). Thus, it is most likely that the suppressive effect on IgE production is mediated by IFN-γ produced by Vα14 NKT cells. The results obtained thus far favor the notion that IFN-γ produced by activated Vα14 NKT cells inhibits Th2 cell differentiation, and results in suppression of antigen-specific IgE production. Consequently, the role of ligand-activated Vα14 NKT cells on Th2 cell differentiation was examined more precisely through the use of an in vitro induction culture system 58 59 . Naive CD4 T cells obtained from (B6 × BALB/c)F1 mice or Ly5.1 B6 mice were stimulated with immobilized anti-TCR mAb in the presence of IL-4 to allow Th1 and Th2 cell differentiation in vitro. Several doses of Vα14 NKT cells from α-GalCer–treated Vα14 NKT mice with normal Ly5.2 B6 background were added in the induction culture, and the intracellular production of IFN-γ and IL-4 in K d -positive T cells or Ly5.1 T cells was assessed as shown in Fig. 6 . No detectable alloreactivity of Vα14 NKT cells from NKT mice against (B6 × BALB/c)F1 splenic T cells was detected (data not shown). The numbers of T cells harvested were similar in these different culture conditions (data not shown). In this culture system, an IL-4 dose-dependent increase in the generation of Th2 cells was observed . However, the addition of activated Vα14 NKT cells in the induction culture inhibited IL-4–producing Th2 cell differentiation in a cell–dose-dependent manner . In addition, the number of IFN-γ producing Th1 cell differentiation was significantly enhanced in the presence of activated Vα14 NK T cells. The addition of nonactivated Vα14 NKT cells from vehicle-treated Vα14 NKT mice did not have any effect on Th1/Th2 cell differentiation (data not shown). These results clearly indicated that Th2 cell differentiation was inhibited by the addition of preactivated Vα14 NKT cells. Finally, we addressed whether the inhibition of Th2 cell differentiation induced by ligand-activated Vα14 NKT cells was mediated by IFN-γ. Anti–IFN-γ mAb was added to the induction cultures containing responder CD4 T cells and activated Vα14 NKT cells. As shown in Fig. 6 C, the inhibition of Th2 cell differentiation induced by Vα14 NKT cells was completely rescued by the addition of anti–IFN-γ mAb. Thus, similar to the mechanisms governing IgE suppression in in vivo experimental system , IFN-γ appeared to be an effector molecule for the inhibition of Th2 cell differentiation induced by activated Vα14 NKT cells in vitro. In this report, we describe in vivo studies that demonstrate that Vα14 NKT cells are not required for antigen-specific IgE responses induced by Nb infection and OVA immunization . This conclusion is supported by in vitro studies indicating that the addition of activated Vα14 NKT cells to in vitro Th1/Th2 cell differentiation cultures resulted in the inhibition rather than the induction of Th2 cell differentiation . These results are in agreement with those reported by others 17 51 52 53 . Smiley et al. reported that the anti-IgD-induced IgE responses were not impaired in CD1-deficient mice in which NKT cell development is largely inhibited 17 . Similarly, β2-microglobulin–dependent T cells, including NKT cells and conventional CD8 α/β T cells, were reported to be nonessential for Th2 responses induced by immunization with different protein antigens or after infection with certain microorganisms 51 52 53 . However, in contrast, Yoshimoto et al. reported that β2-microglobulin–dependent T cells were important for anti-IgD–induced IgE production, and that NK1.1 + thymocytes restored the defect of anti-IgD–induced IgE production in β2-microglobulin–deficient mice 49 50 . Regardless of the reasons that may explain the discrepancy with the results obtained with β2-microglobulin–deficient mice, our data clearly demonstrate that Vα14 NKT cells are dispensable for IgE responses, at least those induced by Nb infection and OVA immunization. In addition, our results suggest that Vα14 NKT cells are not the major cell source of IL-4 that is required for Th2 cell differentiation. Although several cell types have been proposed as candidates for the source of IL-4 that initiates certain Th2 responses 61 62 63 64 65 66 , the precise mechanisms of how these cells are activated and produce IL-4 leading to Th2 cell differentiation remain to be elucidated. More interestingly, by using α-GalCer that is a specific stimulating ligand for Vα14 NKT receptor, we found a unique regulatory role of Vα14 NKT cells on Th2 cell differentiation. We observed a selective in vivo suppression of IgE production in mice treated with α-GalCer during OVA priming or Nb infection. A mild but reproducible suppression of the IgG1 response was also observed . In contrast, IgG2a response was not suppressed and in fact a slight enhancement was detected, suggesting an inhibition in the generation of antigen-specific Th2 cell differentiation. Importantly, Vα14 NKT cells produced large amounts of IFN-γ in the serum when mice were treated with α-GalCer , and the suppression of IgE was not detected in either NKT-KO or IFN-γ–deficient mice . Thus, it is most likely that the selective IgE suppression observed in α-GalCer–treated mice was due to an impaired Th2 cell differentiation induced by large amounts of IFN-γ secreted from activated Vα14 NKT cells. Consistent with this, the inhibition of Th2 cell differentiation induced by activated Vα14 NKT cells appeared to be an IFN-γ–mediated consequence . A similar suppressive effect on IgE production by IFN-γ was reported in several other experimental systems 67 68 69 70 71 72 . IFN-γ produced by γ/δ T cells suppressed IgE responses in OVA-specific responses 71 and cutaneous contact sensitivity system 72 . In addition, since IFN-γ is known to be produced by CD8 + α/β-TCR T cells, a possible inhibitory role for these cells in the regulation of IgE responses has been proposed 73 . Recently, Kitamura et al., in collaboration with us, reported a study addressing the role of IL-12 in the production of IFN-γ from α-GalCer–activated NKT cells 74 . The majority of IFN-γ production induced by α-GalCer and dendritic cells was found to be inhibited by the addition of anti–IL-12 mAb to the culture, indicating the involvement of IL-12 in the IFN-γ production. IL-12 appeared to be produced by dendritic cells only when they interacted with α-GalCer–activated Vα14 NKT cells. The IL-12 in turn enhanced the IFN-γ production of the activated Vα14 NKT cells. In addition, transcriptional upregulation of IL-12 receptor was detected after α-GalCer administration 74 . Thus, it is conceivable that IL-12 plays a significant role for the IFN-γ–mediated suppressive effect on IgE responses. Our results suggest that, upon stimulation with certain ligands such as glycosylphosphatidylinositol-anchored protein 21 expressed on parasites or other microorganism, Vα14 NKT cells become IFN-γ–producing cells, leading to the inhibition of Th2 cell differentiation and suppression of IgE responses. In addition, our recent experiments have suggested that a bacteria-derived material, LPS, is able to stimulate Vα14 NKT cells to produce a large amount of IFN-γ (data not shown). Clearly, further analyses are required for addressing the physiological consequences of Vα14 NKT cell–mediated regulation of IgE response. However, it is noteworthy that the successful activation of Vα14 NKT cells and subsequent inhibition of Th2 responses, as described in this report, may open new avenues for research aimed at developing treatment for Th2-dependent diseases, such as systemic autoimmune diseases, chronic graft versus host diseases, and allergic diseases. | Study | biomedical | en | 0.999996 |
10499918 | Z176 (IgG2b) mAb was obtained by immunizing a 5-wk-old BALB/c mouse with the NK clone SA260 (surface phenotype: CD3 − CD16 + , CD56 + , NKp46 + , NKp44 + , p70/NKB1 + , CD94/NKG2A + ) as described previously 16 . The following mAbs were produced in our laboratory: JT3A (IgG2a, anti-CD3), BAB281 (IgG1, anti-NKp46), Z231 and KS38 (IgG1 and IgM, respectively, anti-NKp44), Z199 (IgG2b, anti-NKG2A), Z27 (IgG1, anti-p70/NKB1), KD1 and c127 (IgG2a and IgG1, respectively, anti-CD16), and c218 and A6-90 (IgG1 and IgG2b, respectively, anti-CD56). The MCA531 mAb (IgM, anti-CD20) was purchased from Serotec. The D1.12 mAb (IgG2a, anti–HLA-DR) was provided by Dr. R.S. Accolla (Università di Pavia, Pavia, Italy). The HP2.6 mAb (IgG2a, anti-CD4) was provided by Dr. P. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain). To obtain PBLs, PBMCs derived from healthy donors were isolated on Ficoll-Hypaque gradients and depleted of plastic-adherent cells. PBLs were either used directly or were incubated with anti-CD3 (JT3A), anti-CD4 (HP2.6), and anti–HLA-DR (D1.12) mAbs for 30 min at 4°C, followed by immunomagnetic depletion with goat anti–mouse coated Dynabeads (Dynal; 30 min, 4°C; reference 17). NK or T cell clones were obtained by limiting dilution in the presence of irradiated feeder cells, 1.5 ng/ml PHA (GIBCO BRL), and 100 U/ml rIL-2 (Proleukin; Chiron Corp.) as described previously 18 . NK cell clones were tested for cytolytic activity against the FcγR + P815 murine mastocytoma cell line in a 4-h 51 Cr-release assay as described previously 2 . The E/T ratio used was 8:1 in all instances. For one- or two-color cytofluorimetric analysis (FACScan™; Becton Dickinson), cells were stained with the appropriate mAbs followed by PE- or FITC-conjugated isotype-specific goat anti–mouse second reagent (Southern Biotechnology Associates) 17 . Sepharose-protein A–coupled Z176 and Z199 mAbs or cyanogen bromide Sepharose (Amersham Pharmacia Biotech)-coupled Z27 mAb were used to immunoprecipitate specific molecules from 1% NP-40 lysates of cells surface labeled with 125 I (NEN) as described previously 19 . Immunoprecipitates were analyzed by discontinuous SDS-PAGE either undigested or digested with N -glycosidase F or O -glycosidase (Boehringer Mannheim) 20 . For two-dimensional peptide mapping (2DPM) analysis, the purified proteins were digested with pepsin, and peptides were analyzed by electrophoresis in the first dimension (Multiphor II; Amersham Pharmacia Biotech) followed by chromatography in the second dimension 20 . NK cells (10 8 ) were stimulated or not with 100 μM sodium pervanadate 19 , and 1% NP-40 lysates were immunoprecipitated with the Z176 mAb. Samples were analyzed in discontinuous SDS-PAGE, transferred to Immobilon P (Millipore Corp.), and then probed with antiphosphotyrosine mAb (PY20-HRPO; Transduction Laboratories) or anti–SHP-1 and SHP-2 phosphatases (PTP1C and PTP1D, respectively; Transduction Laboratories). The Renaissance Chemiluminescence kit (NEN) was used for detection. cDNA was synthesized from RNA prepared from two IL-2–activated polyclonal NK cells and inserted in the expression vector VR1012. The cDNA library, fractionated in 10 pools of 200,000 different inserts, was transfected into COS-7 cells by DEAE-dextran method and immunocytochemical staining using the Z176-specific mAb and sib selection 21 . DNA sequencing was performed using d-Rhodamine Terminator Cycle Sequencing kit and a 377 ABI automatic sequencer (Perkin Elmer-Applied Biosystems). COS-7 cells were transfected with VR1012–AIRM1 construct by DEAE-dextran method 4 . After 48 h, cells were trypsinized and analyzed by immunofluorescence staining for the expression of p75/AIRM1 molecules. Transfected cells and human RBCs were washed twice with serum-free DMEM. The COS-7 cell/RBC ratio used in the experiments was 1:20; the adhesion assay was performed for 30 min at 4°C. The binding of RBCs to COS-7 cells was quantified by counting the percentage of COS-7 cells that bound more than seven erythrocytes. Neuraminidase treatment was carried out by incubating RBCs with 0.1 U/ml of Vibrio cholera neuraminidase (Behringwerke AG) for 3 h at 37°C followed by two washes with DMEM. For cellular adhesion blocking experiments, 10 6 AIRM1-transfected COS-7 cells were incubated with 0.5 ml Z176 mAb supernatant for 30 min at 4°C followed by two washes with DMEM before the adhesion assay. The Somatic Cell Hybrid blot (BIOS Laboratories), containing 20 multi-chromosomal somatic human/hamster cell hybrids plus 3 control genomic DNAs (human, hamster, and mouse) digested with EcoRI, was used to assign the AIRM1 gene to a specific chromosome. A 1203-bp cDNA probe, obtained digesting VR1012–AIRM1 construct with SalI and PstI restriction enzymes, was used to perform high stringency hybridization 22 . Analysis of cross-specific conservation of AIRM1 gene was performed using Zoo-Blot™ from Clontech. This Southern blot contained genomic DNA from humans, Rhesus monkey, Sprague-Dawley rat, BALB/c mouse, dog, cow, rabbit, chicken, and Saccharomyces cerevisiae yeast. Washes were carried out under low stringency conditions 23 . RNA extracted using RNAzol (Cinna/Biotecx) and oligo (dT)–primed cDNA was prepared from polyclonal NK cell populations and clones by standard techniques. The set of primers AIRM1-up (containing the ATG initiation codon; 5′ TCC AAC CCC AGA TAT GCT G) and AIRM1-down (designed in the 3′ untranslated region; 5′ ACA AGC CCG AGC CTC TGC) were used to amplify the AIRM1 open reading frame. 30 cycles of PCR (30 s at 95°C, 30 s at 60°C, and 30 s at 72°C) were performed using TAQ-GOLD (Perkin Elmer-Applied Biosystems) after a preactivation of 15 min at 95°C. The amplification products obtained from polyclonal NK cells populations were purified from gel, subcloned into pcDNA3.1/V5/His TOPO™ vector using the Eukaryotic TOPO TA Cloning ® kit (Invitrogen), and sequenced. Mice were immunized with the NK cell clone SA260 (surface phenotype: CD3 − CD16 + , CD56 + , NKp46 + , NKp44 + , p70/NKB1 + , CD94/NKG2A + ), characterized by a strong cytolytic activity against the P815 murine mastocytoma cell line. After cell fusion, mAbs were analyzed for their ability to inhibit the cytotoxicity mediated by NK cell clones in a classical redirected killing assay against the FcγR + P815 cell line. By using this screening procedure, we isolated the Z176 mAb (IgG2b) that inhibited the cytolytic activity of the majority of the NK cell clones analyzed. Fig. 1 shows four representatives of such clones, including the immunizing SA260 clone. In three of these clones, the addition of Z176 mAb (but not of an isotype-matched anti-CD56 mAb) resulted in inhibition of the spontaneous cytolytic activity against P815 cells . Clone D414 is representative of the infrequent NK cell clones in which no inhibitory effect could be detected. Immunofluorescence and FACS ® analysis of the same clones revealed that Z176 mAb reacted with clones SA260, LM15, and LM8 but not with clone D414. Similar data were obtained in a large panel of NK cell clones, thus suggesting that the Z176 mAb–reactive molecule is expressed and delivers inhibitory signal in the majority of, but not all, human NK cells. We next analyzed the cell surface distribution of the Z176 mAb–reacting molecule in PBLs by two-color immunofluorescence and FACS ® analysis. As shown in Fig. 2 , all CD56 + cells were stained by the Z176 mAb. In contrast, Z176 mAb did not stain CD3 + T cells or CD20 + B cells. Only in some individuals was a minor fraction (<5%) of T cells found to be Z176 + (see donor B). Although not shown, further analysis of the Z176 surface expression in NK cell–enriched fractions of PBLs (upon depletion of CD3 + HLA-DR + cells) confirmed that most NK cells reacted with Z176 mAb while Z176 − NK cells were rather infrequent. Analysis of a large panel (>100) of T cell clones (both α/β + and γ/δ + ) showed that Z176-reactive molecules are not expressed by T lymphocytes after cell activation or clonal expansion (not shown). Finally, Z176 mAb did not stain EBV-transformed B cell lines (including Raji, Daudi, and C1R) or T cell lines (such as JA3, HSB-2, CEM, MOLT-4, H9, and Jurkat). Remarkably, it also failed to stain NK cell lines, including NK3.3, NKL, and YT (not shown). A polyclonal NK cell population was surface labeled with 125 I and immunoprecipitated with Z176 mAb. This antibody immunoprecipitated a molecule with a molecular mass of ∼75 kD (p75) both under nonreducing (not shown) and reducing conditions . To analyze their glycosylation pattern, p75 molecules were treated with different enzymes. The molecular mass was not modified by treatment with O -glycosidase (not shown). In contrast, digestion with N -glycosidase revealed a protein backbone of ∼48 kD , thus suggesting a relatively high N -glycosylation pattern. Finally, in two-dimensional peptide mapping (2DPM) analysis, p75 displayed a digestion pattern that was different from those of known inhibitory receptors, including p70/NKB1 and NKG2A (not shown). Altogether, these data support the notion that p75 is a novel surface molecule with inhibitory function expressed by the majority of human NK cells. It is well known that inhibitory receptors, including p58 (CD158), p70/NKB1, CD94/NKG2A, and LIR-1/ILT-2, are characterized by the presence of ITIM sequences in their cytoplasmic tail. ITIMs, upon tyrosine phosphorylation, recruit SH2-containing phosphatases such as SHP-1 and SHP-2. To assess whether the p75 molecule could also belong to the ITIM-bearing receptor family, a polyclonal NK cell population, treated or not with sodium pervanadate, was immunoprecipitated with Z176 mAb. Samples were probed with antiphosphotyrosine, anti–SHP-1, or anti–SHP-2 mAbs. Fig. 3 b shows that treatment with sodium pervanadate leads to p75 tyrosine phosphorylation (left panel) and association with SHP-1 (right panel). It is of note that, under the same conditions, no association with SHP-2 could be detected (not shown). These data strongly suggest that p75 contains at least one typical ITIM in the cytoplasmic tail. A cDNA library, prepared from RNA derived from 2 polyclonal NK cell populations, fractionated in 10 different pools, was transiently transfected into COS-7 cells. After 48 h, cells were tested for reactivity with Z176 mAb by immunocytochemical staining. The plasmidic DNA of the positive pool was amplified in Escherichia coli , fractionated in smaller subpools, and transfected into COS-7 cells. Six rounds of transfections and screening allowed the isolation of an individual cDNA termed AIRM1. As shown in Fig. 4 a, COS-7 cells transfected with VR1012–AIRM1 construct were brightly stained with Z176 mAb in cytofluorimetric analysis. Cell transfectants were then surface labeled with 125 I, and cell lysates were immunoprecipitated with Z176 mAb. As shown in Fig. 4 b, the immunoprecipitated molecule displayed a molecular mass slightly lower than that immunoprecipitated from NK cells. This difference in molecular mass could reflect a noncomplete N -glycosylation in COS-7 cell transfectants. Indeed, upon treatment with N -glycosidase, the molecule immunoprecipitated from AIRM1 transfectants displayed a 48-kD protein backbone identical to that of the p75 molecule isolated from NK cells. Fig. 5 shows the nucleotide sequence of AIRM1 and its predicted amino acid translation (467 aa). The 5′ noncoding region consists of 64 nucleotides, and in the 3′ noncoding sequence a possible polyadenylation signal (AAUAUA) preceded the poly A tail. The putative protein appears as a type I transmembrane molecule belonging to the Ig-SF. An 18 aa leader peptide precedes a 335 aa extracellular portion characterized by an NH 2 -terminal V-type domain followed by two C2-type domains. Six putative O -glycosylation sites and eight putative N -glycosylation sites are present in the extracellular portion. The predicted polypeptide mass is ∼48 kD, thus corresponding to the protein backbone of the p75 molecule immunoprecipitated by Z176 mAb from both NK cells and p75/AIRM1 COS-7 transfectants. The 23 aa hydrophobic transmembrane portion is followed by a cytoplasmic tail (91 aa) containing two tyrosine residues. Remarkably, Tyr 437 is part of a typical ITIM motif (435–440 = IQYAPL). Finally, other consensus sequences for putative phosphorylation sites are present in the cytoplasmic tail: a 3′–5′ adenosine monophosphate (cAMP)-dependent protein kinase phosphorylation site, three protein kinase phosphorylation sites, and five casein kinase 2 (CK2) phosphorylation sites. Comparison of the aa sequence of p75/AIRM1 with those of known proteins in the EMBL/GenBank/DDBJ database revealed significant similarity with the placenta antigen CD33L1 24 , as well as with the myeloid lineage molecule CD33 25 . In particular, both the IgV domain and the transmembrane region of p75/AIRM1 display a high degree of aa identity (55 and 61%, respectively) with CD33 molecule. On the other hand, the IgC2a and IgC2b domains display a remarkable similarity with those of CD33L1. In this context, the highest degree of aa identity (73%) was found between the IgC2a domain of p75/AIRM1 and that of CD33L1. The IgV and IgC2a domains of p75/AIRM1 contain two Cys residues at position 41 and 174. Cys residues located at the same relative positions are also present in different sialoadhesins 26 27 and are likely to be involved in the formation of interdomain bridges. Altogether, these results suggest that the p75/AIRM1 molecule may represent a novel member of the sialoadhesin family. To identify the human chromosome carrying the p75/AIRM1-encoding gene, genomic DNA samples derived from a panel of human/hamster somatic cell hybrids were analyzed with a 1203-bp AIRM1-specific probe. We could localize the AIRM1 gene on human chromosome 19, since only the somatic cell hybrids containing this chromosome were positive in Southern blot analysis . Moreover, the simple pattern of hybridization observed suggests that AIRM1 is a single gene or a few copy genes. It is of note that other members of the sialoadhesin family, including CD33 28 and CD33L1 24 , myelin-associated glycoprotein (MAG 29 ), CD22 30 , and Siglec-5 31 , have been mapped on human chromosome 19. Reverse transcriptase (RT)-PCR analysis was performed on polyclonal NK cell populations and clones, derived from five different donors. Using the set of primers AIRM1-up and AIRM1-down, we could detect, not only in polyclonal populations but also in NK cell clones, three amplified products of ∼1.4, 1.1, and 0.7 kb, respectively (not shown). These cDNA fragments were subcloned and sequenced. The 1.4-kb product was found to correspond to the AIRM1 cloned cDNA. Moreover, the sequence analysis, performed in five different donors, revealed only an allelic variant of AIRM1 cDNA characterized by two silent substitutions (codon 105, ACC instead of AAT; and codon 452, GGT instead of GGA). Different members of the sialoadhesin family, including CD22 32 33 and Sialoadhesin 34 35 , have been shown to mediate sialic acid–dependent binding to RBCs. As shown in Fig. 8 , COS-7 cells transfected with the AIRM1 cDNA efficiently bound to RBCs. AIRM1-specific rosette formation occurred both at 4°C and at 37°C (not shown). Pretreatment of RBCs with neuraminidase abrogated their binding to AIRM1 transfectants, suggesting that adhesion occurs through a sialic acid–dependent mechanism. In addition, binding to RBCs was inhibited when AIRM1 transfectants were preincubated with Z176 mAb (but not with an isotype-matched mAb), thus indicating a direct involvement of p75/AIRM1 in the RBC–AIRM1 transfectant interaction. In this study, we describe a novel inhibitory receptor, termed p75/AIRM1, which is expressed by resting and activated human NK cells and may play a role in the regulation of their function. Molecular and functional analysis revealed a novel member of the sialoadhesin family; thus, p75/AIRM1 differs from the other major inhibitory receptors expressed by NK cells that are characterized by their specificity for MHC class I molecules. p75/AIRM1 is a transmembrane glycoprotein characterized by one IgV and two IgC2-type Ig-like domains in the extracellular portion that displays similarity with members of the sialoadhesin family. This family is composed of structurally and functionally related molecules, including MAG, Schwann cell myelin protein (SMP), Sialoadhesin, CD22, CD33, CD33L1, and Siglec-5. All of these proteins are characterized by a restricted cell expression pattern. In particular, MAG 36 and Schwann cell myelin protein (SMP) 37 are found in the nervous system on oligodendroglia and Schwann cells, respectively; CD22 is expressed on a subset of B lymphocytes 38 , sialoadhesin on a subset of macrophages 39 , CD33 40 and Siglec-5 31 on cells of the myelomonocytic lineage, and CD33L1 in placenta 24 . Similar to these molecules, p75/AIRM1 also displays a restricted cell expression pattern, being essentially confined to NK cells. Other than their ability to bind sialic acid, limited information is available on the function of the molecules of the sialoadhesin family. The only molecule reported to display inhibitory function (i.e., similar to p75/AIRM1) is CD22 that is expressed on B cells, in which it may downregulate the B cell receptor–mediated cell triggering. CD33 is selectively expressed by hemopoietic cells, in which it represents an important marker in normal differentiation as well as in leukemia typing. In view of the similarity with p75/AIRM1, which includes the presence of an ITIM sequence in the cytoplasmic tail, it is important to reinvestigate the role of CD33, especially with respect to its possible inhibitory effect on hemopoietic cell function and/or differentiation. Experiments along this line are in progress in our laboratory. p75/AIRM1 is encoded by a gene localized on human chromosome 19. Remarkably, genes coding for other members of the sialoadhesin family map on chromosome 19 as well. This may suggest that all of these molecules, including p75/AIRM1, may have evolved through duplication of a common ancestral gene. It is of note that other inhibitory receptors involved in the regulation of NK-mediated cytotoxicity, including KIRs, LIR/ILT, and LAIR-1, have also been mapped on this chromosome. Although not shown, the AIRM1-specific probe, under low stringency conditions, hybridized with genomic DNA from Rhesus monkey, thus suggesting a cross-species conservation between humans and monkeys. DNA sequencing of seven cDNA clones obtained by RT-PCR experiments, performed in five different donors, revealed only an allelic variant characterized by two silent substitutions. However, considering both the relatively low number of samples analyzed and the fact that all donors belonged to the Caucasoid race, a polymorphism of the p75/AIRM1 gene cannot be ruled out. RT-PCR analysis, in addition to the 1.4-kb AIRM1 transcript, also allowed the identification of two alternatively spliced products of 1.1 and 0.7 kb, respectively. Sequence of these products revealed that the 1.1-kb fragment encoded a putative protein identical to the p75/AIRM1 but lacking the IgC2a domain (from codon 146 to codon 238) . The 0.7-kb amplified product contains two cDNA fragments that are both carrying an early stop codon at position 146 . Consistent with the structural similarity between p75/AIRM1 and other sialoadhesins, p75/ARM1 was also found to bind RBCs. This binding was specifically inhibited by Z176 mAb as well as by the neuraminidase treatment of RBCs. These data suggest that p75/AIRM1 may function as a receptor which recognizes its putative ligand(s) in a sialic acid–dependent manner. Carbohydrate-binding proteins are known to play a role in a wide variety of biological processes involving specific cell–cell interactions. As suggested for CD22 41 , it is possible that sialic acids may be required for correct orientation and presentation of the epitope (on the ligand) recognized by p75/AIRM1. Cross-linking of p75/AIRM1 in human NK cells delivers an inhibitory signal resulting in downregulation of spontaneous NK cell–mediated cytotoxicity. An inhibitory effect could also be detected on the NK cell triggering mediated by activating receptors such as CD16, NKp46, and NKp44 (not shown). Coherent with its ability to mediate inhibition, the cytoplasmic tail of p75/AIRM1 was found to contain an ITIM that recruited the SHP-1 phosphatase upon tyrosine phosphorylation. This, in turn, is likely to inhibit downstream molecular events that are critical for the induction of NK cell–mediated cytotoxicity. One may ask the meaning of the inhibitory effect of p75/AIRM1 in NK-mediated function, and what could be the functional relationship with KIRs. KIRs appear to play a predominant role in the discrimination between HLA class I + cells and cells that do not express sufficient amounts of HLA class I, such as tumor- or virus-infected cells. Since p75/AIRM1 does not appear to recognize HLA class I molecules, it is possible to speculate that this receptor may play a role in recognition of still undefined sialylated proteins, possibly present in normal cells that physiologically express low amounts of HLA class I molecules. The expression of ligand(s) for p75/AIRM1 may protect these cells from the NK-mediated attack. It is evident that the identification of the p75/AIRM1 ligand(s) will greatly help to clarify this issue. In addition, it is possible that p75/AIRM1 may function during stages of NK cell differentiation from immature precursors in which cells have acquired cytolytic potential but have not yet expressed HLA class I–specific inhibitory receptors. Indeed, cells with these phenotypic characteristics have recently been identified in our laboratory (our unpublished data). In conclusion, we have identified, characterized, and cloned a novel inhibitory receptor primarily confined to human NK cells which may play a role complementary to that of KIRs in the regulation of NK cell function. | Study | biomedical | en | 0.999998 |
10499919 | Antibodies against P-selectin included affinity-purified polyclonal antiserum produced in rabbits immunized with P-selectin purified from platelet lysates 17 and the mAb WAPS 12.2 (Zymed Labs., Inc.). Anti-GP Ibα mAbs used were: WM23 and AK3, both of which bind within the mucin-like macroglycopeptide 18 ; SZ2, which recognizes an epitope within the anionic/sulfated tyrosine region of GP Ibα bounded by residues Tyr276 and Glu282 16 ; and AK2 (RDI Research Diagnostics), which binds within the GP Ibα NH 2 terminus and inhibits ristocetin- and botrocetin-induced von Willebrand factor binding 16 . Two von Willebrand factor mAbs were used in these studies: 6G1, which inhibits the von Willebrand factor–GP Ibα interaction (our unpublished data), and 2C9, which does not inhibit the binding of von Willebrand factor to GP Ibα and was used as a control mAb 18 . HECA-452 is a rat mAb (IgM) that recognizes a FucT VII–dependent carbohydrate 19 20 . Glycocalicin was isolated from outdated human platelets by a method modified from that of Hess et al. 21 . Erythrocyte-free platelets were isolated from 10 liters of outdated platelet-rich plasma (obtained from the Blood Transfusion Centre, Cambridge, England) by centrifugation and washed with buffer A (13 mM Na 3 citrate, 120 mM NaCl, and 30 mM glucose, pH 7.0). After another centrifugation, the platelet pellet was suspended in 500 ml of buffer B (10 mM Tris/HCl, 150 mM NaCl, and 2 mM CaCl 2 , pH 7.4) and sonicated. The resultant suspension was then incubated at 37°C for 30 min to allow the calpain released from the platelets during sonication to cleave membrane-bound GP Ibα, releasing glycocalicin. After ultracentrifugation to remove cell components, the glycocalicin-containing supernatant was applied to a wheat germ Sepharose 4B column. Bound crude glycocalicin was eluted with 2.5% N -acetyl- d -glucosamine and 20 mM Tris/HCl, pH 7.4. Further purification by ion exchange chromatography (on Q-Sepharose Fast-flow column; Pharmacia) was needed to remove residual contaminants. Glycocalicin was eluted with a linear salt gradient of 0–0.7 M NaCl in 20 mM Tris/HCl, pH 7.4. Chinese hamster ovary (CHO) cells expressing a phosphatidylinositol glycan–linked form of P-selectin were a gift from Dr. C. Wayne Smith (Baylor College of Medicine, Houston, TX) 22 . These cells were grown in medium comprising equal parts DMEM and F12 medium (GIBCO BRL) supplemented with 5% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin. CHO and L cells expressing the GP Ib-IX-V complex and its components have been described previously 23 . In brief, CHO αβIX cells express GP Ibα, GP Ibβ, and GP IX, the three polypeptides that are minimally required for efficient cell surface expression of GP Ibα. L αβIXV cells additionally express GP V, and CHO and L βIX cells express GP Ibβ and GP IX but lack GP Ibα and GP V. The conditions for their growth have been described 23 24 . Before each experiment involving cells that express GP Ibα, the surface level of this polypeptide was first determined by flow cytometry using AK2 as previously described 25 . In brief, cells grown in a monolayer were detached with 0.54 mM EDTA, washed with PBS, and resuspended in PBS containing 1% BSA. The cells were then incubated with AK2 (1 μg/ml) for 60 min at room temperature. After being washed to remove unbound antibody, the cells were incubated with an FITC-conjugated rabbit anti–mouse secondary antibody (Zymed Labs., Inc.) for 30 min at room temperature. The cells were then analyzed on a FACScan™ flow cytometer (Becton Dickinson) stimulating with laser light at 488 nm and collecting light emitted at >520 nm. To induce core-2 branching and α(1,3)-fucosylation of the GP Ibα carbohydrates in CHO cells, the cells were cotransfected with plasmids containing cDNAs encoding C2GnT (core 2 β(1,6)- N -acetylglucosaminyltransferase), (pCDNA1/C2GnT, a gift from Dr. Minoru Fukuda, La Jolla Cancer Research Foundation, La Jolla, CA) and FucT VII (provided for these studies by Dr. John Lowe, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI) using a modified SRa vector containing a hygromycin resistance marker 19 . Transfections were by liposome-mediated DNA delivery (LipofectAMINE; GIBCO BRL). A total of 1 μg of plasmid was used in each transfection, with the FucT VII plasmid transfected either alone or as an equal mixture with the plasmid containing the C2GnT cDNA. The cells were selected by growth in hygromycin-containing medium, for which a resistance marker is contained in the FucT VII plasmid. FucT VII expression was evaluated by flow cytometry with the antibody HECA-452, which recognizes a FucT VII–dependent epitope. Labeling and analysis were as described above for GP Ibα, except that an FITC-conjugated rabbit anti–rat antibody (Zymed Labs., Inc.) was used to label cell-bound HECA-452. The wells of 96-well microtiter plates (Nunc Immulon, Inc.) were coated with 50 μl of purified glycocalicin (20 μg/ml in PBS, pH 7.4) for 2 h at room temperature. The wells were rinsed four times over 20 min with DMEM containing 1% BSA. CHO cells and CHO cells expressing P-selectin (CHO-P) were detached from culture dishes using 5 ml of 0.54 mM EDTA in PBS, washed once in PBS and once in DMEM, and then incubated at 37°C for 30 min in DMEM containing 0.5 mCi of 51 Cr per 10 7 cells. Cells were then washed three times by centrifugation and resuspension and then resuspended to a density of 2 × 10 6 cells/ml in DMEM containing 1.0% BSA and preincubated at room temperature for 15 min with either 10 mM Tris/HCl, pH 7.4, in 0.15 M NaCl (TS buffer) alone or with TS buffer containing appropriate amounts of sulfated glycans or other blocking reagents. Aliquots of 0.1 ml of the cell suspension were incubated at room temperature in four replicate microtiter wells for 15 min. The media and nonadherent cells were then removed, and the wells were rinsed three times with DMEM containing 1% BSA. 51 Cr-labeled cells that remained adherent to the wells were lysed in a solution containing 1% Triton X-100, and their radioactivity was assayed in a gamma counter. For studies that included anti–GP Ibα blocking antibodies, the antibodies were incubated with the immobilized glycocalicin for 15 min at room temperature (each antibody at 40 μg/ml) before the addition of the radiolabeled cells. Each experiment was performed in triplicate; average counts from one representative experiment are reported. CHO-P cells detached with EDTA were resuspended in α–minimal essential medium (α-MEM; GIBCO BRL) containing 1% BSA and aliquoted to a concentration of 2 × 10 6 cells/ml. Purified glycocalicin or soybean trypsin inhibitor were then added to the cell suspensions at various concentrations and shaken gently for 15 min. Polyclonal anti–P-selectin antibody was then added to each tube (final concentration, 2 μg/ml), and the cells were incubated for an additional 45 min. The cells were then washed twice in PBS, and FITC-conjugated goat anti–rabbit antibody was added to a concentration of 8 μg/ml and incubated for 45 min. Antibody binding was then assessed by flow cytometry. CHO cells were labeled with membrane dyes, CellTracker™ Orange (5-(and -6)-(((4 chloromethyl)benzoyl)amino)-tetramethylrhodamine) for CHO-P cells and CellTracker™ Green (5-chloromethylfluorescein diacetate) for CHO αβIX and CHO βIX (both dyes from Molecular Probes, Inc.). For this, the cells were detached from the culture dishes with 0.54 mM EDTA, centrifuged, and resuspended in culture medium. The dyes were each added to the cell suspension at 12 μM and incubated at 25°C for 60 min in the dark, with gentle shaking. After labeling, the cells were washed twice in PBS and resuspended in α-MEM, 1% BSA to a concentration of 6 × 10 6 cells/ml. 500-μl aliquots of CHO-P cells were then mixed with equal volumes of the other cell lines, and the mixture was shaken on a table-top shaker at six cycles per second for 10 min. Heterotypic aggregates of green and orange cells were quantitated by fluorescence microscopy. These experiments were also done with CHO αβIX cells pretreated with mocarhagin, a metalloprotease from the venom of the South African spitting cobra, Naja mocambique mocambique . This protease selectively cleaves the GP Ibα NH 2 terminus, including a region containing three sulfated tyrosine residues 16 . Before the aggregation experiment, the CHO αβIX cells were incubated for 30 min with mocarhagin (20 μg/ml) and then washed twice with PBS. Human P-selectin was purified from outdated platelets as described previously 26 . Detergent, Triton X-100, was removed from the P-selectin sample immediately before use by passage through an Extracti-Gel D column (Pierce Chemical Co.). To prepare the P-selectin matrix, glass coverslips (No. 1, 24 × 50 mm; Corning Glass Works) were coated with a solution containing 50 μg/ml P-selectin for 4 h at 37°C followed by an additional 1-h incubation with PBS containing 1% BSA to block nonspecific binding sites. The coated coverslips were then washed with 0.9% NaCl to remove unbound P-selectin. Human umbilical cords were collected and processed for primary endothelial cell culture as described previously 27 . In brief, human umbilical cords were rinsed with PBS and then treated with collagenase type II (10 μg/50 ml PBS; Worthington Biochemical Corp.) for 40 min at room temperature. Enzymatically dissociated human umbilical vein endothelial cells (HUVECs) were collected by centrifugation (10 min, 200 g ) and then resuspended in M199 medium (GIBCO BRL) supplemented with 10% FBS and 1% glutamine. The cells were then plated onto glass coverslips that had previously been coated with a 1% gelatin solution for 30 min at 37°C. Cells were maintained at 37°C with 5% CO 2 and 95% humidity and reached confluence in ∼4–5 d. To induce surface expression of P-selectin, monolayers of confluent HUVECs were treated with 25 μM histamine (Sigma Chemical Co.) for 12 min at room temperature 27 . Stimulated cells were used immediately. The flow chamber system included a parallel plate flow chamber, an inverted stage phase-contrast microscope (DIAPHOT-TMD; Nikon Inc.), and an image recording system. The parallel plate flow chamber was composed of a polycarbonate slab, a silicon gasket, and a glass coverslip held together by vacuum in such a way that the coverslip forms the bottom of the chamber. The coverslips were first coated with either soluble P-selectin or a monolayer of HUVECs. The chamber was maintained at 37°C by an air curtain incubator attached to the microscope. The wall shear stress was created by drawing PBS through the chamber with a Harvard syringe pump and was proportional to the fluid viscosity and flow rate and inversely proportional to the width of the chamber and height of the gap created by the gasket 28 . In studies of GP Ib-IX-V–mediated cell rolling on P-selectin, 0.6 ml of a suspension of CHO αβIX, CHO βIX, L βIX, or L αβIXV cells (500,000 cells/ml) was injected into the chamber, and the cells were incubated for 1 min with immobilized P-selectin or 2 min with stimulated endothelial cells. At the end of the incubation, PBS was perfused through the chamber at a flow rate of 1.6 ml/min, generating a wall shear stress of 2 dynes/cm 2 (shear rate ∼182/s). Cell rolling through a single view field was recorded in real time for 4–6 min on videotape. The video data were then analyzed off line using Inovision imaging software (IC-300, Modular Image Processing Workstation; Inovision Corp.) to quantify the number and velocities of the rolling cells 27 29 . Cells considered to be rolling were those that translocated over the matrix or HUVECs while maintaining constant contact. The rolling velocity was defined as the distance a cell traveled during a defined period (μm/s). To examine platelet rolling on activated HUVECs, platelets prepared as described below were resuspended to 2 × 10 8 cells/ml in PBS with 1 mM CaCl 2 , 0.5 mM MgCl 2 , and 20% erythrocytes by volume. The HUVECs were stimulated as above, and the platelet suspension was allowed to incubate for 2 min at 37°C before perfusing the chamber with the platelet suspension at a flow rate of 1.6 ml/min, generating a wall shear stress of 2 dynes/cm 2 (shear rate ∼120/s). Blood was drawn from healthy human donors into 1/6 volume of acid citrate dextrose (85 mM Na 3 citrate, 111 mM dextrose, and 71 mM citric acid). The blood was centrifuged at 170 g for 15 min at 25°C to separate platelet-rich plasma from erythrocytes and leukocytes. The platelets were then pelleted from platelet-rich plasma by centrifugation at 800 g for 10 min at 25°C. The platelets were then washed by suspension in CGS buffer (13 mM sodium citrate, 30 mM glucose, and 120 mM sodium chloride, pH 6.5), centrifuged again, and resuspended to 2 × 10 8 platelets/ml in PBS containing calcium and magnesium. All solutions except for the final resuspension buffer contained 1 μM prostaglandin E1 to prevent platelet activation. The data were analyzed either by the unpaired Student's t test or by one-way analysis of variance (ANOVA), depending on the nature of the data. As a first step in determining if the GP Ib-IX-V complex interacts with P-selectin, we examined the binding of CHO-P cells to the soluble extracellular portion of human GP Ibα (called glycocalicin) immobilized on plastic. Glycocalicin was purified from lysates of human platelets and therefore contained all of the posttranslational modifications potentially required to constitute a platelet P-selectin receptor. The binding of CHO-P cells to glycocalicin was approximately fivefold greater than the binding of untransfected cells ( n = 4, P < 0.001, Student's t test), suggesting a specific interaction between P-selectin and GP Ibα . We then tested the ability of different reagents to block this interaction. The P-selectin interaction with PSGL-1 is dependent on two structural modifications of PSGL-1: tyrosine sulfation within a negatively charged sequence at its mature NH 2 terminus and fucosylation, sialylation, and core-2 branching of its O-linked carbohydrate. P-selectin interacts with the carbohydrate motif through its C-type lectin domain in a calcium-dependent fashion. We therefore tested whether EDTA would inhibit the interaction of P-selectin with GP Ibα. Surprisingly, and in contrast to the interaction with PSGL-1, it did not . However, antibodies against both GP Ibα and P-selectin did inhibit the interaction. Of the GP Ibα antibodies, the greatest inhibition of binding was observed with SZ2 (∼55% inhibition; n = 3, P < 0.03, one-way ANOVA, Dunnett's method). This antibody has been shown to recognize an epitope within the tyrosine-sulfated anionic region of GP Ibα 16 , suggesting that this region plays an important role in the recognition of P-selectin. The other two GP Ibα mAbs, WM23 and AK3, directed against the GP Ibα mucin core 18 , inhibited binding to a lesser extent. Binding was also almost completely inhibited by an affinity-purified rabbit polyclonal antibody against P-selectin ( n = 3, P < 0.002, Student's t test). Two findings indicated a potential similarity between P-selectin's interaction with GP Ibα and its interaction with heparin: its calcium independence 17 30 and the involvement of the GP Ibα anionic/tyrosine sulfated region. This region of GP Ibα contains three sulfated tyrosine residues 14 16 and interacts with another of GP Ibα's ligands, thrombin, through thrombin's heparin-binding exosite 31 32 . We therefore studied the effect of heparin and other proteoglycans on CHO-P adhesion to glycocalicin. Heparin and the related proteoglycan fucoidin both partially inhibited binding . In contrast, chondroitin sulfate, a control proteoglycan that does not block the P-selectin–PSGL-1 interaction, similarly did not affect the P-selectin–GP Ibα interaction ( P > 0.1). We next tested the ability of glycocalicin to interfere with the binding to CHO-P cells of a polyclonal antibody against human P-selectin. This antibody blocks the P-selectin–PSGL-1 interaction 17 and, as shown in Fig. 1 B, also blocks the adhesion of CHO-P to glycocalicin. Glycocalicin blocked antibody binding in a dose-dependent manner, with a 50% inhibiting concentration (IC 50 ) of ∼25 μg/ml . The results suggest that most of the binding determinants for the polyclonal antibody lie within a restricted region of P-selectin close to the GP Ibα binding site. CHO cells synthesize simple core-1 O-linked glycans 33 and are incapable of synthesizing PSGL-1 in a form competent to bind P-selectin unless the appropriate carbohydrate-modifying enzymes are also expressed 34 . Thus, CHO cells provide a good cell system to test whether carbohydrate modification of GP Ibα is required for its interaction with P-selectin. The major carbohydrate structure of platelet GP Ibα 35 36 37 is very similar to that reported for PSGL-1 3 , the only difference being the lack of an α(1,3)-fucosyl linkage in GP Ibα. We expressed the GP Ib-IX-V complex in CHO cells alone, with FucT VII, or with both FucT VII and the core-2 branching enzyme (C2GnT). We first assessed whether the cells carry the necessary modification by immunostaining with the antibody HECA-452, which recognizes FucT VII–dependent carbohydrate epitopes 19 . Whereas the untransfected cells demonstrated only a low background fluorescence, all of the cell lines transfected with both C2GnT and FucT VII demonstrated high levels of the HECA-452 epitope . We then evaluated the ability of these cells to form heterotypic aggregates with cells expressing P-selectin. Cells expressing the GP Ib-IX-V complex and its components were fluorescently labeled with the membrane-permeable dye CellTracker™ Green, and the CHO-P cells were labeled with CellTracker™ Orange. Equal quantities of the two cell types were mixed and incubated with gentle shaking, and the heterotypic aggregates containing both green and orange cells were quantitated. Cells expressing GP Ibα demonstrated much greater heterotypic aggregation than the control untransfected CHO cells or CHO cells expressing a partial complex lacking GP Ibα (CHO βIX cells) . Expression of the carbohydrate-modifying enzymes led to only a small further increment in the formation of heterotypic aggregates, indicating that carbohydrate modification of GP Ibα is less important to the interaction with P-selectin than is the modification of PSGL-1. This finding is consistent with the lack of calcium dependence of the interaction. Calcium is required for the interaction of P-selectin with certain carbohydrate ligands but not with sulfated glycans such as heparin sulfate and fucoidin, which apparently involves sulfated moieties 17 26 30 . The involvement of the GP Ibα NH 2 terminus in binding P-selectin was further implicated by studies with the cobra venom metalloprotease mocarhagin . This protease removes the GP Ibα NNH 2 terminus to Glu282, including much of the anionic sulfated region 16 . Mocarhagin pretreatment of CHO αβIX cells reduced heterotypic aggregation by ∼50% ( n = 3, P < 0.01, one-way ANOVA, Tukey's test). The P-selectin–mediated rolling of platelets on inflamed endothelium requires rapid on and off rates. Thus, for the GP Ib-IX-V complex to constitute a physiological counterreceptor for P-selectin, it should be able to support a similar rolling interaction. To test this possibility, we perfused cells expressing full or partial complexes over coverslips coated with P-selectin in a parallel plate flow chamber at a shear stress of 2 dynes/cm 2 . Cells were observed to adhere to and roll on P-selectin . The rolling interaction was independent of cell type, as both CHO and L cells expressing the complex rolled at similar velocities, whereas control cells lacking GP Ibα did not adhere or roll . We next tested whether the GP Ib-IX-V complex could also support cell rolling on stimulated endothelial cells. Endothelial cells were collected from human umbilical veins and grown directly on gelatin-coated coverslips. When confluent, they were activated with 25 μM histamine and the coverslip was immediately placed in the parallel plate flow chamber. CHO αβIX cells were then perfused over the monolayer. Negative controls were CHO αβIX cells perfused over untreated endothelium and CHO βIX cells perfused over activated endothelium. CHO αβIX cells adhered to and rolled on the activated endothelial monolayer but not on the unstimulated endothelium . A low level of CHO βIX adhesion to activated endothelium was noted, but this did not exceed the level of adhesion of these cells to the unstimulated endothelium. Adhesion of the CHO αβIX cells was blocked by the polyclonal antibody against P-selectin . As a final, and more physiological, test of the GP Ib-IX-V complex–P-selectin interaction, we studied the effects of antibodies to GP Ibα and P-selectin on platelet rolling on stimulated endothelium . Platelet suspensions (2 × 10 8 platelets/ml and 20% washed erythrocytes in PBS containing calcium and magnesium) were perfused over histamine-stimulated endothelium either without treatment or in the presence of antibodies against GP Ibα (SZ2 and AK2, alone and in combination), P-selectin (polyclonal anti–P-selectin), or von Willebrand factor (6G1). The number of platelets rolling was much greater on activated than on unactivated endothelium. Treatment with the two GP Ibα mAbs markedly inhibited adhesion and rolling of the platelets, with AK2 being a more potent inhibitor, reducing the number of rolling cells to the level seen on unstimulated endothelial cells. (We did not use AK2 in the experiments examining CHO-P adhesion to glycocalicin because it does not bind to immobilized glycocalicin.) Anti–P-selectin decreased platelet rolling to levels even below those of the unstimulated endothelial cells, suggesting a background level of endothelial cell activation. The anti–von Willebrand factor mAb, 6G1, did not significantly affect platelet rolling. Here we demonstrate a specific interaction between a platelet adhesion receptor, the GP Ib-IX-V complex, and P-selectin. This interaction represents a potential new paradigm for platelet adhesion, where the same receptor (GP Ib-IX-V) can support platelet adhesion to endothelial cells under the appropriate conditions of endothelial activation and to the subendothelial matrix when the endothelial cells have been removed by injury. Several interesting parallels exist between GP Ibα and PSGL-1 as cell adhesion receptors. Both are membrane mucins with elongated structures formed by the presence of a heavily O -glycosylated region rich in threonine, serine, and proline residues 38 39 . In both receptors, this mucin-like region separates the ligand-binding region from the plasma membrane 40 41 , and in both it contains tandemly repeated sequences that account for polymorphism in the human population because of variable numbers of the tandem repeats 42 43 . The major carbohydrate structures of the two receptors are very similar, with only a fucose in α(1,3) linkage in the major PSGL-1 carbohydrate chain differentiating it from the major carbohydrate species of GP Ibα. Furthermore, both receptors contain a highly acidic region with sulfated tyrosines that is important for ligand binding and lies immediately NH 2 -terminal to the mucin-like region 4 5 6 7 14 15 16 . The major ligands for both receptors (von Willebrand factor and P-selectin) are synthesized only in megakaryocytes and endothelial cells and are stored in the same subcellular compartments, the α-granules of platelets and megakaryocytes and the Weibel-Palade bodies of endothelial cells. Despite all of these similarities, the interactions of the two mucins with P-selectin are quite different. Whereas PSGL-1 requires carbohydrate modification with core-2 branching and α(1,3)-fucosylation and the presence of calcium for its interaction with P-selectin, GP Ibα requires neither. This lack of a requirement for carbohydrate modification is consistent with the findings of Frenette et al. 8 that the platelet P-selectin counterreceptor does not require modification by FucTs for function. The different carbohydrate requirements of the two P-selectin counterreceptors may not be absolute but rather may represent a difference in the relative importance of the two P-selectin binding determinants (carbohydrates and sulfated tyrosines). GP Ibα does not contain the fucosylated moiety that is so vital for PSGL-1; on the other hand, its anionic region has a much higher concentration of negative charge than does the analogous region of PSGL-1, as well as three tyrosines that are fully sulfated 14 16 . This region of GP Ibα is known to bind tightly to thrombin and does so by interacting with the same region of thrombin that binds heparin 31 32 . Thus, the anionic sulfated region of GP Ibα, with its high negative charge and sulfate groups, appears to resemble heparin. Several lines of evidence point to the importance of this heparin-like region of GP Ibα in the interaction with P-selectin. First, the calcium independence of the interaction resembles the interaction of P-selectin with heparin, as does the pattern of blockade with heparin and fucoidin, but not with chondroitin sulfate 17 . Second, the interaction is inhibited by SZ2, an mAb that binds within the region and also partially blocks botrocetin-induced von Willebrand factor binding, a phenomenon known to be sensitive to the removal of tyrosine sulfates 15 16 . Finally, mocarhagin, the cobra venom metalloprotease that removes most of the heparin-like region, also inhibits binding. Thus, the GP Ibα–P-selectin interaction is almost completely independent of carbohydrate, although the failure of SZ2 or mocarhagin to fully inhibit the interaction suggests that carbohydrate may have a minor role. The possible involvement of other regions of GP Ibα in the interaction is also suggested by the inhibition by the mAb AK2 of platelet rolling on stimulated endothelial cells. This antibody binds within the leucine-rich repeats of GP Ibα 16 . However, because the three-dimensional relationship of the leucine-rich repeats with the sulfated region in three dimensions is not known, we cannot definitively make a case for the involvement of the leucine-rich repeats in the interaction. The potential involvement of all of the regions of GP Ibα will have to await more definitive studies with mutant polypeptides. The adhesion of platelets to regions of vessel wall injury mediated by von Willebrand factor can occur at very high shear stresses, approaching 100 dynes/cm 2 . The interaction between platelets and endothelium, on the other hand, seems to occur at much lower shear stresses, although evidence exists that platelets can interact with endothelium at shear stresses above those able to support the interaction of leukocytes with the endothelium 44 . The studies reported here were carried out at venular shear stresses and do not address the possibility that the GP Ibα–P-selectin interaction may be able to support platelet rolling at shear stresses much higher than those that support leukocyte rolling. The demonstration of an abundant receptor on the platelet surface for P-selectin brings into sharp focus the potential roles of this receptor–counterreceptor interaction in both physiological and pathological conditions. During hemostasis, the interface between the GP Ib-IX-V complex and P-selectin may be important in the interaction between activated and unactivated platelets. Activated platelets with surface-exposed P-selectin could associate with and activate previously unactivated platelets, thus providing a mechanism for propagation of platelet thrombi. Failure of this mechanism could explain the mild bleeding diathesis of P-selectin–deficient mice 45 and could contribute to the bleeding tendency in patients with Bernard-Soulier syndrome, the genetic disorder resulting from deficiency of the GP Ib-IX-V complex 46 . Nevertheless, this mechanism is likely to be only of secondary importance, as no defect in aggregation has been found in the platelets of patients with Bernard-Soulier syndrome (although a subtle defect might not be detected by conventional methods of performing aggregation). Under certain circumstances, platelets may help neutrophils and other white cells to exit the vasculature by providing an additional ligand for recognizing activated endothelial cells. Such a role has been demonstrated for platelets in lymphocyte trafficking, where activated platelets bound to the lymphocytes can provide a ligand (P-selectin) for the endothelial addressins of lymph nodes 47 . Platelets interacting with activated endothelium may themselves become activated, releasing factors that may influence the behaviors of both the endothelial cells and underlying smooth muscle cells. When such a process is unchecked, such as during sepsis, the platelets may contribute to the deleterious effects of the underlying condition. In this regard, recent findings suggest that activated platelets also form stable attachments with endothelial cells mediated in part by other platelet GP receptors, such as the integrin GP IIb-IIIa 48 . At this point, however, the potential biological consequences of platelet interaction with endothelium are speculative. Nevertheless, with the demonstration of such a specific interaction involving endothelial selectins 9 and the current identification of a platelet receptor for these selectins, the endothelium can no longer be considered refractory to the overtures of the platelet. | Study | biomedical | en | 0.999997 |
10499920 | Isolation of YT cell granule contents (GC) and purification of granzyme B was as described 21 . Purified caspase-3 and -8 and CrmA (cytokine response modifier 1) were gifts from Nancy Thornberry (Merck Research Labs., Rahway, NJ). cDNAs for CENP-B, fibrillarin, topoisomerase I, and post meiotic segregation (PMS)1/PMS2 were gifts from Drs. Ann Pluta (University of Maryland, Baltimore, MD), John Aris (University of Florida, Gainesville, FL), Barbara White (University of Maryland, Baltimore, MD), and Bert Vogelstein (Johns Hopkins University), respectively. Autoantibodies to PMS1 and PMS2 are found in 3–5% of patients with autoimmune myositis 22 . The patient serum recognizing ribosomal protein P was a gift from Dr. Keith Elkon (The Hospital for Special Surgery, NY, NY). All data shown represent 2–20 separate experiments. HeLa cells were passaged in 10% heat-inactivated calf serum using standard tissue culture procedures. To induce apoptosis, cells were incubated with 1,650 J/m 2 UVB and incubated overnight 3 . In Fig. 1 A, the gel samples used in the lanes marked “Apoptotic Cells” consisted of pooled adherent and floating populations. HeLa lysate was prepared as described 6 and incubated at 37°C for 1 h with either 0.8 nM purified caspase-3 and 5 mM dithiothreitol (DTT) or purified granzyme B and 2 mM iodoacetamide (IAA). In the experiment shown in Fig. 7 B, GC were added to lysates to induce cleavage of autoantigens; the amount of GC used was based on its granzyme B activity (1 μl of GC preparation contained the same activity as 1 μl of purified granzyme B). To inhibit granzyme B activity in HeLa lysates, equimolar amounts of CrmA and granzyme B (in GC) were preincubated at 37°C for 5 min before adding to HeLa lysates. All samples were electrophoresed on 10% SDS-PAGE, with the exception of Ki-67 (7.5% SDS-PAGE). Proteins were then transferred to nitrocellulose or polyvinylidene difluoride and immunoblotted with patient sera recognizing Mi-2, PARP, SRP-72, U1-70kD, topoisomerase I, Ro-52, Ro-60, ribosomal protein P, Ku-80, or mAb to Ki-67 (Sigma Chemical Co.). Proteins were detected using horseradish peroxidase–labeled secondary antibodies and chemiluminescence (Pierce Chemical Co.). HeLa lysate was prepared as described 23 , except that DTT was omitted from the lysis buffer. To inactivate endogenous caspase activities, 1 mM IAA was added to the lysates and incubated for 15 min at 4°C. 5 mM DTT was then added in the absence or presence of 50 nM caspase-8, and the reactions were incubated for 1 h at 37°C. Before using the “IAA poisoning protocol” in the caspase-8 experiments, the validity of this experimental approach to inactivate endogenous caspases present in lysates while still permitting exogenously added caspases to cleave was tested. As PARP and U1-70kD are well characterized caspase-3 substrates (generating signature fragments of 89 and 40 kD, respectively ), their ability to be cleaved by caspase-3 in IAA-treated lysates was used in the following defined test system: (i) HeLa lysates containing 0.8 nM caspase-3 and 5 mM DTT were incubated for 1 h at 37°C; the extent of PARP (66%) and U1-70kD (25%) cleavage obtained under these conditions represented maximum possible caspase-3 activity. (ii) 1 mM IAA was added to HeLa lysates prepared in the absence of DTT. After incubating for 15 min at 4°C, 0.8 nM caspase-3 was added and the lysates were further incubated (1 h at 37°C); PARP and U1-70kD were not cleaved under these conditions. (iii) HeLa lysates were incubated as in (ii), except that 5 mM DTT was added simultaneously with caspase-3 after the IAA poisoning step. Both PARP and U1-70kD were cleaved (the amounts of cleavage were 80 and 70%, respectively, of that noted in approach [i]), confirming that exogenous caspases are active in these lysates in which endogenous caspases have previously been inactivated if subsequently added with DTT. Abolition of the contribution of endogenous caspases under these circumstances accounts for the decreased total substrate cleavage observed. [ 35 S]methionine-labeled CENP-B, fibrillarin, PMS1, and PMS2 were generated by coupled in vitro transcription/translation 23 . Cleavage reactions were performed in buffer A consisting of 10 mM Hepes, pH 7.4, 2 mM EDTA, and 1% NP-40 (5 mM DTT was added to the reactions containing caspases). Reactions contained the amounts of caspase-3 and granzyme B indicated in the figure legends and were incubated at 37°C for either 15 or 60 min, as specified. After terminating the reactions by adding gel buffer and boiling, samples were electrophoresed on 10% SDS-PAGE (CENP-B, PMS1, and PMS2) or 12% SDS-PAGE (fibrillarin), and their fragments were visualized by fluorography. HeLa cells were labeled for 2 h with [ 35 S]methionine/cysteine, and the lysates were immunoprecipitated with patient sera recognizing histidyl tRNA synthetase, RNA polymerase II large subunit, PMScl, or alanyl tRNA synthetase, followed by protein A–agarose. The beads, containing washed, radiolabeled endogenous proteins were resuspended in buffer A in the presence or absence of granzyme B and incubated for 15 min at 37°C. The following amounts of granzyme B were used: 46 nM histidyl and alanyl tRNA synthetases and 23 nM RNA polymerase II and PMScl. Samples were electrophoresed on 10% SDS-PAGE, and radiolabeled proteins and their fragments were visualized by fluorography. HeLa lysates were incubated with granzyme B for 1 h at 37°C and then electrophoresed on 8 or 10% SDS-PAGE and immunoblotted with a rabbit polyclonal antibody to Cdc2p34 (Santa Cruz Biotechnology) or mAbs to vinculin or β-tubulin (Sigma Chemical Co.). Cleavage of purified human lactoferrin, apotransferrin, and thrombin (all from Sigma Chemical Co.) was performed by incubating 20 μg of each substrate in the absence or presence of 30 nM granzyme B for 60 min at 37°C. The reactions were terminated by adding gel application buffer, and the samples were electrophoresed on 10% SDS-PAGE and visualized by Coomassie blue staining. Similar amounts of purified La were well cleaved under identical conditions (data not shown). k cat / K m values were determined as described 21 23 using endogenous substrates in cell lysates, radiolabeled endogenous immunoprecipitated substrates, or radiolabeled substrates generated by coupled in vitro transcription/translation. The percent cleavage of each substrate was determined by densitometry; these values were fitted to the first order rate equation, \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*}{\mathrm{\%\;substrate\;cleavage}}=100{\times} \left \left(1 \right - \left {\mathrm{e}}^{-{{\mathit{k}}_{{\mathrm{cat}}}{\cdot} \left \left[{\mathrm{E}}\right] \right }/{K_{{\mathrm{m}}}{\cdot}\;{\mathrm{time}}}}\right) \right {\mathrm{,}}\end{equation*}\end{document} to calculate k cat / K m . For several of the autoantigens listed in Table , k cat / K m values were obtained using two or all three of these methods, and equivalent results were obtained (reference 21; data not shown). K562 cells were incubated with YT cell GC as described 21 , except that the incubations were done in HBSS containing 10 mM Hepes, pH 7.4. Cells were incubated for 2 h at 37°C in the absence or presence of added GC, before terminating reactions by adding 2× SDS–gel buffer directly to cell suspensions. Similar experiments were performed using cultured HeLa cells, human umbilical vein endothelial cells, human myoblasts, and Jurkat cells with identical results (data not shown). LAK cells were prepared and incubated with K562 target cells at a 3:1 ratio in the absence or presence of 100 μM Ac-DEVD-CHO or 50 μM Z-IETD-fluoromethylketone (FMK; Calbiochem Corp.) as described 21 . Incubation of either LAK or K562 cells individually with these inhibitors did not affect the autoantigens analyzed (data not shown). cDNA clones encoding fibrillarin, La, U1-70kD, Mi-2, PMS1, PMS2, and topoisomerase I were used as templates for mutagenesis by overlap-extension PCR to generate clones containing putative granzyme B site P 1 Asp→Ala substitutions 21 . [ 35 S]methionine-labeled polypeptides were generated by coupled in vitro transcription/translation and used as substrates for granzyme B cleavage as described above. We initially determined whether and how efficiently a variety of well-defined autoantigens across the spectrum of systemic autoimmune diseases were cleaved by purified granzyme B ( Table ). We used autoantibodies of known specificity to immunoblot lysates of HeLa cells that had been incubated in vitro with or without granzyme B. Lysates were pretreated with IAA (which covalently modifies the active site cysteine of caspases) to prevent endogenous caspase activity. Interestingly, several autoantigens that have previously been shown to be cleaved by caspases during apoptosis were also efficiently cleaved by granzyme B, generating unique fragments in every case. These substrates included U1-70kD, topoisomerase I, SRP-72, PARP , and NOR-90 (data not shown). We also identified Mi-2, Ki-67, PMS1, PMS2, and La as additional autoantigens that are cleaved by both caspases and granzyme B; again, distinct fragments were generated by the two proteases . Whenever possible, efficient cleavage by granzyme B and generation of novel fragments was confirmed using both endogenous substrates in lysates (detection by immunoblotting) and in vitro–translated substrates (detection by fluorography; this was done for U1-70kD, PARP, NuMA, topoisomerase I, La, PMS1, and Mi-2). The k cat / K m values varied between 1.3 × 10 4 M −1 .s −1 (U1-70kD) and 1.6 × 10 6 M −1 .s −1 (topoisomerase I) ( Table ). Several substrates were cleaved by granzyme B at multiple sites , generating a complex profile of cleavage fragments. Previous studies have identified several autoantigens that are not susceptible to cleavage by caspases during apoptosis 7 9 . Using the immunoblotting system described above and/or cleavage assays using in vitro–translated substrates, we demonstrated that many of these autoantigens are efficiently cleaved by granzyme B. These molecules included fibrillarin, Ku-70, and RNA polymerase II large subunit. . As some well-defined autoantibodies do not recognize their antigens by immunoblotting, we also addressed the susceptibility of radiolabeled endogenous substrates to cleavage by granzyme B in vitro. To perform these studies, HeLa cells were radiolabeled with [ 35 S]methionine/cysteine, and proteins were immunoprecipitated using human autoantibodies. Protein A–agarose beads containing washed, precipitated proteins were resuspended in buffer supporting the activity of granzyme B and incubated in the absence or presence of added purified granzyme B. To confirm the validity of this approach, we tested several different autoantigens known to be cleaved by granzyme B, as well as several autoantigens and nonautoantigens that are not cleaved by granzyme B. The cleavage profiles obtained after incubating granzyme B with (a) lysates (followed by detection with immunoblotting) or (b) immunoprecipitated radiolabeled antigens (followed by detection with fluorography) were compared. Identical results were obtained using these two methods for cleaved autoantigens (topoisomerase I, Mi-2, RNA polymerase II large subunit, Ku-70, PARP, La, and NOR-90), uncleaved autoantigens (Ku-80, Ro-60kD), and control substrates (β-tubulin, vinculin). Using this immunoprecipitation approach, we demonstrated that several additional autoantigens (PMScl, RNA polymerase I and II large subunits, histidyl tRNA synthetase, isoleucyl tRNA synthetase, and alanyl tRNA synthetase) were indeed cleaved by granzyme B, generating unique fragments . Using identical immunoprecipitation conditions, these substrates were not cleaved by caspase-3, whereas Mi-2 cleavage by caspase-3 occurred normally (data not shown). Several autoantigens were not susceptible to cleavage by either caspases or granzyme B . These included Ro-52kD and -60kD, ribosomal protein P, histones, Sm proteins, threonyl tRNA synthetase, glycyl tRNA synthetase, and Ku-80. Susceptibility to cleavage by granzyme B was a highly specific feature of autoantigens; nonautoantigens were either not cleaved by granzyme B or were cleaved but without producing novel fragments 25 26 27 . The following human molecules were not cleaved by granzyme B: α1-antitrypsin, β-tubulin, apotransferrin, C3 (β chain), carbonic anhydrase, CDC2 p34, C-reactive protein, glutathione S -transferase, glycogen phosphorylase, hemoglobin, IgG, lactoferrin, lysozyme, orosomucoid, thrombin α chain, thrombin β chain, and vinculin. Procaspases 3 and 7, which have not been found to be autoantigens during screening of >500 autoimmune sera by immunoblotting (Casciola-Rosen, L., and A. Rosen, unpublished data), are efficiently cleaved by both granzyme B and caspase-8, generating identical fragments 25 26 27 . Thus, in addition to the three autoantigens we have previously described as being cleaved by both caspase-3 and granzyme B (DNA-PK cs , NuMA, PARP), these studies identify an additional nine autoantigens that are cleaved by both proteases but at different sites. Furthermore, another nine autoantigens are cleaved exclusively by granzyme B and not by caspases. Therefore, 21 autoantigens targeted across the spectrum of human systemic autoimmune diseases are efficiently cleaved by granzyme B, generating unique fragments not observed during other forms of cell death. To confirm that similar autoantigen fragments are generated in intact cells during granule-induced cell death, we exposed K562 cells to YT cell GC and analyzed the biochemical status of the autoantigens by immunoblotting. In those cases where autoantigens are substrates for both caspases and granzyme B, signature fragments of both proteases were generated . The amount of granzyme B–specific fragments generated was enhanced in the presence of 100 μM Ac-DEVD-CHO, a caspase inhibitor (see below). Autoantigens known to be cleaved only by granzyme B were also cleaved in the K562/YT granule system; the granzyme B–specific fragments of Ku-70, RNA polymerase II large subunit , and PMS1 (data not shown) were generated. We next determined whether autoantigens cleaved by granzyme B in vitro are cleaved during killing of Fas-negative target cells by LAK cells. Granzyme B–specific fragments of Mi-2, topoisomerase I, U1-70kD, and SRP-72 , as well as PMS1, Ku-70, and La (data not shown), are generated during this form of cell death. For the antigens shown in Fig. 6 , which are directly cleaved by both caspase-3 and granzyme B, the amounts of granzyme B–specific fragments are determined by the relative efficiency of cleavage by the two proteases ( Table ). Thus, granzyme B–mediated cleavage of topoisomerase I is ∼50-fold more efficient than cleavage by caspase-3, and granzyme B–specific fragments are the most prominent . Where the efficiency of substrate cleavage by granzyme B and caspase-3 are similar (e.g., DNA-PK cs and Mi-2), both caspase- and granzyme B–specific fragments are generated. Inhibition of caspases abolishes the caspase-specific fragments only. In contrast, where substrates are cleaved ∼200-fold more efficiently by caspase-3 than by granzyme B (PARP, U1-70kD), no granzyme B–specific fragments were observed in the intact cell killing assay unless caspases were inhibited by adding Ac-DEVD-CHO . Production of unique autoantigen fragments in the LAK cell assay was decreased by inhibitors of granzyme B 28 . Thus, 50 μM of Z-IETD-FMK greatly diminished cleavage of NuMA, Mi-2, and topoisomerase I, as evidenced by the increased amount of intact antigen detected by blotting . This was accompanied by an abolition of the unique cleavage fragments of NuMA (175 kD) and topoisomerase I (98, 75, and 72 kD) and markedly inhibited the production of granzyme B–specific fragments of Mi-2 (48 kD). This pattern of inhibition correlated well with the efficiency of cleavage of these substrates by granzyme B ( Table ). As noted above, production of granzyme B–specific fragments of autoantigens was either unchanged (Mi-2) or enhanced (PARP, U1-70kD, SRP-72) by treatment with the caspase-specific inhibitor, Ac-DEVD-CHO . Similar results were also obtained when 143 nM CrmA was used to inhibit granzyme B activity in cell lysates in which granzyme B–specific fragments of U1-70kD and PARP were abolished , with concomitant recovery of intact antigen. Although both Z-IETD-FMK and CrmA also inhibit group III caspases, unique fragments of these autoantigens are not generated by these proteases . The specificity of granzyme B has recently been defined using a positional scanning combinatorial tetrapeptide library 29 30 . The protease has a preference for I, V, or L in P 4 , E, G, or S in P 3 , and P, S, N, A, Q, H, T, V, E, or D in P 2 , with a near absolute preference for D in P 1 . This specificity and the sizes of the fragments generated by granzyme B cleavage were used to predict cleavage sites. Using site-directed mutagenesis to make a series of P 1 Asp→Ala substitutions in several of the granzyme B substrates, we addressed the effects of mutation on the efficiency of cleavage by the protease and thus defined the granzyme B cleavage sites in fibrillarin, Mi-2, topoisomerase I, PMS1, PMS2, La, and U1-70kD . The granzyme B cleavage sites in PARP and DNA-PK cs were defined previously 21 31 . In every case, the cleavage site sequence is in accord with the specificity determined by the combinatorial library. The P 2 and P 3 residues in these cleavage sites were quite restricted, with prominent representation of P, A, and S in P 2 and G, E, T, D, and S in P 3 . Interestingly, many of these residues are preferred by granzyme B but not by group III caspases 29 . Using fragment sizes to predict likely granzyme B cleavage sites in other autoantigens, we readily identified probable cleavage sites in these proteins ( Table ). Other than the consensus tetrapeptide sequence preceding the cleavage site in these autoantigens, there were no obvious similarities in primary sequence either upstream or downstream of this site. Interestingly, several granzyme B substrates also have consensus tetrapeptide sequences that are not cleaved, indicating that additional conformational information influences susceptibility to cleavage at these sites. In addition to the granule exocytosis pathway, CTLs can also induce target cell proteolysis and apoptosis through ligation of target cell Fas by CTL Fas ligand 32 . As caspase-8 is prominently activated when CTLs induce target cell death through the Fas pathway 33 and because group III caspases have a very similar substrate specificity to granzyme B 29 , we determined if caspase-8 could generate the same proteolytic fragments of endogenous autoantigens in cell lysates that are generated by granzyme B. As caspase-8 efficiently activates precursor effector caspases in cell lysates (which in turn efficiently cleave downstream substrates), we first irreversibly inactivated these caspase precursors with IAA. Exogenous caspase-8 was then added in the presence of excess DTT, and substrate cleavage was assayed by immunoblotting. We initially confirmed that caspase-8 was active in the lysate system by using a coupled proteolysis system to demonstrate cleavage of downstream caspase-3 substrates . None of the autoantigens cleaved by granzyme B was cleaved by caspase-8; data for Mi-2, topoisomerase I, Ku-70, and RNA polymerase II large subunit are shown in Fig. 9 . We have demonstrated that components of 21 out of 29 well-defined autoantigens are susceptible to efficient cleavage by granzyme B, generating unique fragments. Although the cleaved molecules differ markedly in subcellular location, function, and extended primary sequence, they share two features: (a) all are autoantigens targeted by a high titer autoantibody response in human autoimmune diseases, including systematic lupus erythematosus, Sjogren's syndrome, diffuse and limited scleroderma, and autoimmune myositis; and (b) molecules are unified by containing a granzyme B cleavage site not susceptible to cleavage by caspase-8. The status of a molecule as an autoantigen and its unique susceptibility to cleavage by granzyme B but not by caspase-8 are therefore highly related . The positive predictive value of susceptibility to unique cleavage by granzyme B and status as an autoantigen is 100% for the 48 molecules studied, whereas the negative predictive value is 73%, indicating that additional mechanisms play a role in selection of some molecules as autoantigens. It is noteworthy that most of the uncleaved molecules are nucleoprotein complexes (e.g., components of nucleosomes and small nuclear ribonucleoproteins); we are in the process of defining whether these molecules undergo distinct structural modifications during unique forms of apoptotic cell death. Indeed, there are several other components of CTL granules that might play a role in this regard. Interestingly, the autoantigens susceptible to cleavage by granzyme B include antigens that are targeted across the spectrum of autoimmune rheumatic diseases. The striking correlation of specific autoantibody response with unique biologic phenotype (e.g., Mi-2 in dermatomyositis and topoisomerase I in diffuse scleroderma) raises the question of how this disease specificity arises if a common mechanism (CTL granule–induced death) is responsible for targeting of this group of molecules. Although this remains unclear, the immunizing tissue and initiating stimulus may play important roles in focusing subsequent, self-sustaining injury. Several of the cleaved molecules are also cleaved by effector caspases during apoptosis but at different sites in each case. Frequently, the granzyme B and caspase-3 cleavage sites are close to each other in the primary sequence. The striking linkage of susceptibility to cleavage by both caspases and granzyme B in these substrates (but at different sites) suggests that the two different families of apoptotic proteases recognize two distinct structural features of a common motif that have been targeted during independent evolution of the apoptotic cysteine and serine protease families. The likelihood that a functional motif has been targeted by the apoptosis-specific proteases is further underscored by the observation that the granzyme B cleavage sites in several molecules (e.g., fibrillarin, PARP, and PMS1) are highly conserved, even in drosophila and yeast. This striking conservation of sequence at granzyme B cleavage sites in organisms in which CTLs have not yet evolved implies that an important, as yet undefined function is served by these regions that is altered by proteolysis. This new, extended family of granzyme B substrates therefore provides a powerful tool with which to explore the evolution and biological functions of the aspartic acid–specific apoptotic proteases and the specific mechanisms underlying CTL granule–induced cell death. Using a combinatorial scanning tetrapeptide library, the specificity of granzyme B and caspases has recently been determined 29 30 and divides the caspases into three distinct groups. Group II caspases have a DXXD specificity and act downstream in apoptosis as effector proteases, cleaving substrates that have homeostatic and structural functions 34 . In contrast, the group III caspases prefer tetrapeptide substrates with I, V, or L in P 4 , E, D, or Q in P 3 , and H, I, T, W, or V in the P 2 position 29 . Whereas granzyme B has a similar specificity to the group III caspases, it has a broader substrate specificity in the P 3 and P 2 positions. Thus, granzyme B will robustly cleave substrates containing G or S in P 3 (not tolerated by group III caspases) and prefers P, A, N, and Q in P 2 (none of which are tolerated by group III caspases). Interestingly, 10 of 11 proven granzyme B cleavage sites in autoantigens contain residues that are preferred by granzyme B but are poorly tolerated by group III caspases (molecules contain P , A , or S in the P 2 position; four cleavage sites also contain G or S in P 3 ; Table ). Consistent with this observation is the demonstration that none of these substrates could be cleaved by caspase-8 . In contrast, procaspase-3 and -7, which are not autoantigens, are efficiently cleaved by both caspase-8 and granzyme B at the same sites, generating identical fragments 33 . This marked skewing of granzyme B cleavage sites in autoantigens away from P 2 and P 3 residues that are preferred by both granzyme B and group III caspases strongly suggests that unique cleavage by granzyme B plays a role in selection of targets in this spectrum of autoimmune disease. We have previously demonstrated that DNA-PK cs , NuMA, and PARP are cleaved by both caspase-3 and granzyme B and that the most prominent fragments generated in lysates and intact cells reflect the relative cleavage efficiencies of the two proteases 21 . The studies reported here extend those observations to numerous additional autoantigens . Where cleavage of a substrate by granzyme B is equal to or more efficient than that by caspase-3, granzyme B–specific fragments are generated in the LAK/K562 system (e.g., DNA-PK cs , Mi-2, and topoisomerase I; Table ). In contrast, where cleavage by granzyme B is less efficient than cleavage by caspases (e.g., PARP, U1-70kD, SRP-72, and topoisomerase I [72- and 74-kD fragments]), effective generation of the granzyme B–specific fragments is only seen in intact cells when caspase activity is inhibited. This observation focuses attention on defining potential immunizing microenvironments and proimmune insults in which such circumstances may arise in vivo. Relevant possibilities include conditions where viral or endogenous caspase inhibitors are expressed 35 36 37 38 39 40 , as well as in long-lived cells or tumor cells that express low levels of specific effector caspases 41 42 . Human sy stemic autoimmune diseases represent a highly complex disease spectrum, with numerous variables affecting individual susceptibility, initiation, and tissue targets. By demonstrating that the autoantigens targeted across the spectrum of these diseases are unified by their susceptibility to efficient cleavage by granzyme B, with the generation of unique fragments not produced during any other form of cell death, these studies focus attention on the role of the CTL granule pathway in initiation of autoimmunity. Where substrates are cleaved by both caspases and granzyme B, effective generation of unique granzyme B fragments is dependent upon relative inhibition of the caspases . We therefore propose that during proimmune 43 intracellular infections occurring in a microenvironment in which caspase activity is under relative inhibition, production of unique granzyme B fragments is favored. In susceptible individuals in whom clearance of apoptotic material might be impaired 44 45 , suprathreshold amounts of these fragments accumulate and are effectively captured and presented by dendritic cells 46 47 48 49 . The resulting immune response is directed against products of CTL granule–induced death, generating an autoamplifying injury characteristic of these self-sustaining diseases. The recent observation of tumor-specific CTLs in paraneoplastic cerebellar degeneration suggests that such a mechanism may be more broadly applicable to other autoimmune syndromes 50 . | Study | biomedical | en | 0.999996 |
10499921 | A bone marrow (BM) aspirate was collected in preservative-free ammonium heparin (1,000 U/ml) from a 2 y, 202-d-old captive-reared rhesus macaque 1 mo after experimental inoculation with a macrophage-tropic variant of SIV mac239 . The BM aspirate was dispersed, and isopynic gradient-purified (Ficoll-Paque; Pharmacia) BM mononuclear cells were seeded into T-25 culture flasks and grown in the presence of endothelial serum-free media (GIBCO BRL) supplemented with 10% fetal bovine serum, 1% l -glutamine, 1% penicillin/streptomycin/neomycin, and 30 μg/ml endothelial cell growth supplement (GIBCO BRL). Primary BM mononuclear cell cultures developed cytopathic effects (CPE) after 10–12 d and were rapidly frozen in liquid N 2 and thawed, and then supernatants were clarified by centrifugation and filtered through a 0.45-μM membrane. The filtered BM mononuclear cell culture extracts were inoculated on primary rhesus monkey fibroblast cultures 24 . Fibroblast cultures developing CPE were scraped free into medium, pelleted at 400 g , washed in PBS, and suspended in cold Ito and Karnovsky's fixative (2.5% glutaraldehyde, 0.5% picric acid, 1.6% paraformaldehyde, 0.005% ruthenium red) in 0.1 M sodium cacodylate buffer, pH 7.4, for 2 h. Ito and Karnovsky's–fixed cells were washed in cacodylate buffer, postfixed in 1% OsO 4 and 0.8% K 3 Fe (Cn) 6 in cacodylate buffer for 1 h, rinsed in distilled H 2 O, and prestained in 2% aqueous uranyl acetate for 1 h. Fixed and prestained cells were dehydrated in a graded series of acetone, embedded in Epon 812 epoxy resin, polymerized at 60°C, and sectioned at 60 nm on an MT 5000 ultramicrotome. Copper grid–mounted sections were stained with lead citrate and uranyl acetate and viewed on a Phillips 300 electron microscope. Cell-free virus isolated from the primary BM mononuclear cell culture was serially passaged three times in primary rhesus fibroblasts to generate viral stocks, and the stocks were titered on primary rhesus fibroblasts before animal infection studies. For the isolation of vDNA, infected primary rhesus fibroblasts from two 850 cm 2 roller bottles were harvested and the cell debris removed by low speed centrifugation. The cell pellet was resuspended in culture medium, sonicated to release intracellular virus particles, and centrifuged to pellet cell debris. Virus was pelleted from supernatants by high speed centrifugation in a Beckman JA-14 rotor (Beckman Instruments, Inc.) for 1 h at 9,000 rpm and purified through a six-step sorbitol gradient ranging from 20 to 70% spun in a Beckman SW41 rotor for 2 h at 18,000 rpm. Gradient-purified virus was diluted in balanced buffered salts solution and pelleted through a 20% sorbitol cushion. The virus pellet was resuspended in Tris/EDTA buffer (TE; 10 mM Tris/HCl, pH 8.0, and 1 mM EDTA) and lysed in TE with 0.6% SDS and proteinase K (2 mg) at 37°C for 5 h. vDNA was isolated by CsCl 2 gradient centrifugation in a Beckman Ti 75 rotor at 38.4 × 10 3 rpm for 72 h, collected, and dialyzed against TE. PCR amplification of the herpesvirus polymerase gene was performed on purified vDNA using the following degenerated herpesvirus (HV) primers: HV-5, 5′-GCTCTAGATT(C,T)GA(C,T)AT(A,C,T)GA (A,G)TG(CT)-3′; and HV-6, 5′-GGATTCGG(G,A)AA(C,T) TC(G,A)TA(A,C,G,T)AC(A,C,G,T)TC-3′. The conditions for PCR amplification were as follows: 94°C for 2 min (1 cycle); 94°C for 1 min, 37°C for 1.5 min, 3-min ramp to 72°C, 72°C for 1.5 min (10 cycles); 94°C for 1 min, 50°C for 1 min, 72°C for 1.25 min (30 cycles); and 72°C extension for 10 min (1 cycle). Each PCR reaction used 0.1 μg of DNA, 50 pmol of each primer, 1 U of Vent polymerase, 40 μM of each deoxynucleotide triphosphate, 10 mM KCl, 10 mM Tris/HCl, pH 8.8, 10 mM (NH 4 ) 2 SO 4 , and 0.1% Triton X-100 in a final volume of 50 μl. Amplification was performed in a Perkin-Elmer 2400 Thermocycler. PCR products were run out on a 1% agarose gel; DNA fragments were isolated and restriction endonuclease digested with XbaI and EcoRI for cloning into pSP73 (Promega Corp.). Included in the degenerate PCR assay were DNA samples from rhesus CMV- and rhesus Epstein-Barr virus (EBV)-like lymphocryptovirus–infected cells 24 25 . DNA sequencing was performed with Applied Biosystems (ABI) PRISM Dye Terminator Cycle Sequencing Ready Reaction Kits with AmpliTaq DNA polymerase as per the manufacturer's instructions. The DNA sequence was then determined on an ABI 373A sequencer in the Molecular Biology Core at the Oregon Regional Primate Research Center (ORPRC, Beaverton, OR). All plasmids were sequenced in both directions to verify DNA sequences. Open reading frames (orfs) were identified with MacVector (version 5.0; Oxford Molecular Group), and amino acid sequences were aligned by the CLUSTAL method 26 used with the MEGALIGN program provided with DNASTAR biocomputing software for the Macintosh. Viral genomic DNA (5 μg) was restriction endonuclease digested with EcoRI, run out on a 1% agarose gel, and transferred to nitrocellulose. For use as a probe, the KSHV thymidylate synthase (TS) gene was generated by PCR amplification from DNA isolated from BCBL-1 cells 27 with oligonucleotide primers corresponding to a specific region of the KSHV genome encoding TS (nucleotides 20,236–20,650) 28 using standard PCR conditions. Immobilized vDNA was probed with random-primed, [ 32 P]dCTP-labeled KSHV TS for 16 h at 42°C. Hybridized membranes were then washed using conditions of moderate stringency (0.2× SSC and 0.1% SDS at 50°C) for 1 h, and bound probe was visualized by exposing DuPont-NEN reflection film to the washed membrane at −80°C with a DuPont-NEN reflection screen. The corresponding vDNA fragment found to hybridize was isolated and further digested with restriction endonuclease BamHI, which yielded a 3.4-kb fragment that was found to hybridize to the KSHV TS probe. This fragment was cloned into pSP73 and subjected to DNA sequence analysis as described above. Orfs were identified using MacVector software for the Macintosh, and the orfs were compared with SwissProt and PIR protein databases for homologous orfs with BLASTX. Alignments to identified proteins were generated either by the GAP program from the Wisconsin GCG analysis package (version 9.1; Oxford Molecular Group) or by the CLUSTAL program provided with the DNASTAR program. Sequences for RRV 17577 IL-6, TS, and MIP are available from EMBL/GenBank/DDBJ under accession number AF087411. DNA extractions from peripheral blood and tissues were performed with a Puregene DNA isolation kit essentially as described by the manufacturer (Gentra Systems). PCR amplification for detecting the presence of the RRV genome was performed with the following oligonucleotide primers: vMIP-1, 5′-CCTATGGGCTCCATGAGC-3′; and vMIP-2, 5′-ATCGTCAATCAGGCTGCG-3′. PCR amplification for detecting the rhesus EBV genome was accomplished with oligonucleotide primers derived from published sequences of the latent membrane protein (LMP)-1 29 . PCR amplification of the rhesus β-globin gene was used as a control for the amount and quality of DNA used for each reaction 30 . With the exception of β-globin, the conditions for PCR were 94°C for 2 min (1 cycle); 94°C for 0.5 min, 50°C for 0.5 min, 72°C for 0.5 min (30 cycles); and 72°C extension for 5 min (1 cycle). For the rhesus β-globin PCR, the annealing temperature was raised to 55°C. Each PCR reaction used 0.1 μg of total DNA, 50 pmol of each primer, 1 U of Vent polymerase, 40 μM of each deoxynucleotide triphosphate, 10 mM KCl, 10 mM Tris/HCl, pH 8.8, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , and 0.1% Triton X-100 in a final volume of 50 μl. The PCR reactions were run out on a 1% agarose gel, transferred to nitrocellulose, and probed with a [ 32 P]ATP-labeled oligonucleotide primer specific for vMIP (vMIP-3, 5′-ATATTAAACACTCGCCGC-3′), rhesus EBV LMP-1, or β-globin. Hybridizations were performed overnight at room temperature in 6× SSC, 0.1% SDS, and 10 mg/mL Escherichia coli tRNA. Southern blots were then washed with 2× SSC and 0.1% SDS twice at room temperature, followed by two washes for 1 h in 2× SSC and 0.1% SDS at 47°C (vMIP), 45°C (β-globin), or 52°C (rhesus EBV LMP-1), and bound probe was visualized by exposing DuPont-NEN reflection film to the washed membrane at −80°C. RRV 17577–infected cells were solubilized with 0.5% NP-40 and 1% sodium deoxycholate in PBS and clarified in a Beckman SW28 rotor at 23,500 rpm for 1 h at 4°C. The clarified supernatant was used as antigen for coating 96-well Maxisorp plates (500 ng/well; Nunc, Inc.). ELISAs were then performed essentially as described 31 . All aspects of the animal studies were performed according to institutional guidelines for animal care and use at the ORPRC. Rhesus monkeys were inoculated intravenously with cell-free supernatants containing the equivalent of 5 ng of SIV mac p27 prepared from Cos-1 cells transfected with a full length SIV mac239 molecular clone 32 . Before experimental SIV inoculation, PBMCs from the four monkeys were prescreened for in vitro susceptibility to SIV mac239 infection essentially as described 33 . 8 wk after SIV infection, animals 18483 and 18570 were inoculated intravenously with 5 × 10 6 PFU of RRV 17577. Two additional animals, 19092 and 19286, were also inoculated with 5 × 10 6 PFU of RRV 17577 as RRV-only controls. At sequential time points, animals were sedated with ketamine hydrochloride (10 mg/kg body weight) and given complete physical examinations. Venipunctures were performed, and EDTA- or preservative-free ammonium heparin–anticoagulated blood samples were collected and separated into plasma and PBMCs for analysis. Serial peripheral LN biopsies were performed, and PBMCs and dispersed LN mononuclear cells were cultured in 1640 medium (GIBCO BRL) supplemented with 20% fetal bovine serum, 1% l -glutamine, and 1% penicillin/streptomycin/neomycin to determine if continuously replicating or transformed cells were present. Plasma was monitored for SIV p27 during the first 4 wk after infection using the SIV p27 antigen capture ELISA (Coulter Immunology). Duplicate plating of 2 × 10 5 PBMCs or LN mononuclear cells on primary rhesus fibroblasts and PCR analysis of PBMCs and LN mononuclear cell DNA was used for RRV isolation and the detection of RRV DNA, respectively. Animals that became moribund were killed, and complete necropsies were performed. Tissues were snap-frozen in liquid N 2 for PCR analysis or fixed in neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for light microscopy. Fresh PBMCs and dispersed mononuclear cells from lymphoid tissues were collected for leukocyte subset analyses, primary culture, and virus isolation via cocultivation with primary rhesus fibroblasts or cryopreserved for PCR analysis. Alterations in subsets were monitored sequentially after experimental inoculations by FACS™ analysis (Becton Dickinson). T lymphocyte subsets were labeled with OKT4 (CD4; Ortho Diagnostic Systems, Inc.) and B9.11 (CD8, Coulter Immunology) mAbs. B lymphocytes were labeled with B-Ly-1 (CD20; Coulter Immunology) mAb. The CD23 and CD40 B cell activation markers were identified with EBVCS4 (provided by Dr. B. Sugden, University of Wisconsin, Madison, WI) and G28-5 (provided by Dr. J. Ledbetter, XCYTE Therapies, Seattle, WA) mAbs, respectively 34 35 . Total serum Ig (IgG and IgM) concentrations and κ and λ light ratios in total serum Ig were determined by nephelometry, and the Coombs' test (direct antiglobulin) was performed to determine if hemolytic anemia in one of the animals was immune mediated (Quest Diagnostics, Inc.). Primary cell cultures established with BM mononuclear cells from macaque 17577 developed CPE in the fibroblast-like adherent cells after 10–12 d in culture and were harvested for passage. Primary rhesus monkey fibroblast cultures were inoculated with filtered extracts from the primary BM cultures. 10–12 d after inoculation, fibroblast cultures developed CPE characterized by granular focal lesions followed by cell rounding and sloughing that generated holes in the monolayer . Transmission electron microscopic examination of the infected fibroblasts revealed 150–200-nm virus particles with characteristic dense cores resembling herpesviruses in the extracellular spaces as well as immature and mature virus particles in the nuclei and cytoplasm, respectively . This herpesvirus isolate was subsequently passaged, purified, and titered on primary rhesus fibroblasts, and vDNA was purified from cell-free virions for molecular characterization. The initial molecular characterization of the virus was conducted using degenerate PCR analysis targeting a conserved region of the vDNA polymerase gene (FDIEC-VYEFP) from beta- and gammaherpesviruses. Purified vDNA, which was capable of producing infectious virus from CaCl 2 -transfected fibroblasts, was subjected to PCR, and the resulting fragments were cloned and sequenced. As controls, we included DNA from rhesus CMV– and rhesus EBV–infected cells as well as DNA from uninfected cells. This comparative analysis revealed that the virus was a gammaherpesvirus, as the deduced amino acid sequence was more closely related to rhesus EBV than rhesus CMV ( Table ). Inclusion of amino acid sequences from other known gammaherpesviruses, such as KSHV and HVS, supported this assignment. Moreover, comparative analysis with the recently described RRV strain H26-95 revealed that the herpesvirus isolate recovered from rhesus macaque 17577 was an RRV. To further characterize RRV strain 17577, we screened vDNA, restriction endonuclease digested with EcoRI, by Southern blot analysis for the presence of a TS gene, an unspliced cellular gene homologue that is conserved in KSHV and HVS but not EBV. Using a KSHV TS gene fragment as a probe, a single DNA fragment was found to hybridize under conditions of moderate stringency (data not shown). This DNA fragment was subsequently isolated and further digested for subcloning and DNA sequence determination. DNA sequence analysis of a 3.4-kb fragment revealed three orfs, two with striking homology to KSHV and a single orf having 100% identity with RRV and limited homology to cellular IL-6 ( Table ). One orf encodes a homologue of KSHV MIP-II with 30.9% amino acid sequence identity, as defined by the GAP program provided with the Wisconsin GCG analysis package, and the second orf encodes a homologue of both cellular and vTS, with 66 and 64.6% amino acid sequence identity with KSHV and HVS, respectively. The third orf revealed 100% identity with RRV strain H20-95 IL-6, supporting our initial finding that the 17577 virus is an RRV. Rhesus macaque 17577 was humanely killed 503 d after SIV infection and found to have multicentric LPD involving both lymphoid and nonlymphoid tissues. Attempts to propagate continuously replicating or transformed cells from mononuclear cells dispersed from BM and LNs were unsuccessful. Histopathologically, there was marked follicular lymphoid hyperplasia in LNs, spleen, and BM. The follicular elements were dominated by giant, often irregularly shaped, secondary, reactive germinal centers. Follicular architecture in the cortices of LNs was accentuated by expanded cellular interfollicular zones dominated by plasma cells and vascular and stromal elements . The medullary cords were densely populated by plasma cells, and arborizing cords of plasma cells extended into the paracortical zones. Follicular lymphoid hyperplasia was particularly striking in the BM . The LPD lesions in nonlymphoid tissues were principally nodular, pleomorphic lymphoid masses composed of cytologically normal lymphocytes, plasma cells, and macrophage-like cells that effaced normal tissue architecture . To determine if the LPD lesions in macaque 17577 harbored RRV, oligonucleotide primers specific to the RRV MIP homologue were designed to detect vDNA by PCR amplification and Southern blot analysis. By semiquantitative PCR analysis, vDNA sequences were found to be present at >590 copies/0.1 μg of BM-derived DNA . As most animals held in captivity are naturally infected with RRV 18 36 , BM aspirates were obtained from other animals to determine if RRV is a common resident of BM. BM samples were collected from two SIV-infected animals without peripheral lymphadenopathy, and two additional samples were obtained from animals that were killed due to disease unrelated to SIV infection or LPD. Analysis of these samples revealed no detectable signal, suggesting that RRV DNA in the BM-derived DNA from these four animals was either absent or below the limits of detection . Finally, the BM-derived DNA from macaque 17577 was analyzed for rhesus EBV–specific sequences, as rhesus EBV has been shown to be associated with LPD in SIV-infected animals 37 . No signal corresponding to rhesus EBV DNA sequences was detected , indicating that rhesus EBV was not present in this lesion. To test whether RRV strain 17577 can cause disease in its natural host, six juvenile rhesus macaques were selected for experimental inoculation studies. All six macaques were ∼1.5 yr old and shown to be negative for RRV infection over a 6-mo period by absence of RRV-specific serum antibody as measured by whole-virus ELISA, failure to detect RRV sequences in peripheral blood leukocytes by PCR analysis, and failure to isolate RRV from PBMCs cocultured with primary rhesus fibroblasts. As >75% of immunocompetent animals in our colony are seropositive for RRV by 3 yr of age and do not develop spontaneous LPD, we initially inoculated four of the animals with SIV mac239 to provide a background of immunosuppression before experimental RRV inoculation. 8 wk after SIV infection, two of the four animals inoculated with SIV were inoculated intravenously with 5 × 10 6 PFU of cell-free RRV 17577. The two remaining SIV-infected animals were maintained as SIV-only infection controls. Two additional animals were infected with RRV 17577 to serve as RRV-only infection controls. Sequential FACS™ analysis of PBMCs from the experimental animals revealed limited CD4 + lymphocyte depletion (mean values = 900–1,100/μl) 2 wk after SIV infection in the SIV-infected animals, followed by a rebound and sustained CD4 + lymphocyte counts in the normal range (mean values = 1,500–2,000/μl), whereas CD8 + lymphocyte counts increased slightly compared with the preinoculation time points. Significant differences in the number of CD20 + B lymphocytes were observed among the three groups of animals. As early as 6 wk after RRV infection, the number of CD20 + B lymphocytes increased dramatically in the two RRV/SIV-infected macaques (mean values = 2,900–3,360/μl) and persisted for 34 wk in animal 18483 and 14 wk in animal 18570 . In contrast, the SIV-infected control animals exhibited persistently low numbers of CD20 + B lymphocytes (mean values = 200–300/μl) beginning 2 wk after SIV infection . The number of circulating CD20 + B lymphocytes increased slightly 2 wk after infection (mean values = 950–1,000/μl) in the RRV-only controls . Further FACS™ analysis revealed that the PBMCs from the two RRV/SIV-infected macaques expressed the CD40 activation marker coincident with CD20 but not CD23, which is expressed after rhesus EBV infection (reference 38 ; data not shown). RRV was readily recovered from PBMCs of the two RRV/SIV-infected animals as early as 4 wk after RRV infection in macaque 18570 and 8 wk after infection in macaque 18483. Subsequently, positive virus cultures were obtained repeatedly from these animals for the duration of the study . The PBMCs from the two RRV/SIV-infected animals were also shown to harbor RRV DNA 4 wk after RRV inoculation in macaque 18483 and 14 wk after infection in macaque 18570, as determined by PCR and Southern blot analysis for the vMIP gene. Interestingly, no substantial difference in cell-associated viral load was discernible in PBMCs throughout the course of infection by PCR or virus isolation (data not shown). The two SIV-only control macaques were consistently negative for RRV by both methods of detection . The RRV-only control macaques were positive for RRV by both methods of detection 2 wk after infection and remained positive through 6 wk after infection . The humoral response to RRV infection was assessed sequentially using a whole-virus ELISA. One of the two RRV/SIV-infected animals failed to develop a measurable antibody response to RRV, whereas a low antibody response to RRV was detected in the second RRV/SIV-infected macaque 4 wk after RRV infection . Both of the SIV-only controls remained seronegative for RRV for the duration of the study , whereas the two RRV-only controls developed strong sustained anti-RRV antibody responses beginning 2 wk after infection . Early symptoms of SIV infection included fever, mild skin rash, and malaise, coincident with primary SIV p27 antigenemia in the four SIV-infected macaques by 2 wk after SIV infection. 10 wk after RRV infection, the two RRV/SIV-infected macaques developed marked peripheral lymphadenopathy, which persisted for >56 wk in macaque 18483 and 31 wk in macaque 18570. Additionally, the two RRV/SIV-infected macaques developed marked, persistent splenomegaly with spleens ∼10 times normal size. 30 wk after RRV infection, macaque 18570 developed severe hemolytic anemia that was unresponsive to treatment with prednisolone and was killed 286 d after RRV infection. At necropsy, there was marked generalized lymphadenopathy, hepatomegaly, splenomegaly, diffuse erythroid hyperplasia in the BM, and membranous glomerulonephritis. Analysis of necropsy tissues for vDNA indicated that virus was present in all lymphoid organs . Of particular interest is the absence of vDNA in the BM, which also did not exhibit lymphoid hyperplasia observed in the BM of macaque 17577 from which RRV 17577 was isolated. Only slight transient peripheral LN enlargement was seen in the SIV- and RRV-only control macaques, and splenomegaly was not observed. Histopathologic examination of peripheral LN biopsy samples revealed marked differences between the three groups. 27 wk after RRV infection, LNs from the two RRV/SIV-infected macaques had marked angiofollicular lymphoid hyperplasia characterized by giant, secondary, reactive germinal centers that were frequently irregular in shape and lacked distinct mantle zones. Medullary cords and paracortical areas were infiltrated by sheets of plasma cells. The interfollicular zones were accentuated and relatively hypocellular, with increased vascularity and plasma cell infiltration . The lymphoid hyperplasia was persistent and multicentric. Virtually all of the LNs recovered from macaque 18570 at necropsy were profoundly enlarged. The morphologic changes were similar to those observed in the LN biopsy 27 wk after RRV infection, with the exception that the necropsy LN samples contained a mix of both secondary reactive and tertiary regressive germinal centers, with vascular hyalinization in most LNs. Plasma cells dominated the medullary cords, paracortex, and interfollicular zones. The spleen of macaque 18570 also contained large, irregularly shaped lymphoid follicles that lacked mantles and were composed of a mosaic pattern of hyalinized cell-poor areas and cellular areas of normally proliferating germinal center lymphocytes. Cords and islands of mature plasma cells were prevalent in the red pulp (data not shown). In contrast, LNs from the control SIV animals, collected 35 wk after SIV infection, had profoundly atrophied lymphoid follicles and hypocellular paracortices with condensed stromal elements characteristic of SIV-induced lymphoid atrophy . LN biopsies from the two RRV control macaques 9 wk after infection had normal histologic features . Immunophenotyping of LN mononuclear cells revealed significant differences in the number of CD20 + B lymphocytes in the LNs from the two RRV/SIV-infected macaques compared with LNs from macaques in the SIV- and RRV-only control groups. The four animals in the two control groups had twofold increases in the number of LN CD20 + B lymphocytes compared with preinoculation samples, whereas the two RRV/SIV-infected animals had fourfold increases in the number of CD20 + B lymphocytes compared with the preinoculation control samples and twofold increases over the postinfection samples from the two control groups . Attempts to culture continuously replicating or transformed cells from dispersed LN mononuclear cells and PBMCs were unsuccessful. Coinfection with SIV and RRV resulted in a marked increase in serum IgG ( Table ). Two RRV/SIV experimentally infected macaques, as well as macaque 17577 from which RRV strain 17577 was recovered, developed marked hypergammaglobulinemia (serum IgG >2,000 mg/dl). κ and λ Light chain levels in total plasma Ig increased from mean preinfection values of 96 and 121 mg/dl in 17577 and the two RRV/SIV experimentally infected macaques to 149 and 212 mg/dl, respectively. The pre- and postinfection mean κ and λ light chain ratios were 0.8 and 0.7, respectively, indicating that the increase in plasma light chain concentrations was not associated with appreciable skewing. Postinfection total plasma Ig κ and λ levels decreased in the single SIV-only animal tested , also without appreciable skewing. Terminal serum from macaque 18570 was also positive in the direct antiglobulin (Coombs') test, strongly suggesting that the hemolytic anemia in this animal was immune mediated. Modestly elevated serum IgG (1,233 mg/dl) was noted in SIV-only control macaque 18540. Increases in serum IgG were not observed in the second SIV-only control macaque or the two RRV-only control macaques (serum IgG <700 mg/dl). Normal polyclonal electrophoretic profiles were obtained from all serum samples (data not shown). In this study, we describe the isolation of RRV strain 17577 from an SIV-infected rhesus macaque that eventually developed LPD. Like the RRV isolate previously described by Desrosiers et al. 18 , strain H26-95, the isolate described here encodes a vIL-6–like cytokine gene. Moreover, this isolate also contains a vMIP-like homologue with amino acid sequence identity to KSHV vMIP-II. Sequence analysis of the entire RRV strain 17577 genome has recently been completed and reveals that the genome is essentially colinear with KSHV and possesses similar genes with the potential for transformation, regulation of apoptosis, or growth control as KSHV 40 . In particular, RRV possesses a gene, referred to as R1, in the same position as saimiri transforming protein and K1 of HVS and KSHV, respectively. BLAST analysis with R1 shows that the NH 2 terminus is similar to that of the Fc receptor, suggesting that R1 is a transmembrane receptor and therefore may be involved in signal transduction and transformation. RRV stain 17577 is also >99% identical at the DNA level to the regions of the RRV strain H26-95 genome available for analysis. The majority of base differences are found in the midregion of orf 8, glycoprotein B, and predicted to encode both conserved and nonconserved changes in the protein. Geographical variation in KSHV is increasingly documented in association with different forms of KS and KSHV infection rates and raises the possibility that sequence variation may relate to differences in virulence or transmission 41 42 43 44 45 . Interestingly, genotypic differences in glycoprotein B of human CMV are postulated to be associated with increased virulence in BM transplant recipients 46 . This same region has not been analyzed from different isolates of KSHV derived from AIDS-related KS, classical/endemic KS, or LPD, and the significance of the sequence variation in the RRV glycoprotein B gene is unknown. RRV strain 17577 was recovered from the BM of a naturally infected rhesus macaque that developed profound, persistent angiofollicular peripheral lymphadenopathy after experimental infection with a macrophage-tropic variant of SIV mac239 . Subclinical multicentric involvement of lymphoid tissues and solid organs was found at necropsy 503 d after SIV infection. PCR analysis of BM tissue exhibiting lymphocytic hyperplasia revealed that RRV DNA was present in high copy number but not in BM from normal macaques or in macaque 18570. We speculate that the difference in viral load in the BM of animal 18570 may be due to the lack of LPD in the BM, compared with the florid LPD lesions observed in the BM of macaque 17577. As a causal role for RRV in the development of these lesions cannot be established by PCR analysis alone, we undertook experimental inoculation studies in rhesus macaques to determine whether RRV is just a ubiquitous agent in nonhuman primates or a pathogen capable of inducing disease manifestations in its natural host. The absence of B cell hyperplasia in the SIV- or RRV-only controls and the presence of high RRV loads in hyperplastic lymphoid tissues from SIV and RRV–coinfected macaques indicate that B cell hyperplasia is strongly associated with RRV. A role for RRV in B cell hyperplasia or LPDs is consistent with the biology of most or all other gammaherpesviruses (KSHV, EBV, MHV-68, HVS). The index macaque and the macaques experimentally infected with SIV mac239 and RRV 17577 developed profound, persistent, multicentric angiofollicular lymphadenopathy resembling the multicentric plasma cell form of MCD either in the absence or presence of HIV infection 47 . The hypergammaglobulinemia in the index macaque and both experimental RRV/SIV-infected macaques and the fatal immune-mediated hemolytic anemia in one of the two experimentally RRV/SIV-infected macaques are also clinical features that closely parallel those observed in KSHV-infected AIDS patients and HIV-1–seronegative patients with MCD 48 49 50 . Both SIV and HIV-1 induce in vivo B lymphocyte hyperactivation, as shown by hypergammaglobulinemia, circulating immune complexes, and sometimes elevated levels of autoantibodies 49 51 52 . However, the consort of profound hypergammaglobulinemia, immune-mediated hemolytic anemia, and progressive lymphadenopathy reported here are unusual disease manifestations in SIV mac -infected macaques, and few cases are reported 20 39 53 54 . The RRV infection status in these animals is not known, as RRV was only recently identified. The absence of hypergammaglobulinemia, immune-mediated hemolytic anemia, and persistent angiofollicular lymphadenopathy in our SIV mac239 -only and RRV-only infection control macaques suggest that RRV coinfection may be an important factor in the immunopathogenesis of retrovirus-induced immunodeficiency disease in macaques. This cofactor role has long been recognized for EBV in human AIDS patients, and a similar role is evolving for KSHV. Despite the significant evidence of B cell hyperplasia in the peripheral blood, LNs, and spleens of the RRV/SIV coinfected animals, they do not appear to have overt clonal abnormalities in their B cells, nor were we able to establish continuously replicating primary B lymphocyte cultures from them. This differs considerably from rhesus EBV infection, where we have been able to generate continuously replicating cultures of rhesus EBV–positive B cells in vitro from PBMCs collected from naturally infected macaques (our unpublished results). The evolution of clonal abnormalities in LPDs is complex 55 , and although monoclonality was not evident in the LPD manifested in the RRV/SIV-coinfected animals, minor clonal abnormalities may be present. Recent studies suggest that differential expression of rhadinovirus cytokine and oncogene homologues may play an important role in the development of B cell hyperplasia and other abnormal cellular proliferations 15 48 56 57 . Although one of the experimental RRV/SIV-infected macaques developed fatal immune-mediated hemolytic anemia, none of the macaques developed lymphoma, and continuously replicating lymphoid cells could not be obtained from the RRV-positive, hyperplastic lymphoid tissues or peripheral blood. Immortalized KSHV + cell lines have also not been established from healthy KSHV + carriers or KS and MCD patients, with the exception of a continuously replicating KSHV + /EBV − cell line obtained from the peripheral blood of an HIV-seronegative patient with PEL who exhibited a phenotype similar to PEL-derived cell lines 58 . Thus, like KSHV, RRV may require other transforming events or cofactors for lymphomas to develop. Consistent with the association between high RRV load and B lymphocyte hyperplasia reported here, we have recently shown that healthy RRV carrier macaques harbor RRV in their CD20 + B lymphocytes in vivo 59 . Recent studies support the hypothesis that KSHV vIL-6 plays an important role in the pathogenesis of PEL and MCD 48 57 , and a similar role for the RRV IL-6–like cytokine is under investigation. Preliminary analysis of the RRV IL-6–like cytokine reveals that it can functionally substitute for cellular IL-6 in IL-6 bioassays 60 . IL-6 is a cytokine with several functions, including the ability to activate B cells into immunoglobulin-producing cells. More importantly, IL-6 dysregulation appears to be a factor in the development of MCD and multiple myeloma 61 62 . The experimental infection protocol used here, SIV mac239 infection before RRV infection, was designed to provide RRV a replicative advantage, as most herpesviruses, e.g., CMV and EBV, manifest disease in immunocompromised hosts. Moreover, serological analysis of the animals at ORPRC and other primate facilities reveal that >90% of immunocompetent animals are seropositive for RRV. Despite the high prevalence rates, few animals develop LPD, implying that the host immune response is capable of controlling the virus infection. What role SIV mac239 infection plays in the disease manifestations reported here is not clear. One possible mechanism is that SIV-induced immunosuppression may be important for sustained RRV infection and replication. Some have proposed a similar cofactor role for HIV-1 in AIDS-related KS and PEL, whereas others believe, at least in the histogenesis of KS, that HIV-1 invokes an inflammatory response, promoting an increase in inflammatory cytokines that sustain KS lesions 15 . We believe that RRV is likely an opportunistic infection in the SIV-infected animals, given the absence of similar disease manifestations in animals experimentally inoculated with RRV or SIV alone. Finally, in addition to providing an animal model for some aspects of KSHV pathogenesis, coinfection studies such as those described here should be important for AIDS pathogenesis. Preliminary results suggest that the presence of RRV attenuates SIV replication. The KSHV MIP-II protein has been shown to inhibit CCR-dependent HIV infection in vitro 63 64 . Like HIV, genetically divergent strains of SIV utilize CCR5 and other orphan seven-transmembrane receptors for viral entry 65 66 67 68 69 . With the availability of manipulatable molecular clones, we expect that RRV will be extremely useful as an animal model for studying aspects of both KSHV and AIDS pathogenesis. One significant advantage of RRV is that it can be propagated and plaque titered in cultured cells, allowing molecular dissection of genes postulated to be related to pathogenesis. Studies are in progress to determine the role of the vIL-6 homologue and other viral genes in KSHV-induced disease and in this animal model of B cell hyperplasia. | Study | biomedical | en | 0.999995 |
10499922 | Eight adults with a symptomatic viral illness after sexual exposure to HIV were referred to our clinic for evaluation. Seven subjects had acute HIV infection, as defined by detectable HIV RNA in the plasma and nonreactive HIV antibody test or indeterminate Western blot (see Table ). One subject presented with a positive HIV enzyme immunoassay and a positive Western blot (two bands; p24 and gp120/160 were positive) in the setting of exponential decline of plasma HIV RNA. All study subjects signed an informed consent approved by the University of California San Diego Human Subjects Committee. Baseline (day 0) demographic information, risk assessment for HIV exposure, clinical features, and duration of signs and symptoms compatible with an acute retroviral syndrome were recorded. The date of HIV infection was ascertained from patient history by Dr. S.J. Little. For individuals unable to identify a single high-risk exposure, the range of potential exposure dates was recorded and the midpoint in this range was assigned as the HIV exposure date. This was an observational study, and participants chose to initiate antiretroviral therapy in conjunction with their physicians. Subjects 001 and 002 subsequently interrupted their antiretroviral therapy against advice 160 and 34 d, respectively, after treatment initiation . Frequent whole blood samples were obtained for isolation of plasma and PBMCs. Samples were collected every 1–2 d for weeks 1 and 2, twice per week for week 3, weekly for weeks 4–6, and every 4 wk thereafter. Whole blood was collected in acid-citrate dextrose tubes and processed within 6 h of collection. Plasma was separated and stored at −70°C. PBMCs were separated by established density gradient methods and prepared by controlled rate freezing for storage at −150°C. HIV enzyme immunoassay (Abbott Labs.) and Western blot (Cambridge Biotech Corp.) were performed at presentation and repeated at regular intervals until a positive antibody and Western blot were documented. Plasma HIV RNA measures were performed by the Roche Amplicor Assay (Roche Molecular Systems) according to the manufacturer's instructions. The Roche ultrasensitive assay was used to determine HIV RNA copy number on a subset of samples. A virologic “plateau” was defined as at least two measures of HIV RNA, which varied by less than threefold and occurred before the initiation of antiretroviral therapy. Assessment of T cell subsets was performed on whole blood within 24 h of specimen collection by dual color FACS™ analysis (FACScan™; Becton Dickinson Cytometry Systems). Baseline (day 0) T cell subsets were collected within 7 d of the day 0 visit, except for subjects 001 and 002, for which the first CD4 assessments were performed 11 and 37 d after presentation, respectively. Fig. 2 schematically illustrates the viral dynamics of acute HIV infection and is adapted from a study of acute SIV dynamics 12 . This figure demonstrates the viral parameters measured in our study population. These parameters are based on a standard model of infected cell dynamics ( I ) where free virus is used as a surrogate for infected cells: d I d t =β IT −δ I I , where T represents the target cell number, δ I the death rate of infected cells, β the rate of new infection of uninfected cells, and I the number of infected cells. We do not explicitly include free virus; our model assumes that direct cell–cell transmission is the most important mode of spread of infection. If this were not true, we would use the same model, but the definition of β would change to include a constant of proportionality relating free virus density to infected cell density. The inferred growth rate, r 0 , represents the initial rate of rise of viremia per cell per day and is related to the model equation by setting r 0 =β T 0 −δ I so that d I d t = r 0 I and I t = I 0 e r 0 t . The inferred growth rate, r 0 , was measured directly in one of our study subjects (patient identification number [PID] 059). The observed rate of rise of viremia, r̂ , is the measured slope of rising viremia within days of the peak of viremia. Measurements of r̂ are obtained at a time when the slope of the observed rate of rise r̂ of HIV RNA is likely to be slower than that observed initially ( r 0 ). The inferred r 0 assumes that (a) the CD4 cell count before HIV infection ( T 0 ) is 1,000 cells/mm 3 , (b) the efficiency of infection, β, does not change between the time of infection and the observed time, and (c) the life span of an infected cell remains constant. The rationale underlying an inferred calculation for r 0 was that part of the slowdown in growth rate could be attributed to depletion of target cells 13 14 . This assumption is supported by the recent observation that in a comparison of SIV-infected monkeys with and without CD8 + cells, viral dynamics up to and including the time of peak viremia were not affected by the presence or absence of CTLs 15 . Using total CD4 counts as the best available surrogate for target cells and a nominal normal value of 1,000 cells/mm 3 , we corrected the observed rate of rise for the degree of target cell depletion. Based on the observed growth rate for the four study subjects identified with rising viremia r̂ , the following formula was used to calculate an inferred initial growth rate ( r 0 ): 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*}r_{0}= \left \left({T_{0}}/{{\hat {T}}}\right) \right \left \left({\hat {r}}+{\mathrm{{\delta}}}_{I}\right) \right -{\mathrm{{\delta}}}_{I}\end{equation*}\end{document} from the observed growth rate, r̂ , where T̂ represents the CD4 cell count at (or close to) the first viremia observation. If the infected cell death rate had already grown larger by the time we made our observations, this formula would underestimate r 0 by the amount of that change. The initial viral doubling time, t 2 , is calculated using the slope of rising viremia over time. The slope of declining viremia over time is used to measure the rate of decline of viremia during the subsequent spontaneous decline of viremia, α, and the phase 1 (first order) viral decay rate after initiation of potent antiretroviral therapy, δ I . In the two subjects who discontinued antiretroviral therapy against advice, the rate of rise of viremia, r , after interruption of therapy was also measured. The number of secondary infections arising from one infected cell over the course of its life span, when target cells are not depleted, is the basic reproductive number, R 0 . We estimated R 0 using a model that incorporates a fixed time delay, τ, between the infection of a target cell and the subsequent production of progeny virions. This fixed delay model gives R 0 as: \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*}R_{0}= \left \left({T_{0}}/{{\hat {T}}}\right) \right \left \left(1+{{\hat {r}}}/{{\mathrm{{\delta}}}_{I}}\right) \right {\mathrm{e}}^{{\hat {r}}{\mathrm{{\tau}}}}{\mathrm{.}}\end{equation*}\end{document} The actual duration of the intercellular lag between infection of a target cell and production of progeny virions is likely to be between 12 and 24 h, as determined by in vitro and in vivo estimates 16 17 18 . We used a 24-h delay (eclipse period) from the infection of one target cell and the subsequent production of progeny virions for the fixed delay model. A second model without delay was also investigated. Although this is not a biologically plausible model, it was included for comparison with prior studies in macaques 12 . The model without delay is a special case of the fixed delay model with the delay set to zero (τ = 0): \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*}R_{0}= \left \left({T_{0}}/{T}\right) \right \left \left(1+{{\hat {r}}}/{{\mathrm{{\delta}}}_{I}}\right) \right {\mathrm{.}}\end{equation*}\end{document} This model assumes the instantaneous production of progeny virions from the moment of initial infection of a target cell and almost certainly generates minimal estimates of R 0 . These methods of inferring r 0 and R 0 from r̂ and T̂ allow us to combine information on viral growth rate and degree of target cell depletion. They are sensitive to the allotted values of τ and T 0 . Thus, for patient 004, if T 0 takes values of 500, 1,000 or 1,500, then R 0 = 4.5, 9.1, or 13.6, respectively. If τ was 12 rather than 24 h, values of R 0 would be intermediate between those shown for the two different models. Eight subjects (seven men and one woman) were referred to our clinic 2–35 d after the onset of an acute retroviral illness and 12–50 d after high-risk sexual exposure to HIV ( Table ). Patient recall of HIV exposure date placed the interval between HIV exposure and the onset of symptoms between 8 and 47 d in this cohort (mean, 22 d). Seven of the eight subjects initiated potent antiretroviral therapy 26–63 d after their reported HIV exposure ( Table ). All subjects exhibited diffuse lymphadenopathy at presentation. Myalgias, fever, headache, and fatigue were reported in 63–88% of subjects. CD4 cell numbers at initial presentation were highly variable, ranging from 163 to 937 cells/mm 3 ( Table ). The CD4/CD8 T cell ratio was within a normal range (2.0 ± 0.8) in two of eight patients who presented less than 2 wk after HIV exposure but was low (0.2–1.0) in the remaining six subjects identified more than 2 wk after HIV exposure (data not shown). Four of the eight subjects were identified before attaining peak levels of acute viremia, which ranged from 2.5 to 22 million HIV RNA copies per milliliter of plasma . The mean time from reported HIV exposure to the peak measure of viremia was 21 d (range, 12–31 d; Table ). Sequential measures of plasma viremia were obtained to estimate the rates of rising HIV RNA ( r 0 ) before the initiation of antiretroviral therapy . Subject 059 had a serum HIV RNA of 30 copies/ml serendipitously obtained 3 d after his reported HIV exposure. His plasma HIV RNA copy number upon presentation with an acute retroviral illness 9 d later (12 d after reported exposure) was 21,617,100 copies/ml. The observed initial rate of rise of HIV RNA ( r 0 ) was 1.5/day in this subject, which corresponds to a viral doubling time ( t 2 ) of 12 h ( Table ). The observed rate of rise of viremia r̂ for the other three subjects ranged from 0.2 to 1.3/day ( Table ). Because these plasma HIV RNA measurements were collected within days of the peak and likely reflect a reduced exponential growth rate r̂ compared with the initial rate ( r 0 ), we calculated an inferred initial rate . These inferred r 0 values ranged from 1.4 to 3.5/day (mean, 2.0), and the mean doubling time ( t 2 ) was 0.3 d, or 7 h. Comparison of the observed initial rate of rise of viremia ( r 0 ) for subject 059 and the mean inferred rate, r 0 , for the remaining subjects revealed similar estimates of r 0 (1.5 and 2.0/day, respectively) and a mean viral doubling time ( t 2 ) of 10 h. The basic reproductive number ( R 0 ) is the estimate of the number of secondary infections arising from one infected cell over the course of its life span when target cells are not a limiting resource. Using a standard model, which assumes no delay between the infection of a cell and the production of progeny virions, R 0 values ranged from 5.2 to 9.1, with a mean value of 7.1 (95% confidence interval, 4.3–13.2; Table ). Slightly greater values were obtained using a fixed delay model for R 0 , which incorporates a 24-h delay between the infection of a target cell and the subsequent production of progeny. The fixed delay model, which is more biologically plausible, generated estimates of R 0 ranging from 7.4 to 34.0, with a mean value of 19.3 (95% confidence interval, 5.4–54.3). These estimates suggest that ∼19 infected cells will arise from each HIV-infected cell over the course of its life span when CD4 target cells are not limiting. Furthermore, these R 0 estimates predict that an antiretroviral regimen or vaccine, which is at least 95% effective, will prevent sustained viral propagation in the absence of drug resistance or viral latency. These are probably worst case estimates for the required efficacy of a prophylactic vaccine, as they are derived from viremic patients. This estimate of the value of the basic reproductive number must be treated as an attempt to gain an order of magnitude estimate rather than a definitive value. A spontaneous decline of viremia was observed in all subjects before initiation of antiretroviral therapy . The measured rates of decline of viremia (α), measured over the first 10 d of spontaneous viral decay, were highly variable, with a range of 0.1 to 0.8/day (mean, 0.3; Table ). The accompanying t 1/2 of spontaneous decline ranged from 0.9 to 12.3 d (mean, 2.4). An apparent virologic plateau, defined as at least two measures of HIV RNA that varied by less than threefold, was observed a mean of 33 d after the peak measure of viremia in seven of the study subjects (data not shown). In the absence of antiretroviral therapy, these early plateau values for HIV RNA ranged from 7,074 to 436,354 copies/ml (mean, ∼165,000 copies/ml) and were sustained for 2–4 wk in the absence of therapy. A subsequent further spontaneous decline of HIV RNA was noted in subjects 056 and 059, who did not initiate therapy during the plateau period. Seven of eight subjects initiated potent antiretroviral therapy during the first 2 mo after reported HIV exposure. The initiation of potent antiretroviral therapy in these subjects permitted estimation of the death rate of productively infected cells (δ I ) and the t 1/2 of virus during the decline of plasma viremia . Values for the phase 1 viral decay rate (δ I ) were remarkably consistent and ranged from 0.2 to 0.5/day, with clearance t 1/2 of 1.3–3.0 d for the subjects ( Table ). Relapse of viremia was observed after voluntary interruption of antiretroviral therapy in two subjects ( Table ). The mean rate of viral replication upon relapse of viremia ( r ) was 0.4/day, corresponding to a mean viral doubling time ( t 2 ) of 1.4 d. Comparison of the rate of viral replication after withdrawal of treatment ( r ) to the rate of viral replication during initial acute infection ( r 0 ) showed that the initial rate was nearly five times faster (0.4 vs. 1.9/day). A comparison of the viral replication dynamics during primary infection revealed remarkably similar parameters between the macaque model 12 with acute SIV infection and human subjects with acute HIV infection ( Table ). The mean initial rate of rise of viremia ( r 0 ) in 12 SIVsmE660-infected macaques was 2.2/day (range, 1.7–2.7), with a viral doubling time of 0.32 d (8 h). This measure is similar to a mean r 0 value of 1.9/day (range, 1.4–3.5) and doubling time of 0.4 d (10 h) observed in our study population. Both the magnitude and the time to peak of viremia in the macaque and human infections were similar. As observed in HIV-infected humans, a period of spontaneous decay of SIV after the peak of viremia is observed in macaques with primary SIV infection. The mean rate of spontaneous SIV decay (α) was 0.52/day, with a mean clearance t 1/2 of 1.33 d, compared with a mean rate of spontaneous decay (α) of 0.3/day, with a mean viral clearance t 1/2 of 2.4 d in humans. Similar lower limit estimates of R 0 were observed in macaques (5.4) and humans (7.4) using the fixed delay model 12 , with more varied upper limit estimates in the macaques, perhaps attributable to the incorporation of data on target cell availability in the human study. Comparison of phase 1 decay rates and relapse rates of viremia are limited by the very different treatment regimens and durations of therapy used in the different hosts as well as small sample numbers. Viral dynamics of acute HIV infection were described nearly a decade ago 1 2 , yet only recently has the potential value of precisely defining viral and host immune responses in acute HIV infection been appreciated. The increased recognition of the clinical syndrome associated with primary HIV infection coupled with the potential benefits of early intervention with potent antiretroviral therapy have facilitated investigation of acute HIV infection 19 20 . Identifying patients with exponentially increasing viral titers in this study permitted the first estimates of viral doubling time associated with acute HIV infection. The viral doubling time during acute HIV infection was estimated to be 10 h. The abundance of permissive target cells during acute HIV infection and the rapid viral doubling time resulted in extraordinarily high levels of HIV RNA (2.5–44 million copies per milliliter of plasma) within 3 wk of infection. This estimate of the doubling time in acute infection is nearly five times faster than that measured after treatment interruption ( t 2 = 1.4 d) and what has been observed after interruption of treatment of chronic infection ( t 2 = 1.7 d; reference 21). The viral doubling time after treatment interruption may represent an objective parameter to evaluate the restoration of HIV-specific immune responses generated during therapy. A delay before the emergence of detectable plasma HIV RNA after therapy withdrawal or a slower viral doubling time than was observed with our patients may provide a quantitative estimate of acquired host immunity. We cannot, however, exclude the possibility that greater target cell availability during acute infection supports the observed more rapid viral doubling. The quantitation of viral titers during acute infection also permitted estimation of the basic reproductive number ( R 0 ). Any intervention that reduces R 0 to <1 results in less than one secondary infected cell arising from each HIV infected cell. This parameter provides an estimate of how effective an intervention must be to result in extinction of viral replication and thus has significant implications for vaccine design and postexposure therapies. To extinguish ongoing viral replication, any intervention must reduce the basic reproductive number to below one. At least two important qualifications must be considered before claiming that such an intervention will successfully control HIV infection. First, ongoing replication in the presence of the selective pressure of an immune response or drug therapy could permit the outgrowth of resistant (escape) mutants. Consideration of resistance is relevant because both zidovudine 22 23 and multidrug-resistant virus 24 have already been reported in acute HIV infection. Furthermore, mathematical models suggest that drug resistance is more likely to develop if treatment is initiated during the very high titer viremia of acute HIV infection 25 . Second, even during acute infection, latently infected CD4 cells are being generated 26 , which upon later reactivation could provide the spark to rekindle viral replication. Our estimates of R 0 do not consider the impact of long-lived pools of latently infected cells 27 28 , which have been detected within the first week after the onset of an acute retroviral syndrome 26 . Our estimates of the basic reproductive number ranged from 7.4 to 34.0 (mean, 19.3) using the model with fixed delay and suggest that after acute HIV infection, an intervention at least 95% effective will be needed to prevent sustained viral propagation. The presence of drug-resistant virus or an established pool of latently infected cells could make such a target extremely difficult to attain. Our estimates of the basic reproductive number depend on several additional assumptions. First, although biologically implausible, a no delay model was evaluated for the purpose of comparison with prior studies of SIV dynamics in acutely infected macaques. Second, our model assumes that CD4 lymphocytes are a good surrogate for target cells. This amounts to assuming that the fraction of CD4 cells that are target cells is constant, an assumption that is born out by our own preliminary observations (data not shown). The lymphocyte populations, which are productively infected in vivo, have not been precisely defined. Based upon our observations that HIV replication in vitro is selectively favored in CD4 cells of the memory phenotype (CD4/CD45RO) 29 , we and others speculated that activated CD4 cells 30 31 32 33 , such as memory cells expressing the IL-2 receptor (CD4/CD45RO/CD25) might represent a target cell population for HIV infection in vivo. However, in the absence of data defining the rate of production and clearance of these cells (both infected and uninfected), we were unable to fit a model that included these variables and used absolute CD4 counts as the target cell population for our estimates of R 0 . If the population of true target cells for HIV infection was present as a stable proportion of the total CD4 cell population, then our estimates of R 0 would be unbiased. Supporting this assumption, we observed that the proportion of CD4 cells that coexpressed CD45RO/CD25 remained stable during the first 6 mo of follow-up in all study subjects (data not shown). Third, we assumed a preinfection CD4 count of 1,000 cells/mm 3 . This would underestimate R 0 for subjects with preinfection T cell counts ( T 0 ) > 1,000 and overestimate R 0 for subjects with T 0 < 1,000. Spontaneous decline of viremia was highly variable both within and among patients. It is possible that the explanation for this variability is attributable to host immune responses; however, there are yet no strong data to support this assumption. Prior observations by Riggs et al. in a single subject showed that the viral burst size appeared to be stable during the decline of acute viremia, arguing against the hypothesis that the life span of HIV-infected cells was being shortened by effective CTL responses 34 . The rate of viral decay over the first 10 d of declining viremia (α) was identical to the mean phase 1 viral decay rate after initiation of potent antiretroviral therapy (0.3/d). In one patient with peak HIV RNA counts over 10 million, the rate of spontaneous decline was actually faster than that seen after potent antiretroviral therapy (data not shown). This observation is difficult to reconcile with the assumption that during spontaneous decline, some new infections are still being produced, whereas in the setting of potent antiretroviral therapy, virtually all new infections are blocked. The initiation of potent antiretroviral therapy in the setting of acute HIV infection resulted in measures of phase 1 viral decay rates remarkably similar to reported measures in subjects with chronic HIV infection across a range of treatment regimens 18 21 35 36 . In contrast to previous reports 21 37 , no association between baseline HIV RNA or CD4 cell count (or percentage) and the rate of phase 1 viral decay was observed. These data suggest that either an acquired host immune response develops very early in the course of acute HIV infection and remains relatively constant thereafter, or, conversely, that the contribution of host immunity to the clearance of productively infected cells is minimal in both acute and chronic HIV infection 14 34 38 . Frequent measurements of plasma HIV RNA in these subjects demonstrated the appearance of a relative virologic plateau a mean of 33 d after the peak of viremia that has not been identified in prior publications 14 39 . This virologic plateau occurred several months before the virologic “set point” shown to be predictive of disease progression 5 . Additional studies are needed to define the exact timing, duration, and etiology of this observation. Our measures of the initial rate of rise of viremia, magnitude of the peak, and subsequent spontaneous decline of viremia are in close approximation to reported ranges for these variables in a macaque model of primary SIV infection 12 . Although the lower limit estimates for R 0 are similar for the human (7.4) and simian models (5.4), the range for the basic reproductive number in the macaque model was somewhat greater (5.4–68) than in the human model (7.4–34.0). The more limited range of our estimate is related to the use of both the viral growth rate and the degree of target cell depletion to estimate R 0 , whereas the SIV estimate of R 0 used only viral dynamic parameters. Similar viral replication dynamics between humans and macaques support the use of the SIV model to study HIV pathogenesis. Studies evaluating postexposure prophylaxis as well as the establishment of viral latency will almost certainly be addressed more efficiently in the macaque model, where route, inoculum, and viral strain are specified. Because candidate HIV vaccines will be evaluated first in the SIV model, our estimates of the basic reproductive number will provide a rational means to select vaccines that merit testing in humans. | Study | biomedical | en | 0.999996 |
10499923 | Female Balb/c mice were purchased from Janvier and kept under pathogen-free conditions. They were used at 8–12 wk of age. Mice were primed subcutaneously in each limb (50 μg) and in the back of the neck (100 μg) with chicken egg OVA (Sigma Chemical Co.) precipitated in alum 13 . Lymph nodes were obtained on days 0–4 after the primary immunization with OVA. Organs of a particular day were collected together and frozen in OCT compound on dry ice. The blocks were then cut into 10-μm-thick sections, air dried for 1 h, acetone fixed for 10 min, and finally stored at −20°C. The CR-Fc fusion protein was generated as described elsewhere 12 . In brief, the CR domain of the mannose receptor was amplified by reverse transcription PCR and cloned into the pIG expression vector. The resulting plasmid was transfected into Cos-7 cells. The chimeric protein consisting of CR fused to the Fc portion of human IgG1 was purified from supernatants of transfected cells by protein A chromatography and stored at −20°C. To characterize in situ the population binding the CR-Fc fusion protein, triple-color confocal microscopy was used. For this, CR-Fc binding cells were identified with Cy5 (λ em = 670 nm), IgM + B cells with Texas red (λ em = 615 nm), and a third protein with FITC (λ em = 525 nm). These were excited with the 633- and 543-nm HeNe lasers and a 488-nm argon laser, respectively. This triple labeling then offered the most efficient strategy to (a) locate CR-Fc binding cells (blue) in relation to the IgM + B cell follicles (red); and then (b) determine if the blue cells expressed either a specific molecule or were adjacent to a certain cell type (green). The antibodies used for this third variable parameter included the following: N418 (anti-CD11c), M1/70 (anti-CD11b), 53-6.7 (anti-CD8α), T24 (anti-CD90/anti–Thy 1), GK 1.5 (anti-CD4), 14.8 (anti-B220), Syndecan-1 (anti-CD138), Ser-4/3D6 (antisialoadhesin), biotinylated FDC-M2 (anti-FDC), F4/80 (antimacrophage), biotinylated 14.4.4 (anti–MHC class II), and biotinylated NLDC-145 (anti-DEC205; a generous gift of K. Shortman, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). These mAbs, with the exception of NLDC-145, were either obtained from American Tissue Culture Collection and produced in house or, when available, purchased from PharMingen. Secondary antibodies and reagents included Texas red–conjugated rat anti–mouse IgM (Southern Biotechnology Associates), Cy5-conjugated mouse anti–human IgG (Jackson ImmunoResearch Laboratories), streptavidin-Cy5 (Caltag Laboratories), FITC-conjugated mouse anti–rat IgG (Southern Biotechnology Associates), FITC-conjugated mouse anti–rat IgG, FITC-conjugated mouse anti–human IgG (both from Jackson ImmunoResearch Laboratories), and FITC-conjugated goat anti–hamster IgG (Southern Biotechnology Associates). Human IgG (Jackson ImmunoResearch Laboratories) was used as a control for nonspecific binding of CR-Fc. The photomicrographs were computer analyzed using a program developed in house. The program divides the image into 4,096 squares (64 × 64). The mean fluorescence intensity (MFI) is calculated for each of the squares and stored in a matrix. The MFI is then represented as a three-dimension (3D) histogram ( x , y = position of the square, z = MFI). The 3D immunohistograms can represent single parameters as well as an overlay of two or more colors. Mice were injected with OVA precipitated in alum (50 μg s.c. per limb and 100 μg s.c. behind the neck). 24 h later, the draining lymph nodes were collected and the cells recovered using an enzyme cocktail containing collagenase and DNAse 14 . The low buoyant density cells (δ = 1.057–1.065 g/ml) were collected from Percoll gradients, and B cells were depleted with anti-B220–coated Dynabeads (Dynal). The remaining cells were sequentially incubated with CR-Fc (10 μg/ml) and N418 (10 μg/ml), biotinylated mouse anti–human IgG (1:100) and goat anti–hamster FITC (1:100; Southern Biotechnology Associates), and Streptavidin-PE (1:100; Serotec) before separation by flow cytometry. All washes and dilutions of reagents for FACS ® used a solution consisting of PBS containing 10% FCS. For assessing in vivo T cell priming, the N418 + /CR-Fc binding cells were injected subcutaneously into the right side of naive mice. 7 d later, the draining (right side) and nondraining (left side) lymph nodes were collected separately, and the cells were recovered using the enzyme cocktail. The cells (obtained from the nondraining versus draining lymph nodes) were then fractionated using discontinuous Percoll gradients 15 . The T cell band (δ = 1.081–1.085 g/ml) was collected and incubated with a cocktail of anti-CD8 (54.6.7), anti–MHC class II (14.4.4), and anti-B220 (14.8) coated Dynabeads according to the manufacturer's specifications. This method produced >98% CD4 + T cells. The T cells (2 × 10 5 cells/well) were then incubated for 48 h in medium (IMDM, 5% FCS, l -glutamine, penicillin/streptomycin, and 2-ME) with spleen cells (irradiated with 30 Gy; 2 × 10 5 cells/well) in the presence or absence of 10 μg/ml OVA. For the final 24 h of culture, [ 3 H]thymidine was added and incorporation measured using a β-counter (Wallac). N418 + /CR-Fc binding cells were isolated as specified above and cultured overnight with T cells isolated from DO11.10 mice (i.e., transgenic for an OVA-specific T cell receptor 16 ). At 24 h, [ 3 H]thymidine was added, and incorporation was measured 24 h later. For assessing in vivo B cell activation, both the N418 + /CR-Fc binding fraction and the N418 + /CR-Fc nonbinding population (representing interdigitating cells) were isolated as specified above. The CR-Fc binding cells were injected subcutaneously into the right forelimb while the CR-Fc nonbinding cells were injected subcutaneously into the left forelimb of naive mice. 7 d later, three sets of lymph nodes were collected and processed separately: 1 the draining lymph nodes receiving CR-Fc + cells (i.e., right axial and brachial), 2 the draining (i.e., left axial and brachial) lymph nodes receiving CR-Fc nonbinding cells, and 3 the lymph nodes receiving no cells (inguinal and paraaortic). Cells were recovered using the enzyme cocktail and fractionated using discontinuous Percoll gradients 15 . The low buoyant density band (δ = 1.060–1.065 g/ml) was obtained and cultured for 1 h at 37°C to remove adherent cells. The nonadherent cells were then incubated (3 × 10 5 cells/well) in 96-well plates containing medium. After 7 d, the supernatants were collected, and OVA-specific antibody titers were assessed using standard ELISA procedures. The hybridomas 2.4G2 (anti-CD32) and YN1.1.7 (anti-CD54) were obtained from American Type Culture Collection, 7G6 (anti-CD21/35) from Dr. T. Kinoshita (Osaka University, Osaka, Japan), FGK (anti-CD40) from Dr. A. Rolink (Basel Institute for Immunology, Basel, Switzerland), and B3B4 (anti-CD23) from Dr. D. Conrad (Virginia Commonwealth University, Richmond, VA). 1G10 (anti-CD80), GL1 (anti-CD86), and 145-2c11 (anti-CD3) were purchased from PharMingen. The hybridoma Ser-4/3D6 (anti–mouse sialoadhesin) was generated in our laboratory. The secondary reagent was the mouse anti–rat IgG (H+L) F(ab′) 2 fragments conjugated to FITC (Jackson Immunoresearch Laboratories), and the control antibody, rat IgG, was purchased from Sigma Chemical Co. N418 + /CR-Fc binding and N418 + /CR-Fc nonbinding cells were purified using flow cytometry according to the protocol mentioned above. In addition, DCs were derived from mouse bone marrow according to the protocol by Metlay et al. 17 . In brief, the bone marrow was flushed from femurs, pipetted into a single cell suspension, and then incubated at 37°C, 5% CO 2 for 2 d in the presence of GM-CSF. At days 2 and 3, the nonadherent cells were removed after gentle pipetting, discarded, and new medium containing GM-CSF was added to the remaining adherent cells. On day 6, the nonadherent cells were harvested and shown to contain ≥99% N418 + cells by flow cytometry. Cells were incubated at 37°C in 96-well plates (2 × 10 5 cells/well) in 200 μl IMDM plus 5% FCS (GIBCO BRL). Supernatants were harvested at 24 h, and chemokine levels were determined by ELISA using mouse-RANTES (kit MMR00; R&D Systems) and mouse–MIP-1α (kit MMA00; R&D Systems) kits according to the manufacturer's specifications. During an immune response, it has been previously shown that within lymph nodes, cells with dendritic morphology bind the CR-Fc fusion protein 12 . To further investigate their nature, we developed a system involving immunohistochemistry and three-color microscopy. As shown in Fig. 1 , the B cell follicles were visualized using an anti-IgM antibody conjugated to Texas red. CR-Fc binding cells were labeled with Cy5-conjugated reagents. A third marker associated with FITC was then used to determine either the phenotype of the CR-Fc binding cells (double positive = cyan) or the proximity of these cells to other cell types. 3D histograms were then created in order to accurately assess the number and location of single or double positive cells. The coexpression of the murine DC-specific marker, CD11c (using the N418 mab ), was assessed on lymph node sections obtained before (day 0) and 1, 2, 3, and 4 d after a primary immunization. In general, as shown in the histograms of Fig. 1 , CR-Fc binding cells represented a subset of N418 + cells. Before antigenic stimulation , no CR-Fc binding cells were observed below the subcapsular sinus. Within 24 h of antigen injection , CR-Fc single and CR-Fc/N418 double positive cells appeared in the follicle. By day 2, the cells were scattered throughout the follicle, becoming more organized towards the edge of the follicle during days 3 and 4. At day 5, very few CR-Fc/N418 double positive cells were observed except in the subcapsular sinus (data not shown). As a control to address nonspecific Fc-mediated binding, the sections (days 0–3) were incubated with human IgG instead of the fusion protein CR-Fc. No binding was observed on the tissue sections (data not shown). Finally, very few, if any, N418 + interdigitating cells in the paracortex demonstrated the ability to bind CR-Fc . Using this procedure, the expression of several proteins was analyzed ( Table ). The CR-Fc binding cells in the follicles were observed to also express MHC class II and sialoadhesin, the latter being one of the ligands for CR-Fc (Martinez-Pomares, L., and S. Gordon, manuscript in preparation). A subset of cells expressed CD90 (Thy 1), especially the CR-Fc binding cells present at the border of the B cell follicle and paracortex. At days 3 and 4, a minor subset of cells also expressed CD8α and CD138 (Syndecan-1). However, CD11b, Dec205, CD4, FDC-M2, F4/80, and B220 were all absent. Taken together, these results reveal a novel pattern of protein expression for the CR-Fc binding cells that localize in B cell follicles. Interestingly, many CR-Fc binding cells tended to localize at the border of the B cell follicle . As it had recently been reported that adoptively transferred antigen-specific transgenic T and B cells colocalize in this region 11 , we asked whether T cells could be observed in the vicinity of CR-Fc binding cells. As shown in Fig. 2 (see areas within boxes), indeed T cells (CD4 + ) were found to be mingled with B cells (IgM + ) and CR-Fc binding cells at the periphery of the follicle between days 3 and 4 after antigen injection. The kinetics of their appearance in the particular anatomical location and their expression of the murine DC-specific protein, CD11c, suggested that CR-Fc binding cells play a role in lymphocyte stimulation. To address this hypothesis, CR-Fc binding cells were isolated from draining lymph nodes 24 h after subcutaneous injection of OVA and purified by flow cytometry. As shown in Fig. 3 , the CR-Fc binding cells had a high side scatter value and were all CD11c + . Sorting the CR-Fc binding cells using the R1 gate produced a purity of >99%. The cells were then adoptively transferred into naive mice, and 7 d later, the T cells were isolated separately from the draining versus nondraining lymph nodes. As shown in Fig. 4 A, the CR-Fc binding cells were able to prime naive T cells, as no antigen-specific proliferation occurred in T cells isolated from the nondraining side. These results suggested that CR-Fc binding cells were capable of antigen uptake in vivo followed by processing and presentation of peptides to naive T cells. To insure that CR-Fc binding cells could present antigen they obtained in vivo, the cells, isolated in a similar manner as above, were incubated in decreasing concentrations with antigen-specific T cells. As few as 3,000 CR-Fc binding cells were able to significantly stimulate the DO11.10 transgenic T cells to proliferate in vitro. Therefore, not only can CR-Fc binding cells take up antigen in vivo, they can also act as antigen-presenting cells both in vitro and in vivo. As B cells are stimulated to produce the initial wave of antigen-specific Ig in the outer T cell zones, an experiment was designed to ascertain if CR-Fc binding cells played a role in this process. Similar to the protocol mentioned above, CR-Fc binding cells were isolated from draining lymph nodes 24 h after a subcutaneous injection of OVA. In addition, CR-Fc nonbinding cells were also obtained for comparison. The CR-Fc binding cells were then adoptively transferred into the right forelimb of naive mice while the CR-Fc nonbinding cells were injected into the left. 7 d later, the B cells were isolated separately from the right versus left axillary and brachial lymph nodes as well as the inguinal and paraaortic lymph nodes (i.e., the sites receiving no cells). They were then cultured for a further 7 d, at which time the supernatants were harvested. As shown in Fig. 5 , only cells isolated from lymph nodes receiving the CR-Fc binding cells produced antigen-specific antibody. Although only a remote possibility due to the 24-h time point used for isolation, contamination by preplasma cells in the CR-Fc preparations was ruled out by putting these cells in culture for 7 d and assaying for antibody production by ELISA (none was detected; data not shown). In addition, the same results as shown in Fig. 5 were obtained when CR-Fc binding preparations were subjected to a round of B220 depletion using magnetic beads before injection into mice (data not shown). Next, to determine the characteristics of the antigen-specific antibody response in vivo after transfer of CR-Fc binding cells, serum was collected from mice after a single and second injection of cells. As shown in Fig. 6 , a significant increase was observed for OVA-specific IgG1 titers after both the primary and secondary injections. IgM did not increase, although this was clearly observed when B cells were assessed ex vivo . These data suggest that although the B cells that encounter CR-Fc cells can become IgM-secreting cells ex vivo, left in vivo, they will encounter signals that drive IgG1 production and can be induced to produce an increased level of antibody after a secondary application of the stimulus. IgG2a and IgG2b antigen-specific serum titers showed a slight but not statistically significant change. This observation suggests that transfer of CR-Fc cells themselves does not promote the development of microenvironments for downstream Ig class switching events. Double immunolabeling in conjunction with flow cytometry revealed that the CR-Fc binding cells were rich in Fc (CD32) and complement (CD21/35) receptors . In addition, they were positive for intercellular adhesion molecule 1 (ICAM-1, CD54), CD40, and CD86 (B7-2) whereas they expressed little to no CD80 (B7-1). The cells also expressed sialoadhesin (Ser-4/3D6), one of the ligands for CR-Fc. Finally, neither CD3 nor CD23 were expressed. N418 + /CR-Fc nonbinding cells (i.e., interdigitating cells) and N418 + /CR-Fc binding cells isolated 24 h after antigen injection as well as N418 + DCs derived from bone marrow (BM-DCs) were assessed for their capacity to produce T cell chemoattractants. As shown in Fig. 8 , RANTES (for regulated upon activation, normal T cell expressed and secreted) production followed the hierarchy of BM-DC < CR-Fc + /N418 + ≤ CR-Fc − /N418 + (interdigitating cell). In contrast, only N418 + /CR-Fc binding cells produced MIP-1α. Using the CR-Fc binding property, we have identified a population of cells that localize within B cell follicles. These cells appear to be members of the DC family, as they express CD11c in situ and ex vivo and are capable of priming naive T cells. Whether the cells belong to the myeloid or lymphoid lineage is unclear, as CD11b and DEC205, proteins normally associated with the myeloid and lymphoid DC lineage, respectively, were not detected. The lack of F4/80, CD4, and FDC-M2 expression, molecules found on macrophages 18 , germinal center DCs 19 , and FDCs 20 , respectively, further define these as a unique DC subset. As CR-Fc binding DCs appear mainly in the follicles and not the paracortex, these cells may represent a population specifically suited to precipitating the early phases of humoral immunity. These cells provoke antigen-specific antibody responses in vivo after adoptive transfer, and the isotypes (i.e., IgM and IgG1) produced are restricted to that associated with initial antibody responses 8 9 . These data together with the in vitro and in vivo T cell priming results are significant because they suggest that CR-Fc binding cells have a mechanism not only for presenting peptide but also for transporting native antigen to the B cell compartment. Our observations concur with those of Wykes and colleagues, who showed that DCs can capture and retain unprocessed antigen in vitro and in vivo and could transfer this antigen to naive B cells to initiate specific antibody responses 7 . This mechanism would provide an efficient means of initiating early protective immunity when physiological (i.e., low) doses of virus or bacteria enter the body and are encountered by the sentinel DCs. Indeed, low affinity polyclonal IgM is essential for the resistance of mice to a systemic bacterial infection 21 . Hence, DCs moving in from the blood or interstitial sites, quickly providing both peptide and native antigen for lymphocyte stimulation in microenvironments of secondary lymphoid tissues, would prove to be evolutionarily advantageous for survival. The functional profile of these murine-derived CR-Fc binding DCs parallels that of the human CD14 + -derived DCs characterized by Caux and colleagues 22 . The CD14 + -derived DCs have a robust and long-lasting antigen uptake capacity 22 resulting in the induction of efficient T cell proliferation, and demonstrate a unique ability to induce naive B cell differentiation into IgM-secreting plasma cells. In addition, the accessory molecule profiles of the murine and human DC subtypes are similar, as only CD40 and CD86 are necessary for CD14 + -derived DC–induced T cell proliferation while CD80 plays no role. As shown in Fig. 7 , the murine CR-Fc binding DC expresses CD40 and CD86 but is negative for CD80. Assessing the chemokine production by various murine DC subsets demonstrated that CR-Fc binding DCs have a unique phenotype compared with immature (day 6) BM-DCs and interdigitating cells (the remaining population of CD11c + lymph node cells isolated 24 h after antigen injection). Although the significance of a more robust MIP-1α production remains to be shown, this may relate to the CR-Fc binding DC ability to recruit cell types quickly into the tissue 23 . MIP-1α is reported to attract both T cells and other DCs 24 25 . Furthermore, MIP-1α may also act as a B cell chemoattractant, as B cells have been shown to express mRNA for CC chemokine receptor CCR1 26 27 . As other reagents become available to determine the levels of murine chemokine production, we will be able to further define the function of this cell type during immune responses. Is the CR-Fc binding DC a migratory cell, or are we witnessing upregulation of a receptor on a resident population? Based on the ability of the CR-Fc binding DCs to prime naive T cells in lymph nodes after a subcutaneous adoptive transfer as well as their similarity to human CD14 + -derived DCs, we favor the former. The relationship of the murine CR-Fc binding DC to the germinal center DC remains to be further investigated. In the future, the use of imaging systems as described by Robert and colleagues 28 should provide the means to trace the exact fate of CR-Fc binding cells in vivo. | Study | biomedical | en | 0.999999 |
10499924 | Reagents were from Sigma Chemical Co. or GIBCO BRL unless otherwise stated. Pharmacological reagents were obtained from Biomol or Sigma Chemical Co., stored as 1,000× stock solutions in DMSO at −20°C, and diluted into cell culture medium directly before use. The following antibodies were used: rat αvβ3 function-blocking β3 antibody clone F11 30 31 and mouse β3 function-blocking antibody clone 2C9.G2 32 were from PharMingen. Immunoprecipitating, function-blocking, heterodimer-selective αvβ5 antibody clone P1F6 33 was from Chemicon. P1F6 recognizes αvβ5 in many species, including rodents . For immunoblot detection, β5 antibodies were purchased from Chemicon or from Upstate Biotechnology (clone B5-IVF2 34 ), and β3 antibody clone 26 was from Transduction Laboratories. Myeloperoxidase antiserum was from Dako. Mouse αM function-blocking antibody 5C6 35 was a gift from Dr. W.A. Muller (Weill Medical College of Cornell University). Secondary antibodies were from Jackson ImmunoResearch Laboratories. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) detection was performed using the Fluorescein In Situ Cell Death detection kit from Boehringer Mannheim. Surface phosphatidylserine was detected using the ApoAlert Cell Death detection kit from Clontech. J774.A1 murine macrophages, NRK-F49 rat fibroblasts, and rat RPE-J cells were obtained from American Type Culture Collection and routinely maintained in DMEM supplemented with 10 or 4% (RPE) FCS. For binding and phagocytosis experiments, macrophages and fibroblasts were seeded at confluence on glass coverslips for 30 min or overnight before use. RPE-J cells were differentiated on semipermeable filters as described previously 36 . Rat primary cells were isolated from adult Long Evans rats (Richard Harlan, Inc.) immediately after CO 2 asphyxiation. Rat bone marrow–derived macrophages and rat blood–derived monocyte macrophages were prepared and maintained according to published procedures 13 37 . In brief, blood-derived monocytes were isolated by plastic adherence after Ficoll gradient purification of leukocytes from anticoagulant-treated rat blood. Bone marrow cells were flushed aseptically from femur bones of Long Evans rats, washed, seeded, and used without repassaging. All primary macrophages were cultured for 6–7 d before use in IMDM, supplemented with 10% autologous serum; bone marrow–derived cell medium was supplemented with 5% L cell–conditioned medium (Sigma Chemical Co.) as a source of M-CSF 13 . Primary rat neutrophils, which were isolated from fresh rat blood according to established protocols 38 , underwent spontaneous apoptosis over 24 h in culture. HL-60 cells, a gift from Dr. F. Maxfield (Weill Medical College of Cornell University) were maintained in 10% FCS in RPMI. To induce differentiation and apoptosis, HL-60 cells were cultured in the dark at 2 × 10 5 cells/ml in growth medium containing 1 μM all-trans retinoic acid for 6 d. Before binding or phagocytosis assays, apoptotic cells were routinely tested for viability by trypan blue exclusion and phosphatidylserine surface exposure staining with FITC–annexin V. All populations used were >95% viable and >60% annexin V positive. OS were isolated according to established protocols from bovine eyes obtained fresh from the slaughterhouse 39 . OS were stored suspended in 10 mM sodium phosphate, pH 7.2, 0.1 M sodium chloride, 2.5% sucrose at −80°C. Before use, OS were thawed and labeled by addition of 20% vol of 1 mg/ml FITC or 0.2 mg/ml Texas red (both from Molecular Probes) in 0.1 M sodium bicarbonate, pH 9.0, for 1 h at room temperature in the dark, before being washed and resuspended in cell culture medium. Zymosan particles were opsonized with serum complement components as described previously 40 . In brief, 1% zymosan particles in PBS were boiled for 30 min and opsonized in 50% bovine serum in HBSS for 1 h at 37°C. Washed particles were labeled with FITC as described for OS, and stored at 4°C. To study particle binding, phagocytes at confluence were challenged with 10 particles per cell in growth medium (RPE) or serum-free growth medium (other cells) for the duration of the experiment, washed three times with PBS containing 1 mM MgCl 2 and 0.2 mM CaCl 2 (PBS-CM), and fixed in ice-cold methanol. OS were covalently labeled with FITC or Texas red, and thus could be observed directly. Apoptotic cells were visualized by TUNEL staining or by granulocyte-specific myeloperoxidase immunofluorescence staining using FITC- or Texas red–conjugated secondary antibodies. Nuclei were counterstained with DAPI or propidium iodide at 1 ng/ml in PBS-CM. For competition experiments, OS and apoptotic cells were labeled with different fluorochromes. Particle labeling with either fluorochrome yielded identical results. Unlabeled particles competed with fluorescence-labeled particles for binding and phagocytosis by all cell types. For inhibition experiments, GRGDSP or GRADSP peptides (Calbiochem) were used at 1 mg/ml. Effects of peptides and antibodies were concentration dependent as established previously 26 , and maximal effective concentrations of antibodies, 20–50 μg/ml, were used throughout this study. Concentrations of pharmacological reagents were as follows: calphostin C at 100 nM (light activated), cytochalasin D (Cyt D) at 5–20 μM, Gö6976 at 10 nM, hypericin at 5 μM, and latrunculin B at 1 μM. PMA was routinely used at 50 nM; 16–160 nM gave similar results. Cells were pretreated for times indicated in the figure legends with activators or inhibitors before challenge with OS or apoptotic granulocytes in the continuous presence of reagent. Cell viability and morphology remained unchanged, and none of the cell types initiated detectable apoptosis over the course of the experiments. Since experiments were performed on confluent cells, the spreading effect of PMA on macrophages was negligible. Phagocytosis or binding of OS was quantified by fluorescence scanning of fixed samples as described 26 . Samples were scanned with a STORM 860 Imager, at 950 V (blue or red fluorescence setup; Molecular Dynamics). Areas representing the binding by 1–2 × 10 5 phagocytes were selected, and the fluorescent signals were quantified with ImageQuant 1.2 (Molecular Dynamics). Within one experiment, particle counts directly correlated with particle binding. To compare particle binding of different cell types, as RPE cells and macrophages, the fluorescence of propidium iodide (nuclei, red) and the particle-derived FITC fluorescence were both measured in each field. The binding plus internalization index or the binding index (bound particles at early times of phagocytic challenge) were calculated dividing particle counts by nuclei counts, thereby normalizing for phagocyte numbers. Quenching of fluorescence derived from externally bound particles using trypan blue 26 41 allowed determination of the internalization index. Phagocytosis or binding of apoptotic cells was quantified following the same procedure based on TUNEL or myeloperoxidase immunostaining fluorescence emissions; binding indices were determined dividing by nuclei counts of control fields, so as not to count apoptotic nuclei. Microscopic observation revealed that 75% of RPE-J cells and >90% of J774 cells had bound or phagocytosed an average of 5 ± 1 OS after 2 h. Using the double fluorescence scanning method on the same samples, this translated into a mean index combining binding plus internalization of 7.6 ± 0.9 for macrophages and 6.3 ± 0.9 for RPE. After 30 min, 75% of macrophages had bound multiple OS; this translated to a mean binding index of 3.8 ± 0.4. At this time point, <20% of particles had been internalized by macrophages, similar to the 2-h time point of RPE 26 41 . Samples were fixed in ice-cold methanol or 4% paraformaldehyde in PBS-CM and processed as described previously 42 . Samples were observed with a Nikon fluorescence microscope E600. Digital images were acquired with a back-illuminated cooled CCD camera , translated using MetaMorph (Universal Imaging), and recompiled in Adobe Photoshop ® 4.0. Horizontal (x–y) sections were acquired at 0.5-μm steps using a z motor (Prior), and out of focus light was removed using MetaMorph. Integrin cytoskeletal association was assayed by cell fractionation into detergent-soluble and insoluble fractions according to previously described protocols 43 . Confluent cells in 6-cm dishes, preincubated with pharmacological reagents or vehicle as before binding or phagocytosis assays, were extracted in 800 μl 50 mM MES, 5 mM MgCl 2 , 3 mM EGTA, 0.5% Triton X-100, pH 6.4, for 40 s at room temperature (soluble fraction). The remaining cellular material was completely solubilized in an equal volume of RIPA (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 0.1% SDS, 1% sodium desoxicholate, 1% Triton X-100) (insoluble fraction). Equal volumes of soluble and insoluble fractions were compared by 7.5% SDS-PAGE and immunoblotting. For immunoprecipitation of integrins from the different fractions, the soluble fraction was harvested as described above, whereas the insoluble fraction was obtained by scraping the remaining cellular material in an equal volume of 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 1% Triton X-100, 1% NP-40 supplemented with 2 mM each of aprotinin, leupeptin, pepstatin, iodoacetamide, and PMSF, and 1 mM N -ethylmaleimide (IP lysis buffer), vortexing for 30 min at 4°C. This procedure completely solubilized integrin proteins. Immunoprecipitations were performed from this insoluble and the soluble fraction as follows. For metabolic labeling, confluent cells on plastic dishes were starved for 90 min in cysteine, methionine-free DMEM, and labeled with 0.1 mCi/ml 35 S Express (NEN) for 12–14 h. Preincubation with pharmacological reagents or vehicle as control was performed as before binding/phagocytosis assays. To biotinylate surface proteins, cells were incubated with Sulfo-NHS-LC biotin (Pierce Chemical Co.) at 0.5 mg/ml in PBS-CM twice for 20 min on ice. Excess reactive biotin was quenched in 50 mM NH 4 Cl in PBS-CM. Cells were lysed in IP lysis buffer for 30 min. Protein concentration of supernatant lysates was determined according to Bradford 44 . Crude lysates were analyzed by nonreducing 7.5% SDS-PAGE followed by Western blot detection of integrins as published previously 45 . Streptavidin-agarose precipitation has been described in detail elsewhere 46 . Immunoprecipitates were formed incubating precleared lysate with 5 μg P1F6 IgG or nonimmune mouse IgG, with 5 μg rabbit anti–mouse IgG, and with 5 mg protein A–sepharose, each for 1 h at 4°C. Samples were washed four times in 50 mM Tris-HCl, pH 8.5, 0.5 M NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 1 mg/ml egg albumin, 0.1% Triton X-100 eluted in nonreducing or reducing SDS sample buffer, and analyzed on 10% SDS-PAGE followed by fluorography or blotting. Blots were incubated with integrin antibodies, horseradish peroxidase–conjugated streptavidin, or secondary antibodies, followed by ECL (NEN) detection. X-ray films were scanned, and signals were quantified using NIH Image 1.61. Blood monocyte or bone marrow–derived macrophages use αvβ3 integrin receptors to bind apoptotic cells before phagocytosis 7 , whereas RPE cells bind OS via αvβ5 integrins 25 26 . We initially tested the hypothesis that the characteristic choice of different integrin receptors by macrophages and RPE cells was determined by differences in the bound particles. To this end, we exposed the phagocytes to the physiologic ligands of the other cell, i.e., macrophages to OS and RPE cells to apoptotic cells . Rat bone marrow macrophages and the murine macrophage cell line J774, which retains the αvβ3 integrin–dependent apoptotic cell clearance mechanism of noninflammatory monocyte macrophages 13 , efficiently bound and phagocytosed OS . Primary cultures of rat RPE, and rat (RPE-J) and human (ARPE-19) RPE cell lines identified and took up apoptotic granulocytes (and other apoptotic cells; data not shown), but not their nonapoptotic counterparts . Most subsequent studies were performed on J774 macrophages and RPE-J cells, but were confirmed on rat bone marrow–derived macrophages and primary rat RPE cultures. Particle binding and phagocytosis proceeded with kinetics that were characteristic for each cell type. Macrophages bound and internalized OS with the same rapid and linear kinetics with which they take up apoptotic cells . The early phase of RPE clearance of OS is characterized by a lag phase following particle challenge after which OS binding occurs rapidly 26 . RPE cells bound apoptotic cells as slowly as OS . After 2 h of phagocytic challenge, the sum of bound plus internalized OS was similar in macrophages and RPE cells . However, at this time 78 ± 9% of these OS in macrophages had been internalized, compared with only 17 ± 4% internalized OS in RPE cells. Phagocytosis of apoptotic cells bound by RPE cells followed the same slow time course as previously established for OS: we removed unbound apoptotic cells after 2 h and followed the fate of bound cells. Over the next 3 h, 85% of bound apoptotic cells were internalized by RPE cells ( 26 ; and data not shown). To specifically address particle binding, we chose to study 30 min of particle challenge for macrophages and 2 h for RPE cells, both of which corresponded primarily to the recognition/binding phase of particle clearance (see above, and Materials and Methods). Peptides containing the cognate integrin-binding motif, RGD, reduced binding of both particles by either phagocyte . In contrast, function-blocking β3 antibodies only inhibited particle binding by macrophages while αvβ5 antibody P1F6 only blocked RPE recognition . OS and apoptotic cells competed for binding by both macrophages and RPE cells . These experiments indicate that neither macrophage nor RPE binding receptor systems discriminate between ligands of both particles and that these systems involve αvβ3 in macrophages and αvβ5 in RPE cells. We tested three hypotheses that might account for particle binding by different integrin binding receptors in macrophages and RPE cells. Hypothesis 1 was that cell type–specific integrin protein expression determined receptor availability for particle binding. However, Fig. 3 shows that selective integrin expression was not involved, as both β3 and β5 were expressed at similar levels by J774 cells, rat bone marrow–derived macrophages, and RPE-J cells. Immunoprecipitation of αvβ5 from RPE and macrophage lysate using the antibody P1F6, which recognizes only intact heterodimers, and coimmunoprecipitation of β3 integrin with αv integrin confirmed the formation of αvβ3 (data not shown) and αvβ5 receptors . We have shown previously that the steady state distribution of β3 integrins is basolateral in the RPE 26 . Although this does not exclude a temporary presence of αvβ3 at the apical phagocytic surface, this spatial segregation may render it less available for efficient apoptotic cell or OS binding by the RPE than αvβ5, which localizes apically and cytoplasmically. In contrast, double immunofluorescence staining with antibodies recognizing the β3 extracellular domain and with P1F6 antibodies specific for the extracellular face of the αvβ5 receptor complex showed that in nonpermeabilized macrophages, both antigens were localized in the same optical sections of the plasma membrane of a given cell, even if their distribution within the plane of the membrane differed. Like β3 integrins, αvβ5 receptors localized partially to basal attachment sites of macrophages but were also available at their free surface for binding to apoptotic cells or OS. Hypothesis 2, applicable to macrophages, was that their preferred use of αvβ3 for apoptotic cell or OS binding might be based on the faster early kinetics of this pathway over αvβ5-mediated uptake . To facilitate detection of a hidden αvβ5 particle binding activity, αvβ3 receptors were depleted from the free macrophage surface. When compared with control cells plated on laminin (which is not a substrate for either αvβ3 or αvβ5), a large fraction of αvβ3 receptors was recruited to the attached surface in macrophages plated on the specific αvβ3 substrate fibrinogen , resulting in a reduction in particle binding of 40% after 30 min of particle challenge , and a 44% decrease of combined binding and internalization even after 90 min . αvβ5 receptor distribution remained unchanged on fibrinogen, as judged from the similar appearance of αvβ5 immunofluorescence signals on both attached and open cell surfaces on laminin and fibrinogen . However, the remaining binding at 30 min and the combined binding plus internalization at 90 min were still reduced by β3 but not by αvβ5 function-blocking antibodies . Incubation with β3 antibody in the presence of the peptide inhibitor GRGDSP did not have an additive effect , indicating that αvβ3 was the only RGD-sensitive receptor, which mediates apoptotic cell or OS recognition in this system. However, ligand binding by leukocyte β2 integrins may not be inhibitable by RGD peptides 48 . At the 90-min time point, at least 65% of the particles had been internalized . Comparing total and internal particles of laminin- and fibrinogen-seeded macrophages, differences in internalized particles were less pronounced than differences in surface-bound particles (as deduced by subtracting internal from total particles), confirming that attachment to fibrinogen inhibited particle binding but not internalization . Similar results were obtained when cells were plated on β3 integrin antibodies or on vitronectin, a substrate for both αvβ3 and αvβ5 . These experiments indicate that αvβ5 integrin receptors are present in macrophages but are not active in apoptotic cell or OS binding. Hypothesis 3 was that integrin-mediated binding of apoptotic cells or OS was subject to cell type–specific regulation. Several earlier studies have reported that, unlike αvβ3 integrin, αvβ5 is sensitive to pharmacological modulation by protein kinase C (PKC) 33 49 50 . Here, we compared the effects on macrophage and RPE particle binding of generic PKC activation using phorbol esters (PMA), and inhibition by calphostin C, Gö6976, and hypericin. Inhibitors were used at concentrations near IC 50 for PKC. PMA treatment did not alter RPE particle binding; on the other hand, calphostin C, Gö6976, and hypericin reduced binding by RPE cells . Significant inhibition (by 54%) by calphostin C required a preincubation of 3 h. It is possible that RPE cells take up calphostin C more slowly than other cultured cells or maintain lower cytoplasmic concentrations of this inhibitor. Hypericin and Gö6976 significantly reduced particle binding after as little as 30 min of preincubation by 39 and 22%, respectively, but both were also more effective after 3 h of preincubation (69 and 81% reduction). These experiments indicate that the apoptotic cell or OS binding activity of resting confluent RPE cells is drastically reduced by pharmacological inhibition of PKC. A strikingly different scenario was observed in the case of macrophages. PKC inhibition by calphostin C, Gö6976, or hypericin did not alter particle binding, regardless of preincubation time. On the other hand, stimulation of PKC by incubation with PMA increased particle attachment by 45%. This enhanced binding activity was sensitive to RGD-containing peptides, to β3 antibodies, and, importantly, to αvβ5 antibody Fab fragments . Thus, PMA treatment activated αvβ5-mediated particle binding that was dormant in untreated macrophages. The effect of the PMA treatment directly involved PKC, since it was abolished by preincubation of cells with calphostin C for 20 min before PMA and particle challenge. To confirm the PKC dependence of αvβ5-mediated apoptotic cell or OS binding in a different cell context, we carried out similar experiments in NRK-F49 fibroblasts. When challenged with OS for 2 h, these cells bind (but do not internalize) few OS, equivalent to one fifth of the number bound by RPE over the same period , although they express comparable levels of αv, β3, and β5 integrins . Incubation with integrin antibodies had no effect on the basal NRK-F49 particle binding of apoptotic cells or OS (data not shown). However, addition of PMA stimulated particle binding by NRK-F49 fibroblasts by 250% to a binding index of 3.4 ± 0.5, which is an increase to approximately half-maximal binding capacity of professional phagocytes. Strikingly, this induced binding activity was completely sensitive to RGD peptides, was blocked specifically by αvβ5 antibodies, and was not sensitive to β3 antibodies . Over the 2-h time period studied, NRK-F49 fibroblasts retained particles at the surface and did not internalize them, regardless of pharmacological stimulation. These experiments demonstrate that inhibition and activation of PKC regulate the function of αvβ5 integrin as binding receptor for apoptotic cells or OS. To determine if the stimulated αvβ5 integrin–mediated particle binding in macrophages exhibited typical features of OS binding by RPE cells, we compared the temperature sensitivity and kinetics of particle binding and internalization by J774 cells, in the presence or absence of PMA. Total (bound plus internalized) and internalized particles were measured to calculate binding and internalization indices plotted in Fig. 7 . Onset of binding of apoptotic cells or OS to control and PMA-stimulated cells occurred with the same rapid kinetics typical of macrophages . When challenged with particles for 30 min or longer, both control and PMA-stimulated cells increasingly internalized bound material , resulting in a decrease in particles detected bound to the cell surface. J774 cells, which were equilibrated to 18°C before particle challenge, also bound both particles with the same fast kinetics regardless of whether they had been preincubated with PMA or with solvent alone, albeit slightly delayed regarding the 37°C time course . Under these conditions, <10% of total particles were internalized regardless of PMA treatment and time of incubation . Finally, incubation at 14°C or on ice during particle challenge abolished particle binding with or without PMA treatment , while allowing normal binding of complement-opsonized zymosan via macrophage αMβ2, sensitive to αM-blocking antibody 5C6 (data not shown). These experiments demonstrate that apoptotic cell or OS binding by macrophages via αvβ3 or αvβ3 plus αvβ5 integrin shows the same temperature requirement as particle binding via αvβ5 to RPE cells. On the other hand, PMA-stimulated macrophages, which possess an activated αvβ5 integrin, bind apoptotic cells or OS with the same rapid kinetics as control macrophages, which do not use αvβ5, indicating that RPE particle binding does not occur slowly due to an intrinsic property of αvβ5. To gain insight into the mechanism of activation of αvβ5-mediated binding of apoptotic cells or OS by PKC, we searched for biochemical changes associated with PKC activation. Pharmacological activation or inhibition of PKC did not influence αvβ5 receptor levels in either cell type or their steady state expression levels at the cell surface, as revealed by surface biotinylation, αvβ5 immunoprecipitation, and streptavidin blot or enrichment of plasma membrane fractions and β5 immunoblot . Incomplete extraction of plasma membrane proteins with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton serves to distinguish integrin receptors, which are stabilized by actin cytoskeletal elements 43 51 . PKC inhibition in RPE and PKC activation in macrophages correlated with decreased and increased β5 resistance to nonionic detergent, respectively, as shown in Fig. 9 , a and b, suggesting a regulation of β5 association with actin cytoskeletal elements. β5 was 65% insoluble in control RPE, 58% insoluble in PMA-treated RPE, but only 33% insoluble in RPE treated with PKC inhibitor Gö6976. In contrast, β5 was mostly soluble in control macrophages (5% insoluble) but became 78% insoluble after PMA incubation. This effect was greatly inhibited in the presence of Gö6976 (23% insoluble). This effect was specific for β5, as β3 solubility was unaffected by PKC activation/inhibition . β3 integrin was 30% insoluble in RPE and 40% insoluble in J774 cells, regardless of treatment. Pretreatment of RPE or macrophages with the actin microfilament disrupting drug, Cyt D, rendered β3 and β5 completely soluble in RPE and macrophages, confirming that integrin resistance to nonionic detergent extraction in our biochemical analysis reflected their association with actin microfilaments . As expected, PMA treatment did not induce integrin insolubility in cells in which actin microfilaments had been disrupted by Cyt D . We further determined the detergent solubility of intact αvβ5 receptors . Strikingly, the effect of PKC activation or inhibition on αvβ5 heterodimer solubility was most pronounced: 90% of αvβ5 in RPE cells was insoluble but became mostly solubilized (26% insoluble) upon incubation with the PKC inhibitor Gö6976 . In control macrophages, the entire αvβ5 receptor pool was present in the soluble fraction . In contrast, PMA-treated macrophages exhibited 62% insoluble αvβ5 . Attempts to visualize the change in receptor solubility by indirect immunofluorescence staining of αvβ5 or β5 in cells fixed before or after a short pulse of nonionic detergent failed, as, unfortunately, detergent pretreatment abolished all immunoreactivity. Loss of immunoreactivity after detergent treatment was independent of cell type or pharmacological treatment, suggesting that antibody–antigen binding did not tolerate detergent treatment followed by paraformaldehyde fixation. Nonetheless, the biochemical changes observed clearly demonstrate that αvβ5 function as particle binding receptor correlates with its increased resistance to nonionic detergent extraction, which was reversibly regulated by PKC signaling. To directly test the possibility that an intact actin cytoskeleton might be required for αvβ5-mediated particle binding, we challenged J774 macrophages with OS in the presence or absence of PMA and Cyt D. Fig. 10 shows particle binding determined at 18°C to minimize phagocytosis of bound particles. OS bound efficiently to control macrophages even in the presence of Cyt D , in both cases in an β3 integrin–sensitive, αvβ5-independent manner (average β3 antibody inhibition 59% in control cells, 42% in Cyt D–treated cells). Increased OS binding in the presence of PMA exhibited the dual β3- and αvβ5-dependent mechanism (51% inhibited by β3 antibody, 39% inhibited by αvβ5 antibody), and blocking of both integrins with a combination of antibodies resulted in maximal inhibition of OS binding (75%). Strikingly, the addition of Cyt D decreased OS binding to PMA-treated cells by 40%, abolishing the increase induced by PMA. Addition of αvβ5-inhibiting antibody to these cells had no effect, whereas OS binding remained sensitive to β3-inhibiting antibodies (52% inhibition). Similar effects on PMA-induced particle binding only were observed when 1 μM latrunculin B, a different actin-disruptive drug, was substituted for Cyt D (data not shown). When we tested RPE-J particle binding in the presence of 5–20 μM Cyt D, we also found their binding activity reduced. However, the morphology of the epithelium was dramatically altered after 2 h of actin disruption. These experiments demonstrate that, in macrophages and RPE-J cells, binding of apoptotic cells or OS to αvβ5 integrin receptors but not to αvβ3 required intact actin microfilaments. The experiments reported here demonstrate that noninflammatory phagocytosis of apoptotic cells or OS can proceed along routes gated by different integrin-binding receptors, αvβ3 and αvβ5. Several important aspects of the particle recognition functions of these integrins were unraveled. Although RPE cells lack inflammatory phagocytic mechanisms mediated in immune cells by Fc receptors, complement receptors, and glycan receptors, they possess a highly efficient clearance pathway for OS. The RPE phagocytic mechanism for OS and apoptotic cells has a capacity similar to the monocyte macrophage phagocytic mechanism. Our results confirm and extend earlier reports that described the extraordinary specificity of the RPE recognition machinery towards OS 20 and of the machinery used by noninflammatory macrophages for apoptotic over normal cells 4 .OS and apoptotic cells quantitatively compete for both monocyte macrophage and RPE binding, demonstrating that the binding receptors used by either cell type to initiate phagocytosis of these types of particles do not discriminate between them. They also suggest that at least some of the local surface changes elicited by the daily photoreceptor outer segment renewal program are functionally equivalent to the recognition signals exposed during apoptosis. Surprisingly, rat primary and murine J774 macrophages used αvβ3 integrin to bind OS, even though they expressed abundant αvβ5 integrin at the cell surface. The partial reduction in particle binding observed when using αvβ3 inhibitory antibodies and RGD peptides on macrophages confirms earlier reports that they also use nonintegrin binding receptors for apoptotic cells or OS, presumably in parallel mechanisms (for a review, see reference 11). In contrast, RPE cells likely use αvβ5 integrin as primary binding receptor for OS, as αvβ5-inhibiting antibodies abolish 85% of particle binding. RPE cells bound apoptotic cells as efficiently as OS via an αvβ5-dependent pathway, even though they express equal levels of β3 integrins as macrophages. Since β3 integrins in RPE cells are polarized at the basolateral surface of the RPE, their availability for particle binding may be limited. Although we expect this hypothesis to be difficult to test experimentally in epithelial cells, macrophages do not share similar permeability barriers between plasma membrane domains. Indeed, on appropriate immobilized substrates or specific antibodies, β3 integrins were efficiently trapped at macrophage attachment sites, leaving αvβ5 receptors exposed at the particle contact surface of the phagocytes. In spite of their favorable exposure, αvβ5 receptors failed to mediate apoptotic cell or OS binding. These experiments clearly demonstrate that neither the ligand (OS versus apoptotic cells), the exclusive expression of a given integrin receptor (αvβ3 versus αvβ5), nor its localization at the site of particle contact is sufficient to generate the characteristic receptor selectivity of RPE and macrophage particle recognition. Our results show that specific cellular context determines which integrin receptor, αvβ3 or αvβ5, will be activated for recognition and binding of OS and apoptotic cells. Pharmacological studies revealed that the PKC signaling pathway plays a key role in the activation of αvβ5 for particle binding. Interestingly, RPE cells possess a maximally activated αvβ5 integrin–gated binding mechanism that can be blocked by PKC inhibitors but cannot be enhanced by PKC stimulators. In contrast, monocyte macrophages constitutively express a functionally equivalent binding mechanism insensitive to PKC, in which αvβ3 plays an important role, but activate a dormant αvβ5 binding mechanism upon PKC stimulation that acts in parallel with their αvβ3 pathway. Although there is no evidence to date supporting a functional role for the novel αvβ5-mediated particle recognition mechanism in phagocytosis by monocyte macrophages, it is likely that this route may be important in vivo. Indeed, an active αvβ5-dependent phagocytic recognition mechanism for apoptotic cells was recently described in immature dendritic cells 29 , and our results predict that PKC signaling may also be involved in the regulation of αvβ5 binding activity in these cells. Future studies should address the molecular mechanisms underlying αvβ5 integrin activation by cellular inside-out signaling mechanisms involving PKC (for a recent review, see reference 52 ). It is equally important to understand the unavailability of the abundant αvβ3 integrin receptors in the RPE, which, as mentioned above, may be related to their subcellular distribution, and the αvβ3 activation in macrophages. Particle binding involving both αvβ3 and αvβ5 integrins share a requirement of temperatures of at least 18°C, which may be a characteristic property of receptor mechanisms recognizing apoptotic cells and OS. Importantly, our results show that the activation of αvβ5-mediated particle recognition in macrophages by PKC did not result in slower binding or a lag phase after particle challenge, as would have been expected from the very slow kinetics of this process in RPE cells. Furthermore, RPE cells retain their αvβ5 integrin–dependent activity even after successive phagocytic challenges in vivo, and in vitro (Finnemann, S.C., unpublished data). In contrast, macrophages lose their αvβ3 integrin–dependent phagocytic recognition mechanism upon an initial particle challenge 2 . Thus, RPE cells are permanent noninflammatory phagocytes whose clearance mechanism exhibits unique binding characteristics that cannot solely be attributed to intrinsic properties of their primary recognition receptor, αvβ5 integrin. Our findings agree well with earlier investigations which demonstrated that αvβ5 but not αvβ3 integrin function is sensitive to modulation by PKC signaling 33 49 50 53 . However, this is the first report to demonstrate a specific effect of PKC on the association of αvβ5 itself with the actin cytoskeleton. Our biochemical assays did not determine whether PKC induced a direct or indirect association of αvβ5 with actin. Lewis et al. 50 have shown a correlation between PMA activation of αvβ5 and a redistribution or activation of several cytoskeleton-associated molecules, e.g., α-actinin, tensin, and vinculin. These actin-associated proteins may mediate both mechanical as well as signaling roles of integrin receptors. Cytoskeleton-associated proteins and their roles in apoptotic cell or OS binding and subsequent noninflammatory phagocytosis have yet to be studied in detail. Before inflammatory phagocytosis, particle binding to surface Fc receptors or αMβ2 integrins appears to be independent of the integrity of the actin cytoskeleton 54 55 . The hypothesis that binding of the same particle to a different integrin binding receptor, in response to phagocyte- rather than ligand-specified signals, induces different outside-in signaling effects, which ultimately determines properties of later phases of phagocytic clearance, is intriguing and will be the subject of further studies. The unique susceptibility of RPE phagocytes to genetic manipulation by viral or plasmid expression vectors in vitro and in vivo 42 56 will certainly be helpful in future studies designed to characterize the roles of specific actin binding and regulatory proteins in apoptotic cell or OS binding and phagocytosis. | Study | biomedical | en | 0.999996 |
10499925 | We obtained BALF from 11 patients with I-PAP, 2 patients with S-PAP, 14 patients with other lung diseases (3 cases each of sarcoidosis and collagen vascular lung disease; 2 cases each of interstitial pneumonitis, hypersensitive pneumonitis, and eosinophilic pneumonia; and 1 case each of bronchiolitis obliterans organizing pneumonia and lung cancer), and 53 normal subjects. Diagnosis of PAP was based on biochemical analysis of BALF and histopathological findings of lung biopsy. The I-PAP patients had no history of hematological disorders, infectious diseases, or toxic inhalation. Two S-PAP patients were in the blast phase of chronic myelogenous leukemia. 5 of 11 I-PAP patients, 1 of 2 S-PAP patients, and 30 normal subjects underwent venipuncture to obtain sera at various periods after bronchoalveolar lavage. Written informed consent to participate in this study was obtained from all subjects. Recombinant human (rh)GM-CSF was provided by Kirin Brewery Co., Ltd. (Takasaki, Japan), and 125 I–Bolton Hunter–labeled rhGM-CSF was purchased from NEN Life Science Products. rhIL-3 was purchased from R & D Systems, Inc. Biotinylated or nonlabeled rat anti–human GM-CSF mAb (BVD2-21C11 and BVD2-23B6, respectively) was purchased from PharMingen. Peroxidase-labeled rabbit anti–human IgA, -D, -E, -G, and -M polyclonal antibodies were purchased from DAKO Corp. A GM-CSF– or IL-3–dependent cell line, TF-1 12 , was provided by Dr. Kitamura (The Institute of Medical Science, The University of Tokyo, Tokyo, Japan). Proteins in BALF or sera were subjected to SDS-PAGE using gradient gel (2–15%) under nonreducing conditions at 30 mA constant current for 150 min. Separated proteins were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane at 12 V constant voltage for 75 min. The membrane was fixed with 10% (vol/vol) acetic acid and 50% (vol/vol) methanol, stained with Coomassie brilliant blue solution, washed with methanol, and treated with a blocking reagent (PBS supplemented with 1% [wt/vol] BSA and 0.1% [vol/vol] Tween 20) overnight at 4°C. The membrane was then incubated with 0.16 nM 125 I–GM-CSF for 1 h at room temperature. After washing, the membrane was exposed to x-ray film for 4 d. The method of this assay was described previously 11 . In brief, after coating a micro-ELISA plate with anti–human GM-CSF mAb (BVD2-23B6), rhGM-CSF and BALF were incubated in the wells. rhGM-CSF bound to BVD2-23B6 antibody was detected using biotinylated anti–human GM-CSF mAb (BVD2-21C11) and streptavidin conjugated to horseradish peroxidase. Color was developed using tetramethylbenzidine, and the absorbance was measured at 450 nm using a microplate spectrometer . Binding activity was calculated with the following equation: binding activity = 1 − detected GM-CSF (ng/ml)/applied GM-CSF (ng/ml). BALF was centrifuged at 1,000 g for 5 min. To remove lipoid materials, the supernatant was mixed vigorously with an equal volume of 1-butanol, and the mixture was centrifuged at 1,000 g for 5 min. After removing the butanol layer, the procedure was repeated. The aqueous layer was dialyzed against 10 mM ammonium acetate, pH 7.0, and lyophilized. Delipidated BALF was purified by using HiTrap SP column, a cation exchange column (equilibrated with 20 mM ammonium acetate, pH 6.0, and eluted with a linear sodium chloride gradient); HiTrap Q column, an anion exchange column (equilibrated with 20 mM Tris-HCl, pH 9.0, and eluted with a linear sodium chloride gradient); Superose 12 column, a gel filtration column (equilibrated with PBS containing 0.1% [vol/vol] NP-40 and eluted with the same buffer); RESOURCE Q column, an anion exchange column (equilibrated with 20 mM Tris-HCl, pH 9.0, and eluted with a linear sodium chloride gradient); and RESOURCE S column, a cation exchange column (equilibrated with 20 mM ammonium acetate, pH 6.0, and eluted with a linear sodium chloride gradient). All of these columns were from Pharmacia Biotech. Delipidated BALF was applied on recombinant protein A affinity column (Pharmacia Biotech) equilibrated with 20 mM sodium phosphate, pH 7.0. Ig bound to the column was eluted by pH gradient (pH 3.0–7.0). NH 2 -terminal sequencing of protein was performed by the phenyl isothiocyanate method using the HP G1005A NH 2 -terminal protein sequencing system (Hewlett-Packard Bioscience Products). Various concentrations of Ig purified from BALF of an I-PAP patient (39–5,000 ng/ml) were transferred to micro-ELISA plates coated with 1 μg/ml rhGM-CSF, and the plate was kept at room temperature for 1 h. After washing, 0.3 μg/ml of peroxidase-labeled anti–human IgA, -D, -E, -G, or -M polyclonal antibody was added to each well and incubated at room temperature for 1 h. Color was developed using tetramethylbenzidine, and the absorbance was measured at 450 nm. The method of this assay was described previously 13 . In brief, TF-1 cells (2 × 10 4 cells/well) were incubated for 3 d with 1 ng/ml of rhGM-CSF or rhIL-3 and 1 μg/ml of Igs purified from BALF of an I-PAP patient. To the culture, 5 μg/ml of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) was added and incubated. After formation of formazan crystals, isopropanol/HCl was added to dissolve the crystal, and the absorbance was measured at 595 nm. Occurrence of the GM-CSF binding factor in BALF supernatant was studied from 80 donors, including 11 I-PAP patients. As shown in Fig. 1 , blot assay with 125 I–GM-CSF gave a single band with a molecular mass of 180 kD in all I-PAP cases examined. In contrast, no band was detected in S-PAP patients, normal subjects, or patients with other lung diseases such as sarcoidosis, collagen vascular lung disease, interstitial pneumonitis, hypersensitive pneumonitis, and eosinophilic pneumonia. The binding factor in BALF was purified by cation- and anion-exchange chromatography and gel filtration chromatography . For evaluation of binding activity, a competition assay of GM-CSF binding to the mAb (BVD2-23B6) in ELISA was used. The purified protein showed a single band of 180 kD on SDS-PAGE under nonreducing conditions and two bands of 28 and 57 kD under reducing conditions . 125 I–GM-CSF binding to the purified 180-kD protein was confirmed by blot assay , and bound 125 I–GM-CSF was released when treated with citrate buffer, pH 2.0 (data not shown). The 57-kD band was electroblotted onto a PVDF membrane and sequenced directly. The NH 2 -terminal sequence of the 57-kD protein was EVQLVESGGGLVQPGGSLRL, identical to the NH 2 -terminal sequence of human Ig H chain. The data suggest that the GM-CSF binding protein in the BALF is an antibody. To show that the binding factor is actually an antibody, Ig in BALF from an I-PAP patient was isolated using a recombinant protein A column. Ig eluted from the column by changing pH gradient showed 125 I–GM-CSF binding activity . Specificity of binding to GM-CSF was demonstrated by effective competition of 125 I–GM-CSF binding with nonradioactive GM-CSF . 125 I–GM-CSF binding activity was not affected by the presence of nonspecific human Ig (data not shown). To confirm the bioactivity of isolated Ig, its inhibitory effect on growth of the TF-1 cell line was examined using the MTT assay. Growth of TF-1 cells is dependent on either GM-CSF or IL-3. The Ig purified from BALF inhibited GM-CSF–dependent growth of TF-1 cells but not IL-3–dependent growth . The results indicate that the antibody inhibits growth of this cell line by neutralizing the bioactivity of GM-CSF. The isotype of the antibody was determined by antigen capture assay. The Ig captured by GM-CSF reacted with only anti–human IgG, whereas no other antiisotype antibody reacted, indicating that the isotype of the antibody is IgG (data not shown). To know whether the I-PAP patients have circulating antibody against GM-CSF, we performed a blot assay with 125 I–GM-CSF of sera from 36 donors, including 5 I-PAP patients, 1 S-PAP patient, and 30 normal subjects who underwent bronchoalveolar lavage during this study. Serum samples from all I-PAP patients showed a single 180-kD band, whereas no such band was detected in samples from the S-PAP patient and normal subjects . Interestingly, the band was observed even in those from three patients who had entered remission and whose chest x-rays showed no opacity at the time of study. The isotype of the antibody in sera was also IgG (data not shown). These results indicate that the antibody is not limited to the lung but exists systemically in I-PAP patients. We have shown in this paper that autoantibody against GM-CSF is present in the lungs and sera of I-PAP patients but not in those of S-PAP patients, normal subjects, or patients with other lung diseases. Because GM-CSF is a key factor for maintaining the differentiation and proliferation of macrophages, dysfunction of AMs due to the neutralization of GM-CSF bioactivity by an autoantibody is a plausible explanation for the pathogenesis of human I-PAP. In fact, AMs from patients with active acquired PAP are known to be functionally impaired 14 15 . Furthermore, one type of congenital PAP was known to be associated with a defect of the GM-CSF receptor, and in murine models, knockout of the gene for GM-CSF or its receptor induced PAP-like disease. The disease in these murine models was corrected by transgenic expression of GM-CSF in the pulmonary epithelia or bone marrow transplantation from wild-type mice 16 17 . It is increasingly understood that sera in some healthy and diseased individuals contain autoantibodies against cytokines. Natural antibodies have been reported against rh IL-1β, IL-2, IL-8, and TNF-α 18 19 20 21 . Most of these autoantibodies were neutralizing antibodies and interfered with the binding of cytokines to receptors by simple competition 19 20 21 . Recently, it was reported that anti–GM-CSF autoantibody was frequently detected in pharmaceutically prepared human IgG 22 and that 0.3–2% of sera had a low titer of anti–GM-CSF antibody 22 23 . However, it was not reported that autoantibody against GM-CSF is associated with any disease or symptoms. Our results strongly suggest that I-PAP is an autoimmune disease with neutralizing autoantibody against the cytokine. As mentioned above, previous work in the patient case report and in mice has established that impaired production or action of GM-CSF can cause PAP. It is, therefore, reasonable to ascribe causality to the presence of autoantibody in the cases presented here, frequently and solely in the patients with I-PAP. This is, therefore, the first disease for which it can be argued that the cause is an autoantibody to a cytokine. It is not clear at the time of this writing why autoantibody is generated in I-PAP patients. One possibility is that the autoantibody was provoked by a cross-reactive antigen, possibly from infectious pathogen(s). Alternatively, the autoantibody was generated to endogenously regulate GM-CSF bioactivity. Although the functional consequences are predominantly manifest within the lung, our data suggest that the pathophysiologic disorder of I-PAP may originate from the systemic generation of autoantibody against GM-CSF. In this regard, recent clinical trials of GM-CSF administration to acquired PAP are intriguing 24 . The hematopoietic response to GM-CSF in the patients was attenuated. The impaired response was not due to altered expression of GM-CSF receptor on PBMCs. It is possible that the administrated GM-CSF was neutralized by anti–GM-CSF antibody before it reached the target organs. Finally, our observations have therapeutic implications. Whole lung lavage provides temporary remission in most cases, but additional lavage is required in more than 50% of patients 25 . Measurement of the autoantibody in the lung after whole lung lavage may identify patients who require further therapy or who have risk for recurrence. | Study | biomedical | en | 0.999996 |
10499926 | C57BL/6 (B6) and B6-β2m–deficient mice were purchased from The Jackson Laboratory. K b D b double knockout mice were a gift from Dr. Hidde Ploegh (Dept. of Pathology, Harvard University School of Medicine, Boston, MA). Anti–TCR-α/β (H-57), anti–TCR-γ/δ (GL-3), and anti-CD8β (Ly-3.2) antibodies were purchased from PharMingen. Anti-CD8α (53-6.7) was purchased from Sigma Chemical Co. For the isolation of lymphocytes from spleen and lymph node, spleens and lymph nodes were macerated individually using frosted glass slides. The resulting suspension was centrifuged on a Ficoll gradient at 900 g for 10 min. Cells at the interface were collected, and T cells were enriched by passing through a nylon wool column as described previously 14 . iIELs were prepared as described previously 15 . In brief, small intestines were harvested and washed by passing through PBS. Mesentery and Peyer's patches were carefully removed. The intestines were cut longitudinally and then in ∼0.5-cm pieces. Intestinal pieces were agitated in 50 ml of extraction buffer (PBS, 3% FCS, and 10 mM EDTA) for 30 min at 37°C. This slurry was centrifuged at 2 g for 2 min to remove the aggregates. The cell suspension was layered on a discontinuous Percoll (Amersham Pharmacia Biotech) gradient. This gradient was then centrifuged at 900 g for 20 min. Cells at the interface of the 40/70% layer were collected and washed in staining buffer. Lymphocytes from the liver were prepared as described previously 16 . In brief, livers were macerated using stainless steel mesh and suspended in PBS, 3% FCS. They were loaded on a discontinuous Percoll gradient and centrifuged at 900 g for 20 min. Cells were collected from the 40/70% interface, washed, and used for further experiments. Cells were suspended in staining buffer (PBS, 3% FCS, 0.01% sodium azide) at a concentration of 10 7 cells/ml. 100 μl of the suspension was incubated with directly conjugated antibodies for 30 min on ice. Cells were washed twice with staining buffer and fixed with 1% paraformaldehyde. Fluorescence intensities were measured with a FACScan™ (Becton Dickinson). For positive selection, CD8 + T cells require MHC class I proteins, and in this process the CD8 molecules serve as coreceptor 17 . In peripheral CD8 + T cells, the CD8 molecule is expressed as a membrane-bound heterodimeric protein consisting of α and β chains 18 . However, certain CD8 + T cells express an alternate CD8α/α homodimer. Thus, we examined the organ distribution of these unusual T cells. We found that most of the CD8 + T cells in spleen, lymph node, thymus, and liver express CD8α/β, and they all consistently bear TCR-α/β. By contrast, in iIELs most of the T cells express CD8α, but only a small portion of the T cells express CD8β as well . In spleen, lymph node, thymus, and liver, only a very small fraction of CD8α + TCR-γ/δ cells were found. In contrast, in iIELs a large portion of the CD8α/α 1 T cells were found to also be TCR-γ/δ + . In three-color staining, gating on TCR-α/β revealed that in spleen, lymph node, thymus, and liver, almost all of the CD8α-bearing T cells express CD8β, but in iIELs a large number of TCR-α/β cells express only CD8α. Among these, only a fraction (9–11%) are CD8β + . On further analysis of the iIELs, it was found that among iIELs, TCR-α/β and TCR-γ/δ cells are present at almost equal numbers, and a majority of the cells express the CD8 cell surface molecule . Gating on TCR-γ/δ cells among the iIELs revealed that most such cells express CD8α and none express CD8β . β2m-associated MHC class I proteins are present on the cell surface at ∼10 5 –10 6 molecules per cell and are divided into two distinct categories. The classical MHC class I proteins are derived from the genes for K, D, and L, which in B6 mice are called K b and D b (there is no L molecule in the H-2 b haplotype). The nonclassical MHC class I proteins are called class Ib molecules, are the products of Qa, TL, and M regions, and are also coded by the non-MHC genes of the CD1 locus on chromosome 1. To evaluate the type of MHC restriction of both CD8α/α and CD8α/β T cells, we examined the composition of these two groups of cells in K b D b double knockout mice. It was found that most of the CD8α/β TCR-α/β cells disappeared in spleen, lymph node, and liver as well as in iIELs . In iIELs, numbers of CD8α 1 cells were not affected. In the thymus, there were normal numbers of CD4 + CD8 + double positive (DP) cells in K b D b double knockout mice. These are presumed to be of the TCR-α/β lineage. However, there were virtually no CD4 − CD8 + single positive (SP) cells , and all of the DP cells were found to use CD8α/β . Therefore, these DP cells in the thymus are uncommitted immature thymocytes that had not undergone intrathymic selection. Thus, CD8α/α 1 T cells are mostly found in iIELs. This clearly indicates the potential for recognition of nonclassical MHC class Ib molecules by T cells bearing a TCR-α/β and the CD8α/α homodimer. To examine whether this group of CD8α/α T cells required β2m-associated MHC class I or class Ib molecules, we analyzed the composition of CD8α/α cells in β2m −/− mice. It was found that CD8 + T cells are absent in spleen, lymph node, and liver. However, substantial numbers of CD8α + T cells were present in thymus and iIELs . In thymus, all of the CD8α + T cells bear CD4 as well. No CD8 SP (CD4 − CD8 + ) cells were found in these mice. A careful analysis revealed that all CD8α + T cells in iIELs possess a γ/δ TCR . Thus, the development of the vast majority of the TCR-α/β CD8α/β cells requires contact with K b or D b molecules. By contrast, CD8α/α TCR-α/β cells depend on β2m-associated nonclassical MHC class Ib molecules. The development of CD8α + TCR-γ/δ cells in iIELs either does not require MHC class I for their positive selection or they are restricted to β2m-independent MHC class I molecules. The existence of β2m-independent MHC class I molecules is as yet an open question. Recently, a group of stress-induced β2m-independent MHC class I molecules was reported in humans, but the homologous genes are not found in mice 19 . Thus, the diversity of a β2m-independent MHC class I molecule in the gut is a worthy goal for future research on the development of CD8α + γ/δ T cells. | Study | biomedical | en | 0.999995 |
10499927 | Mice were housed at Princeton University under specific pathogen-free conditions and were 10–14 wk old at the time of study. Mutant mice (deficient in TAP-1 24 , K b /D b 25 , CD1.1 [Park, S.-H., and A. Bendelac, manuscript in preparation], CD1.1/1.2 26 , or β2m 27 ) were generated from embryonic stem cells of 129 origin and used after 6–10 backcrosses to C57BL/6. K b /D b /CD1-deficient mice were generated by crossing CD1.1- with K b /D b -deficient mice. In all cases, +/+ or +/− littermates were used as controls, except for β2m- and CD1.1/CD1.2-deficient mice, which were compared with age- and sex-matched C57BL/6 mice. For histological study, duodenal fragments of 1 cm taken 3 cm below the pylorus were properly oriented on filter paper and fixed in Carnoy's fluid for 24–48 h. Paraffin-embedded sections were prepared and stained with periodic acid-Schiff (PAS), and the numbers of IELs per 100 epithelial cells (ECs) were counted under the microscope. The small intestine was separated from Peyer's patches and mesenteric lymph nodes, cut longitudinally, and washed in PBS. Fragments of 0.5–1 cm were incubated for 30 min at 37°C in RPMI 1640 (GIBCO BRL) containing 1% dialyzed FCS (Biofluids), 1.5 mM MgCl 2 , and 1 mM EGTA and supplemented with 1 mM dithiothreitol (Sigma Chemical Co.), under constant shaking. The IELs contained in the supernatant were collected, washed three times in PBS/5% FCS, and passed through a nylon mesh. IELs were further purified by centrifugation over Ficoll-Paque™ (Amersham Pharmacia Biotech). Lymphocytes were first incubated for 30 min with 2.4G2 anti-Fc antibodies in order to block Fc receptors. For triple membrane staining with directly conjugated antibodies, cells were labeled with a combination of antibodies conjugated to PE, FITC, Cy-Chrome, or biotin. Biotinylated antibodies were revealed with Streptavidin Tricolor (Caltag Laboratories). Anti–TCR-α/β, TCR-γ/δ, CD8β CD8α, CD4, CD19, CD44, CD69, B220, CD103, and TCR Vβ were purchased from PharMingen. Fluorescence was analyzed on a FACScan™ (Becton Dickinson). The live gate for acquisition contained >95% CD103 + cells, no CD19 + cells, and <10% CD4 + cells in all cases. Fig. 1 shows a flow cytometry analysis of splenocytes and intestinal IELs from K b /D b -deficient mice and littermate controls. Unlike CD8 + TCR-α/β 1 cells in the spleen, which were virtually all dependent on K b or D b expression, the percentage of CD8 + TCR-α/β 1 IELs was unaffected by the loss of the classical MHC molecules. This striking observation was confirmed on a total of nine K b /D b -deficient mice (33 ± 9% CD8 + TCR-α/β 1 IELs) and eight littermate controls (38 ± 9% CD8 + TCR-α/β 1 IELs). Furthermore, because cell recoveries after isolation of IELs may vary from one sample or one experiment to another, we counted the number of IELs per 100 ECs on histological sections of duodenal samples, and found that the numbers of IELs/100 ECs were comparable in K b /D b -deficient mice (15 ± 3 IELs/100 ECs) and littermate controls (15 ± 3 IELs/100 ECs) ( Table ). Thus, the absolute number of CD8 + TCR-α/β 1 IELs seemed to be conserved. In addition, no change in the frequency of TCR-γ/δ 1 or CD4 + TCR-α/β 1 IELs could be detected in K b /D b -deficient mice . To further investigate the observation that CD8 + TCR-α/β 1 IELs seemed unaffected in K b /D b double-deficient mice, we analyzed the CD8α/α 1 and CD8α/β 1 subsets of TCR-α/β 1 cells separately. These two subsets differ with respect to their ontogeny, recirculation, and function, suggesting that they might exhibit different requirements for MHC ligands as well 4 5 6 . Fig. 2 and Table show that the CD8α/β 1 subset was virtually entirely absent in K b /D b double-deficient mice, whereas the CD8α/α 1 cells were conserved or even increased. As previously noted, there were no significant differences between the absolute numbers of IELs/100 ECs found in the various genetically modified mice used in this study and in their littermate controls. Thus, the frequency of various subsets among total IELs directly reflected their absolute number . These results demonstrate a fundamental difference in the MHC ligands used by the two subsets of CD8 + TCR-α/β 1 IELs. Most CD8α/α 1 cells are independent of classical MHC class I molecules, whereas most CD8α/β 1 cells are dependent on K b /D b . To further investigate the possibility that the development and/or expansion of a CD8α/α 1 TCR-α/β 1 cell population was dependent in any way on classical K b and D b molecules, we examined their surface phenotype in the K b /D b -deficient mice. We found that they expressed the same degree of activation as their littermate controls, as judged by the expression of the activation markers CD44, CD69, and B220 . We also examined the rare subset of CD8/CD4 double-negative TCR-α/β 1 IELs, as it might include precursors of the CD8α/α 1 lineage. Again, no difference could be detected between K b /D b -deficient mice and littermate controls (data not shown). We next analyzed the Vβ repertoire of the CD8α/α 1 population of K b /D b -deficient mice. Table shows that CD8α/α 1 cells expressed a diverse TCR Vβ repertoire, but that the frequency of various Vβs varied considerably from mouse to mouse. This pattern was also found in K b /D b +/− littermates, and most likely results from the presence of oligoclonal expansions, as reported previously 19 20 . Thus, the CD8α/α 1 TCR-α/β 1 IEL subset of K b /D b -deficient mice appears to conserve the activated phenotype and the pattern of oligoclonal expansions that are characteristic of normal IELs, further indicating that CD8α/α 1 TCR-α/β 1 IELs persist unaltered in K b /D b -deficient mice. Although it was previously reported that all of the CD8α/β 1 IELs were dependent on the presence of TAP, some CD8α/α 1 TCR-α/β 1 IELs seemed to be TAP independent 21 22 . We confirmed these results, showing that TAP-deficient mice retained about half the number of CD8α/α 1 TCR-α/β 1 IELs in their littermate controls . In contrast, as reported previously 21 , all CD8α/α 1 IELs as well as CD8α/β 1 IELs depended on β2m. These results suggest two possibilities. Either CD8α/α 1 TCR-α/β 1 cells recognize two distinct nonclassical MHC class I–like molecules, only one of which is TAP dependent, or they recognize one nonclassical MHC class I–like molecule that is partially TAP dependent. The conservation of the nonclassical MHC class I–like molecule CD1d in mammals, and the reports that it is expressed by intestinal epithelial cells of both mice and humans and that CD8 + T cell clones isolated from human IELs were CD1d reactive in a TAP-independent fashion, made CD1d an attractive candidate as the ligand of a subset of the CD8α/α 1 TCR-α/β 1 IELs 23 . Mice have two CD1 genes that are 95% identical, both belonging to the CD1d family 28 29 . However, the CD1.2 gene has a frameshift mutation in the B6 strain that is predicted to abolish cell surface expression and in other strains CD1.2 also seems to be poorly, if at all, expressed on the cell surface 30 31 . We analyzed the IEL population of CD1.1 as well as CD1.1/CD1.2 double-deficient mice. Fig. 2 shows that both the CD8α/α 1 and CD8α/β 1 TCR-α/β 1 IELs were conserved in CD1.1/CD1.2-deficient mice. Results obtained from CD1.1- and CD1.1/CD1.2-deficient mice were comparable and are pooled in Table . Altogether, these results show that neither the CD8α/α nor the CD8α/β subset is dependent on CD1, and suggest that the CD8α/α subset requires an unknown nonclassical MHC-like molecule that is β2m dependent and partially TAP independent. Because of the compensatory expansion of IEL subsets often observed in the various mutant mice (see Table ), there remained the possibility that CD8α/α 1 TCR-α/β 1 IELs were a heterogeneous population made of a TAP-dependent subset restricted by K b /D b and another TAP-independent subset restricted by CD1, but that the ablation of one subset in the corresponding mutant mouse was masked by the expansion of the other. To formally address this possibility, we generated K b /D b /CD1 triple-deficient mice. Fig. 4 shows that the IELs isolated from K b /D b /CD1 triple-deficient mice contained similar proportions of CD8α/α 1 TCR-α/β 1 cells as CD1-deficient or K b /D b -deficient control littermates. These results suggest that neither CD1 nor K b /D b are ligands of CD8α/α 1 TCR-α/β 1 cells, unambiguously demonstrating the existence of a large population of intestinal IELs that is dependent on a nonclassical, non-CD1 MHC class I–like molecule. Although CD8α/α 1 TCR-α/β 1 IELs could be detected in transgenic mice expressing TCR-α/β with defined MHC class I/peptide specificities 32 33 34 , normal nontransgenic CD8α/α 1 TCR-α/β 1 IELs have not been associated with antigen-specific, classical MHC class I–restricted function. The pattern of expression of CD8α/α 1 TCR-α/β 1 IELs in β2m-, TAP-, K b /D b -, and CD1-deficient mice, as reported in this study, clearly demonstrates that most CD8α/α 1 TCR-α/β 1 IELs do not recognize classical MHC class I molecules, and points to nonclassical MHC class I–like molecules that are β2m dependent and partially TAP independent. Since CD1 could be ruled out by the study of CD1-deficient mice, candidate ligands include, but may not be restricted to, TL, which is expressed on intestinal epithelial cells 35 , and Qa1 36 37 , both nonclassical MHC class I molecules that can function in the absence of TAP. There is increasing recognition that several body tissues, especially barrier epithelia of the skin and intestine, and the liver, have specialized immune systems that contain prominent populations of resident T lymphocytes with original, yet poorly understood, antigen specificity and functions. In recent years, two previously orphan families of nonclassical MHC class I–like molecules have become associated with such populations, including MICA/MICB for human intestinal TCR-γ/δ 1 IELs and CD1 for liver NKT cells 3 31 . Thus, the emerging pattern suggests that nonclassical MHC-like molecules with specialized functions are critical in specialized tissue environments. Our results now clearly demonstrate that another major subset of mouse intestinal IELs, the CD8α/α 1 TCR-α/β 1 cells, recognize a nonclassical, non-CD1 type of MHC class I–like molecule. Further studies are warranted to identify the ligand(s) of CD8α/α 1 TCR-α/β 1 intestinal IELs, a key for our understanding of local immunity in the intestine. | Study | biomedical | en | 0.999996 |
10508849 | Secretory lysosomes are a mixture of lysosomes and secretory granules on many different levels. Secretory lysosomes contain both the hydrolases and membrane proteins characteristic of lysosomes as well as the specialized secretory products of different cell types. Functionally, they serve both as the lysosome of the cell and as the secretory granule. The acidic pH is optimal for the action of lysosomal hydrolases and contributes to keeping secretory products inactive before release. As is true for lysosomes, proteins can reach secretory lysosomes by both endocytic and biosynthetic routes as demonstrated by HRP uptake and the delivery of newly synthesized proteins. Morphologically, secretory lysosomes are a mixture of the multilamellar structures characteristic of lysosomes and the dense cores characteristic of secretory granules. Studies on secretory lysosome biogenesis in T lymphocytes indicate that the dense core forms and enlarges within a multivesicular structure . In mature T lymphocytes, there is a high degree of overlap between secretory and lysosomal markers, suggesting that the majority of lysosomes are secretory lysosomes (Stinchcombe, J.C., and G.M. Griffiths, unpublished observation). However, in neutrophils, lysosomal structures lacking VAMP-2 can be identified alongside other VAMP-2–labeled granules , suggesting that these cells may possess both true lysosomes and secretory lysosomes ( Table ). The mechanisms that regulate the sorting of proteins to the secretory lysosomes are also a mixture of those used to target lysosomal proteins and secretory granule proteins in conventional cells, although in hemopoietic cells the proteins are targeted to the same organelle. For example, the soluble secretory granzymes of T lymphocytes follow the mannose-6-phosphate pathway used by lysosomal hydrolases to reach the granules . Other soluble proteins of secretory lysosomes, such as perforin in T lymphocytes, are able to complex with proteoglycans and may be sorted by selective condensation into the dense core, as has been suggested for chromogranins in endocrine secretory cells . Membrane-bound proteins that are expressed only in cells with secretory lysosomes, such as GMP-17 or CTLA-4 , possess the tyrosine-based sorting motifs found in lysosomal membrane proteins that enable selective sorting to the secretory lysosome. Recent data suggest that specialized mechanisms for sorting to secretory lysosomes may also exist. This arises from the observation that the membrane-bound protein of T lymphocytes, Fas ligand, is differentially sorted in hemopoietic and nonhemopoietic cells. The cytosolic tail of this protein preferentially sorts Fas ligand to secretory lysosomes in hemopoietic cells, but is unable to do so in nonhemopoietic cells in which it is expressed directly on the cell surface . Mutagenesis of the tail demonstrates that a proline-rich domain is required for sorting to secretory lysosomes (Bossi, G., and G.M. Griffiths, unpublished observation). One possible mechanism for the differential sorting might, therefore, involve the interaction of this proline-rich domain with an SH3-domain containing protein that is preferentially expressed in cells with secretory lysosomes. The regulated secretion of the lysosomal compartment by hemopoietic cells provides an important pathway for controlling the release of both soluble and membrane proteins in many cells from this lineage. Cells of the immune system have both effector and regulator functions, and often delivery of both soluble and membrane proteins needs to be tightly controlled. For example, the secretory lysosomes of T lymphocytes can contain not only soluble proteins required for destruction of virally infected and tumorigenic targets (such as perforin and granzymes), but also membrane-bound proteins that are essential for controlling the immune response, e.g., Fas ligand and CTLA-4 . Recognition of the target cells via the T cell receptor triggers kinesin-driven movement of the secretory lysosomes along microtubules to the point of membrane contact between the T lymphocyte and target . The secretory lysosome membranes then fuse with the plasma membrane, acting not only to release the soluble proteins (e.g., perforin), but also to deliver the membrane proteins (e.g., Fas ligand) to the site of interaction. Delivery of these proteins via the secretory lysosome allows the T lymphocyte to first store these proteins within the cell and then control the precise location and timing of their release so that they are perfectly focused on the target cell. This is important in ensuring the specificity of the T lymphocyte response and in preventing damage to bystander cells which are not recognized by the T cell receptor. In some cells it appears that not only the lysosome can be released but other prelysosomal compartments can also fuse with and deliver proteins to the plasma membrane. This mechanism is particularly important in hemopoietic cells expressing MHC class II. MHC class II is present within the cell in a prelysosomal compartment, termed the MIIC, and can be relocalized to the cell surface . Since MHC class II presents peptides from extracellular pathogens that need to be taken up and degraded by the cell, the exocytosis of a compartment from the degradative pathway on the way to the lysosome is therefore ideal for efficient antigen presentation. Curiously, secretion of this multivesicular compartment not only translocates proteins of the outer membrane to the cell surface but also results in the release of small internal vesicles, termed exosomes, which may themselves have important biological effects . Several of the key proteins involved in secretion of conventional secretory granules are found in cells with secretory lysosomes. For example, both v- and t-SNARES , as well as Rab proteins and synaptotagmins , have been found to be associated with secretory lysosomes in hemopoietic cells ( Table ). This indicates that some of the machinery involved in regulating the release of secretory lysosomes is common to that used by conventional secretory cells. However, some of the critical components of the secretory machinery are specific to cells with secretory lysosomes. The most compelling evidence comes from the human autosomal recessive disease, Chediak-Higashi syndrome (CHS). In this disease, all lysosomes are abnormally enlarged but with no obvious effect on the endocytic and degradative roles of the mutant lysosomes . Similarly, the secretory function of conventional secretory cells is normal in CHS. What is particularly interesting about CHS is that the cell types that are functionally impaired all seem to be those with secretory lysosomes. In the case of T lymphocytes it has been demonstrated that the secretory lysosomes are unable to be secreted . These observations implicate the defective protein in a unique aspect of secretory lysosome release which is not required for exocytosis of secretory granules in conventional secretory cells. The fact that replacement of cells of the hemopoietic lineage by bone marrow transplantation can be successfully used to treat patients with CHS supports this hypothesis . The gene that is defective in CHS has now been cloned from both humans and mice . The sequences predict homologous cytosolic proteins of ∼400 kD. The gene is expressed in the majority of tissues examined, consistent with the abnormally sized lysosomes found in all CHS cell types. The most direct clue as to the function of the protein comes from experiments in which the wild-type protein is overexpressed in mutant fibroblasts. This results in the production of abnormally small lysosomes, suggesting that the protein is involved in lysosomal fission . One of the most intriguing clues from CHS regarding secretory lysosome biogenesis and secretion is the observation that the defect results in partial albinism. This is most apparent in the mouse model of the disease, the beige mouse, due to its partial albino coat color compared with the wild-type strain from which it arose. The critical link is the melanocyte, which, although not arising from the hemopoietic lineage, also possesses secretory lysosomes. These organelles, known as melanosomes, secrete the pigment melanin which then enters keratinocytes and gives rise to coat color . Defective pigmentation in CHS has two important implications. First, secretory lysosomes are not entirely restricted to cells derived from the hemopoietic lineage. And second, other forms of albinism may reflect defects in secretory lysosome biogenesis and release. Since albinism requires sorting of proteins required in melanin synthesis, as well as polarization and secretory steps, then mutants may reflect different stages of this process. The examples that have already emerged show that this is the case. Recent studies on two human autosomal recessive diseases that give rise to partial albinism have produced some intriguing findings concerning the link between albinism and secretory lysosome biogenesis. The first is Griscelli's syndrome that has a mouse homologue known as dilute . Griscelli's syndrome is clinically related to CHS in that the patients show selective immunodeficiency and partial albinism . The defective gene in the dilute mouse was shown to be the myosin Va heavy chain and the human lesion has been shown to encode the same protein . In wild-type melanocytes, melanosomes are concentrated in the peripheral dendrites from which they are released. However, in dilute mice the melanosomes are more concentrated in the center of the cell. In wild-type cells, myosin Va colocalizes with melanosomes in the dendrites . Recent studies suggest that myosin Va is important in capturing melanosomes that reach the periphery, since overexpression of a dominant negative myosin Va in wild-type cells dramatically depletes melanosome accumulation at the periphery . The selective nature of these diseases, which affect both hemopoietic cells and melanocytes, raises the intriguing possibility that myosin Va may also play a critical role in the secretory lysosome polarization which is required for secretion in many hemopoietic cell types. The second disease to shed light on the link between albinism and cells with secretory lysosomes is Hermansky-Pudlak syndrome (HPS). HPS is an autosomal recessive disease of humans resulting in partial albinism and defects in lysosomal secretion which has several mouse models . Since the defective genes in the different mouse models map to at least 10 different loci, it seems likely that defects in multiple genes result in a similar phenotype . Recent studies demonstrate that HPS and its mouse models reflect defects in a lysosomal sorting pathway. Two different proteins have been identified so far. One has been termed HPS1 and encodes a 79-kD novel transmembrane protein of unknown function . The HPS1 sequence provides few clues as to the function of this protein and there is no significant homology to other known proteins. The other protein that has been found to be defective is the adaptor protein AP3. The mocha mouse is defective in the Δ subunit of AP3, while pearl mice are defective in the β3A subunit . Some HPS patients which show normal expression of HPS1 have also been shown to be defective in the β3A subunit of the AP3 adaptor protein . Several lysosomal membrane proteins are mis-sorted in fibroblasts derived from these patients. Findings from earlier studies of HPS and its related mouse models which indicate that both melanocyte function and lysosome secretion are affected by these mutations, suggest that AP3 may be especially important in transporting proteins to both melanosomes and secretory lysosomes. Intriguingly, all of these mutants have also been reported to be defective in the secretion lysosomal hydrolases from kidneys, another cell type that uses secretory lysosomes . Together, these studies demonstrate that many of these albinism mutants may provide important clues in understanding the different steps of the secretory mechanisms used by cells with secretory lysosomes. Although secretory lysosomes are predominantly used by cells of the hemopoietic lineage, there are clearly some nonhemopoietic cells which use a lysosomal compartment for regulated secretion, such as melanocytes and renal tubular cells . This suggests that the secretion of lysosomes might be a more widespread phenomenon which has simply been enhanced in cells of the hemopoietic lineage. Several observations support this idea. First, lysosomes from nonsecretory cells can be secreted. It has been shown that both Chinese hamster ovary and normal rat kidney cells can be induced to secrete their lysosomes in response to influx of high levels of calcium . Although the percentage of the total lysosomal population responding to the signal is generally low (10%, compared with 60% in cells with secretory lysosomes) and high levels of calcium are required, these observations suggest that there may be secretion-competent lysosomes in these cells. Similarly, trypanosomes seem to be able to trigger calcium-mediated fusion of host cell lysosomes at the cell surface during their invasion into a variety of nonhemopoietic mammalian cells . Again only a sub-population of lysosomes respond to the trypanosomal signal in fibroblasts. These differences in the level of lysosomal secretion may simply reflect variations in the number of secretion-competent lysosomes in different cell types. Second, there is evidence that repair of the plasma membrane may involve a process of lysosomal secretion. Wounding of the plasma membrane studied in many nonhemopoietic cell types results in a calcium flux which causes membranes of endocytic origin to fuse with the plasma membrane . In this way, lysosomal exocytosis could be an important mechanism in wound healing required by all cell types . Studies from Dictyostelium suggest that secretion of lysosomes may represent a primitive secretory system. Dictyostelium are also able to secrete their lysosomal contents and a number of mutants in this process have been isolated, demonstrating that several groups of genes are required for secretion . Many of these genes also appear to be important in the development of the multicellular stage of the Dictyostelium life cycle. Of particular interest is the observation that one of the genes required for cytokinesis bears strong homology to the gene that is defective in CHS (De Lozanne, A., personal communication). Many of the secretory mutants which block polarized membrane delivery in Saccharomyces are also required for cytokinesis and may also be involved in polarized delivery of secretory lysosomes. The existence of genes required for lysosomal secretion in Dictyostelium related to those required for lysosomal secretion in mammals suggests a strong evolutionary conservation of these mechanisms. Taken together, these findings suggest that the mechanism of regulated secretion of lysosomes used by hemopoietic cells is present in many cell types and may represent a primitive secretory system which has simply been enhanced in cells of the hemopoietic system. If this is the case, then it is likely that the specialized secretory granules used for regulated secretion from exocrine and endocrine cells are a later evolutionary development. Intriguingly, although conventional endocrine and exocrine secretory cells contain distinct lysosomes and secretory storage compartments they contain a post-Golgi intermediate, the immature granule, on the pathway to these distinct organelles in which the newly synthesized secretory and lysosomal proteins coexist . This compartment has several features in common with secretory lysosomes: it is acidic, performs some proteolytic functions, and can be simulated to secrete in a calcium-dependent manner . It may be that the immature granule is an evolutionary vestige of a secretory lysosome but that in these cells the secretory and lysosomal functions have subsequently become separated. In contrast, regulated secretory cells of the hemopoietic lineage have optimized a more primitive secretory mechanism and use it to regulate the release of soluble and membrane proteins. | Study | biomedical | en | 0.999996 |
10508850 | MDCK cells were grown as described 4 . Recombinant adenoviruses encoding the wild-type (WT) and mutant ARF6, as well as the clathrin hub, were produced as described 1 . Levels of protein were regulated by the concentration of doxycycline (DX), amount of virus, and length of time after removal of DX. Very high levels of expression produced toxic effects. 0.1 ng/ml DX was included to partially repress ARF6 production. Cells were incubated for 16–18 h to express recombinant proteins, except clathrin hub, for 24–26 h. Unless stated, we used 60–70 pfu/cell for ARF6–WT and ARF–Q67L, or 90–100 pfu/cell for ARF6–T27N. These produced equal levels of the ARF6 proteins, as assayed by immunoblotting. For assays with 125 I-IgA, 0.1 ng/ml DX was included. Using antibodies that specifically recognize the endogenous ARF6 (kind gift of V. Hsu, Harvard Medical School, Boston, MA) the level of exogenous ARF6 was approximately fivefold, relative to endogenous ARF6. For immunofluorescence, we omitted the DX to produce a ninefold overexpression relative to endogenous ARF6. These conditions minimized the possibility of toxic effects and produced an adequate signal for our localization and functional studies. Controls in all experiments included cells that were: not infected; infected but the expression of ARF6, dynamin-I K44A, or clathrin hub was fully repressed by 20 ng/ml DX; or infected with a control virus encoding β-galactosidase (gal). These caused complete loss of the ARF6, dynamin-I, or hub-specific signal in immunofluorescence and biochemical studies. Immunofluorescence was as described 11 . For colocalization of ARF6 with IgA, cells were washed with cold medium and incubated with 300 μg/ml IgA for 60 min. Processing for EM was as described 2 . Cells were observed at a magnification of 19,000 and every third cell was photographed and viewed at 80 kV. A total of 30 randomly selected cell profiles were photographed. The negatives were scanned using Adobe Photoshop at a resolution of 1,000 dpi. Clathrin-coated pits were easily and reproducibly discernible. A total of 148 images were used: control, 38 images; ARF6–WT, 33 images; ARF6–Q67L, 39 images; and ARF6–T27N, 38 images. Two observers counted coated pits within each coded image and gave consistent observations. The length of the apical PM was measured using a ruler on Adobe Photoshop, and the number of each type of coated pit was divided by the length. Endocytosis was assayed as described previously 4 . For endocytosis in the presence of clathrin hub, IgA (300 μg/ml) was bound for 1 h at 4°C and cells were washed over 45 min. Cells were warmed to 37°C for 5 min. Cells were cooled to 4°C and PM-bound material was stripped with trypsin. Trypsin was neutralized with soy bean trypsin inhibitor. Cells were fixed for 40 min on ice and IgA was stained as described. Cells expressing clathrin hub were stained with T7 antibody (Novagen, Inc.). We used a recombinant adenovirus incorporating the tetracycline transactivator system to express WT ARF6 and its mutants. Confocal microscopy showed that ARF6–WT is associated with the AP region of the cell, and not the BL region. Fig. 1 A shows a vertical X-Z section taken through the plane of a monolayer of cells expressing ARF6–WT. ARF6 staining is in green, whereas ZO-1 staining of the tight junctions is in red. Fig. 1 B shows horizontal X-Y sections taken either at the level of the AP PM or through the basal region of the cell . Both ARF6–WT and ARF6–Q67L were localized at, or close to, the AP PM. In contrast, ARF6–T27N gave staining in both the AP and BL regions of the cell. The AP PM tends to bulge upward in our MDCK cells, forming a dome-like structure. We took advantage of this to utilize the higher X-Y confocal resolution to determine if ARF6 was entirely at the PM, or beneath the PM. Cells were costained with antibody to gp135, an endogenous AP PM protein. The X-Y sections in Fig. 1 C were just above the level of the tight junction, at the level indicated by the arrow in Fig. 1 A. In the ARF6–WT expressing cell, this X-Y section shows the gp135 staining of the AP PM as a narrow ring . The ARF6–WT in the same cell is a broader ring. The ARF6–WT staining in the outer portion of the ring was coincident with the gp135, indicating that this portion of the ARF6–WT is at the PM . The inner portion of the ARF6–WT ring is underneath the gp135 staining, i.e., in the cytoplasm beneath the AP PM (arrow). Expression of ARF6–Q67L gave a similar pattern as ARF6–WT. ARF6–T27N gave no ring, but rather, central staining that had little overlap with gp135, indicating that it is mostly absent from AP PM. To examine the effects of ARF6 on endocytosis, we used 125 I-IgA as an endocytic marker, which can be endocytosed via the polymeric immunoglobulin receptor (pIgR) at either PM. Strikingly, overexpression of either ARF6–Q67L or ARF6–T27N stimulated endocytosis of 125 I-IgA from the AP PM . ARF6–Q67L and ARF6–T27N increased endocytosis during the first minute by ∼3-fold or 1.5-fold, respectively. In contrast, AP endocytosis of IgA in cells overexpressing ARF6–WT did not markedly differ from control cells infected with a virus encoding β-gal or uninfected cells. Overexpression of ARF6–WT or either mutant did not significantly alter the rate or extent of BL endocytosis, recycling back to the AP PM, BL to AP transcytosis, or BL to BL recycling (our unpublished data). Therefore, only AP endocytosis was affected by ARF6. In Fig. 2 B, levels of ARF6–Q67L or ARF6–T27N were manipulated by infecting cells with varying amounts of virus. The stimulatory effect of ARF6–Q67L was significantly greater than that of ARF6–T27N at all expression levels examined. In cells simultaneously expressing ARF6–Q67L and ARF6–T27N (achieved by double infection), we observed increased stimulation of endocytosis above levels induced by ARF6–Q67L alone . We next tested if ARF6 could stimulate endocytosis of markers that are not internalized via clathrin-coated pits. Mutation of both cytoplasmic Tyr in pIgR gave a pIgR that is endocytosed at about five percent of the rate of the WT 15 . Fig. 2 D shows that neither ARF6–Q67L nor ARF6–T27N stimulated endocytosis of this mutant. ARF6 did not affect AP internalization of ricin or fluid phase markers (our unpublished data), confirming that ARF6 does not affect nonclathrin endocytosis. To test if ARF6–Q67L stimulates clathrin-mediated endocytosis, we inhibited clathrin function by cytosol acidification or hypertonic media, and found that ARF6–Q67L-stimulated endocytosis was blocked (our unpublished results). We next tested the ability of the K44A mutant of tge dynamin-I mutant to inhibit ARF6–Q67L-stimulated endocytosis 1 . Coinfection with virus encoding dynamin-I K44A inhibited the endocytosis stimulated by ARF6–Q67L, and this inhibition exhibited a dependence on the amount of dynamin-I K44A expressed . Therefore, endocytosis stimulated by ARF6–Q67L is dynamin-dependent. Overexpression of a fragment of clathrin heavy chain, clathrin hub, acts as a dominant-negative inhibitor of clathrin-mediated endocytosis 10 . We made recombinant virus expressing the hub, and coexpressing both the clathrin hub and ARF6 (WT or mutants). Uptake of IgA was analyzed by immunofluorescence microscopy, using the T7 epitope tag on clathrin hub to detect transfected cells. We found an inverse correlation between expression of clathrin hub and internalization of IgA . The inhibition of AP IgA endocytosis by the clathrin hub was true for cells not overexpressing ARF6, as well as cells expressing ARF6–WT and the two mutants . Therefore, AP endocytosis of IgA promoted by ARF6 mutants is clathrin-mediated. As ARF6-stimulated endocytosis is clathrin-mediated, we asked if ARF6 overexpression recruited clathrin to the AP PM. Confocal sections were taken at the level indicated by the solid arrowhead in Fig. 1 A. Cells expressing ARF6–WT have a small increase in AP PM clathrin . Overexpression of either ARF6–Q67L or ARF6–T27N gave a greater increase in clathrin. We next asked if the number or morphology of clathrin-coated pits was altered, using thin-section EM under conditions where clathrin coats were easily identified. We classified clathrin-coated pit profiles as shallow (width of pit > depth), or invaginated (depth ≥ width). Examples are shown in Fig. 4 B. Fig. 4 C shows that overexpression of ARF6–WT gave a modest increase in coated pits, as compared with control. This is in agreement with the small increase in clathrin recruitment seen in Fig. 4 A. Consistent with this, we found that overexpression of ARF6–WT at a higher level than we routinely used stimulated AP endocytosis somewhat (data not shown). Overexpression of either ARF6–Q67L or ARF6–T27N gave a much greater increase of coated pit profiles. Notably, the greatest number of shallow pits was found with ARF6–T27N . In contrast, ARF6–Q67L and ARF6–T27N gave nearly equal numbers of invaginated pits . These results further support the hypothesis that ARF6 affects clathrin-mediated endocytosis at the AP PM. We next investigated if ARF6 is present, even transiently, in coated pits by trapping ARF6 in the deeply invaginated coated pits induced by dynamin-I K44A 9 . As shown in Fig. 5 , there was significant colocalization of ARF6–WT and ARF6–Q67L with endogenous clathrin at the AP PM of cells expressing dynamin-I K44A. This indicates that ARF6 may normally cycle through coated pits, and when coated pit pinching-off is prevented, a significant fraction of the ARF6–WT or ARF6–Q67L can be trapped. We also investigated if IgA bound to pIgR at the AP PM would accumulate in clathrin-coated pits under these conditions. Ordinarily, although endocytic receptors such as pIgR and TfR are concentrated 5–10-fold in clathrin-coated pits, ∼80–90% of these receptors are at steady state located outside of coated pits. Fig. 5 shows that ARF6–WT and ARF6–Q67L significantly colocalize with IgA under these conditions. How does ARF6 influence AP endocytosis? Like other small GTPases, ARF6 probably has multiple effectors, and these might be responsible for the cell type-specific differences in ARF6 function 12 . ARF6 also acts on the cortical actin cytoskeleton ( 18 , 20 ). The actin cytoskeleton underlying the AP PM of epithelial cells has a unique organization, involving actin cores in microvilli and an underlying terminal web. Cytochalasin D inhibits clathrin-mediated endocytosis at the AP PM of MDCK cells 8 . ARF6 may act on the AP actin cytoskeleton to regulate clathrin-mediated AP endocytosis. Both ARF6–Q67L and ARF6–T27N stimulate AP endocytosis, and coexpression of the two proteins produces an even greater stimulation. In macrophages, both ARF6–Q67L and ARF6–T27N inhibit phagocytosis, providing a precedent for both mutant forms of ARF6, giving effects in the same direction 23 . Although both ARF6–Q67L and ARF6–T27N stimulate endocytosis, there are quantitative and qualitative differences in their effects, suggesting differences in their modes of action. ARF6–Q67L is more potent in stimulating endocytosis. Although both ARF6–Q67L and ARF6–T27N give equal increases in the number of clathrin-coated pits, ARF6–T27N gives a greater increase in shallow pits . This suggests that ARF6–T27N may preferentially increase the formation of shallow pits and/or decrease the rate of progression to invaginated pits. In contrast, the greater increase in endocytosis produced by ARF6–Q67L suggests that it acts on a later step in endocytosis. ARF6–WT and ARF6–Q67L can be trapped in clathrin-coated pits when pinching-off of the pits is prevented. ARF6 may normally cycle through coated pits, but then leaves the pits so rapidly that an accumulation in coated pits is not ordinarily detectable. The large stimulation of endocytosis caused by ARF6–Q67L could be due to a direct action on coated pits themselves. In contrast, ARF6–T27N is largely not on the AP PM. Its modest stimulation of AP endocytosis may be due to an indirect effect, e.g., via the actin cytoskeleton. Further work will be needed to distinguish this model from other possibilities. Our results illustrate the importance of comparing the localization and function of proteins in nonpolarized and polarized cells. ARF6 may regulate a specialized endocytic pathway in nonpolarized cells that is cognate to AP endocytosis in epithelial cells ( 7 , 17 , 22 ). As cells develop a polarized AP PM, ARF6 and the specialized endocytic pathway may become restricted to that PM. | Study | biomedical | en | 0.999997 |
10508851 | Human primary fibroblasts (kindly provided by Dr. L.H.F. Mullenders, University of Leiden, The Netherlands) with a normal female karyotype (46, XX) were grown at 37°C under a 2.5% CO 2 atmosphere in Ham's F-10 (GIBCO BRL), supplemented with 15% (wt/vol) heat inactivated FCS (Boehringer Mannheim Corp.), 2 mM l -glutamine (GIBCO BRL), 100 IU/ml penicillin and 100 μg/ml streptomycin (GIBCO BRL). For BrUTP labeling, as well as in situ hybridization, cells were cultured on Alcian blue coated coverslips . Cells were grown to 50–70% confluency before use. HeLa cells were stably transfected with a H2B–GFP vector by Dr. M. Kimura (Sir William Dunn School of Pathology, Oxford, UK). Cells were grown at 37°C under 10% CO 2 atmosphere in DME, supplemented with 10% FCS, and were kept continuously under 0.35 mg/ml G418 (Sigma Chemical Co.) drug selection. To visualize H2B–GFP in living cells, cells were grown on glass-bottom microwell dishes coated with poly- d -lysine (Mattek Co.). Run-on labeling nascent RNA in permeabilized cells by incorporation of BrUTP has been described in detail by Wansink et al. 1993 , Wansink et al. 1994a . In brief, cells were detergent-permeabilized and incubated with a run-on transcription buffer, containing 0.5 mM ATP, CTP, GTP, and BrUTP for 20 min at room temperature. BrUTP incorporated into nascent RNA was visualized by indirect immunofluorescent labeling. Cells were fixed for 15 min at 4°C in 2% (wt/vol) formaldehyde, diluted in PBS. After fixation, cells were permeabilized with 0.5% (wt/vol) Triton X-100 (Sigma Chemical Co.) in PBS for 5 min and incubated with PBS containing 100 mM glycine (Sigma Chemical Co.) for 10 min. Subsequently, cells were incubated overnight at 4°C with a rat mAb raised against BrdU (Seralab) diluted 1:500 in PBS, containing 0.1 μg/ml herring sperm DNA to block nonspecific binding. After several washes in PBS, cells were incubated for 1 h at room temperature with either Cy3-conjugated donkey anti–rat antibody (Jackson ImmunoResearch Laboratories, Inc.), or FITC-conjugated donkey anti–rat antibody (Jackson ImmunoResearch Laboratories, Inc.), diluted in PBS containing 0.1 μg/ml herring sperm DNA. Next, cells were washed in PBS and used for the in situ hybridization procedure. Control experiments, using UTP instead of BrUTP, inhibition by α-amanitin, and RNase-dependent destruction of BrU-containing RNA, have been described previously . In vivo labeling of nascent RNA was carried out as described by Wansink et al. 1993 , Wansink et al. 1994a . In brief, living cells were microinjected into the cytoplasm with 100 mM BrUTP in 140 mM KCl and 2 mM piperazine-N,N′-bis (2-ethane sulfonic acid), pH 7.4. Approximately 5% cell volume was injected. After microinjection, cells were cultured for 10 min at 37°C and subsequently fixed and labeled as described above. For immunofluorescent labeling, cells were fixed for 10 min at 4°C in 2% (wt/vol) formaldehyde in PBS. After fixation, cells were permeabilized with 0.5% (wt/vol) Triton X-100 in PBS for 5 min and incubated with PBS containing 100 mM glycine for 10 min. Subsequently, cells were incubated for 1 h at 37°C with an antibody recognizing acetylated histone H4, an antibody recognizing centromeres, or an antibody recognizing PML. Rabbit polyclonal antibodies (R232 and R252) against acetylated histone H4 (kindly provided by Dr. B. Turner, University of Birmingham, UK) were used. R232 polyclonal antibody recognizes histone H4 acetylated at lysine 8, and antibody R252 histone H4 acetylated at lysine 16 . Human autoimmune serum H33 recognizes centromeres (kindly provided by Dr. W.J. Van Venrooy, Katholieke Universiteit Nijmegen, The Netherlands). To label PML protein in PML antibodies, we used the mouse mAb 5E10 . R232 and R252 antibodies were diluted 1:1,000, anticentromere 1:50,000, and 5E10 1:3 in PBS containing 0.1 μg/ml herring sperm DNA. After several washes in PBS, cells were incubated with appropriate secondary antibodies, using either Cy3- or FITC-conjugated donkey anti–rabbit antibody, goat anti–human antibody, or donkey anti–mouse antibody (Jackson ImmunoResearch Laboratories, Inc.). Secondary antibodies were diluted in PBS containing 0.1 μg/ml herring sperm DNA and incubations were performed for 1 h at room temperature. As a control, cells were incubated in the absence of primary antibody. Subsequently, cells were washed in PBS and used for the in situ hybridization procedure. Painting of the human X chromosome and of chromosome 19 was achieved using chromosome-specific biotin-labeled DNA library probes (Cambio). Probes were denatured at 85°C for 5 min. To suppress labeling of repetitive sequences, which are often not chromosome-specific, denatured probe samples (85°C, 5 min) were self-annealed (2–3 μg/ml, 60–100 min, 37°C) before use. Preannealed probes gave the same result in the FISH procedures as using a large excess of Cot-1 DNA. The probes have been shown to label their respective metaphase chromosomes completely and specifically, showing the specificity of the DNA library probes . In some experiments, we specifically labeled only repetitive sequences. In that case, we used human Cot-1 DNA that was directly conjugated to FITC or Cy3 (kindly provided by Dr. J.C.A.G. Wiegant, University of Leiden, The Netherlands; 20 μg/ml). The FISH procedure was an adaptation of protocols described by Lichter et al. 1988 , Cremer et al. 1988 , Cremer et al. 1993 , and Kurz et al. 1996 . Fixation, pretreatment, and denaturation procedures were subjected to several modifications. The following protocol was found to produce optimal preservation of intranuclear organization of transcription sites and H4 acetylated histones. Cells were rinsed with PBS and fixed for 10 min at 4°C in 4% (wt/vol) formaldehyde diluted in PBS. To facilitate probe penetration, cells were treated with 0.1 M HCl for 10 min and subsequently with a mixture of 0.5% Triton and 0.5% (wt/vol) saponin in PBS for 10 min. After washing in PBS, genomic DNA was denatured by incubating cells in 2× SSC containing 70% formamide at 73°C for 4 min, followed by incubation in 2× SSC containing 50% formamide for 1 min. Hybridization with the biotin tagged probe was allowed to proceed overnight at 42°C. Posthybridization washes with 2× SSC containing 50% formamide and subsequent washes with 2× SSC were performed at 42°C. After blocking in 4× SSC, containing 5% (wt/vol) nonfat dry milk (NFDM; Coberco) for 30 min at room temperature, cells were incubated with FITC or Cy3-conjugated streptavidin, rinsed, and incubated with biotin-conjugated antibody from goat against avidin (Vector Labs, Inc.), subsequently rinsed and incubated once more with FITC or Cy3-conjugated streptavidin. All incubations for fluorescent labeling were performed at room temperature during 20 min in 4× SSC containing 5% (wt/vol) NFDM and 0.1 μg/ml herring sperm DNA. The wash steps after antibody incubations were performed with 4× SSC, 0.05% Tween-20. After fluorescent labeling, DNA staining was performed with 0.4 μg/ml Hoechst 33258 (Sigma Chemical Co.) in PBS, or with 0.5 μM Sytox green (Molecular Probes) in 25 mM Hepes buffer, pH 7.4. As a control, cells were incubated in hybridization buffer in the absence of probe. Slides were mounted in Vectashield (Brunschwig). Slides were kept at 4°C until evaluation and analyzed within 24 h. Images were recorded with a Leica or LSM 510 Zeiss confocal laser scanning microscope both equipped with a 100×/1.23 NA oil immersion lens. With the Leica microscope, a dual-wavelength argon ion laser was used, whereas the Zeiss confocal microscope used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite green and red fluorochromes simultaneously at 488 nm and 514 nm, respectively. Emitted fluorescence was detected in the Leica confocal microscope using a 525 DF10 bandpass filter for FITC and a 550-nm longpass filter for Cy3, whereas in the Zeiss microscope a 505-530-bandpass filter for green signal and a 560-nm longpass filter for the red signal was used. Pairs of images were collected simultaneously in the green and red channels. Three dimensional (3-D) images were scanned as 512 × 512 × 32 voxel images (sampling rate 49-nm lateral and 208-nm axial). Images were corrected for optical cross-talk . Image analysis was performed using SCIL-IMAGE software . Images were subjected to a 3-D image restoration procedure to correct for diffraction-induced distortion, using the Huygens System 2 . The image restoration procedure uses a measured point spread function, which was obtained at precisely the same conditions as the image. To this end, 200-nm fluorescent beads (FluoSpheres; Molecular Probes, Inc.) were imaged. The 3-D image restoration procedure significantly improved the quality of the 3-D images by removing Poisson noise and by deblurring the images. We have developed a modified chromosome painting protocol with improved signal to background ratio, based on the chromosome painting procedures described by Lichter et al. 1988 , Cremer , and Kurz et al. 1996 . It was important to establish that the relatively harsh FISH procedure did not alter nuclear structure in general, and chromosome structure in particular. To investigate the degree of structural preservation, we have carried out four tests. First, we analyzed whether the FISH procedure induced an altered spatial distribution of transcription sites. Second, we investigated the effect of the FISH procedure on the distribution of DNA stained with Sytox green. Third, we analyzed the spatial distribution of acetylated histone H4 before and after the chromosome painting procedure. Histone acetylation is generally correlated with gene activity and reduced acetylation levels with gene silencing . Fourth, we measured in one and the same cell nucleus before and after the FISH procedure the spatial distribution of centromeres and of PML bodies . Visual inspection shows that the spatial distribution of nascent RNA did not change in any recognizable way due to the FISH procedure . Staining of DNA with Sytox green with or without chromosome painting showed that the FISH procedure also did not induce major changes in DNA distribution . After the FISH procedure, the DNA staining pattern seemed slightly more blurred and the overall DNA labeling appeared more intense. Fig. 1E and Fig. F , show immunofluorescent labeling of histone H4 acetylated at lysine 8, before and after FISH labeling. The punctate distribution of acetylated H4 throughout the nucleoplasm did not significantly change after chromosome painting. Only the diffuse, low intensity component in the labeling was somewhat diminished after FISH, making the intense punctate granular labeling more prominent. This may be due to extraction of some histone protein during FISH labeling. These results show that the FISH procedure does not result in major rearrangements in DNA, chromatin, and nascent RNA. Fig. 2 shows the spatial distribution of PML bodies and of centromeres imaged in the same cell before and after FISH. The corresponding optical sections before and after the FISH procedure show that nuclear organization is well-preserved during the FISH procedure. Both the integrity and the spatial distribution of the nuclear bodies and of the centromeres are essentially unchanged. The small changes in spatial distribution can be attributed largely to a slight tilting of the nucleus relative to the substratum, due to the FISH procedure. Together, these results establish that the FISH procedure does not induce significant changes in the spatial distribution of subnuclear structures at the light microscopy level. In particular, we did not observe any collapse, aggregation, unfolding, or other major rearrangement of chromatin in the nucleus. We conclude that, at the resolution level of the light microscope, nuclear structure is not affected by the chromosome painting procedure. A striking feature that emerges from our confocal images after FISH labeling is that chromosome territories display a distinct substructure . Chromosome territories do not appear as compact objects. Rather, they show a modulated intensity distribution inside the territory. Since the chromosome-specific library-probes label the complete metaphase chromosome, it is unlikely that considerable parts of the interphase chromosome remain unlabeled . The chromosome territories contained strongly labeled chromosomal subdomains, surrounded by less intensely labeled areas. The strongly labeled subchromosomal structures have a diameter in the range of 300–450 nm . Intensely labeled parts of a chromosome often seemed interconnected, forming thread-like, folded structures. A similar, distinct substructure in nuclei stained for DNA with Sytox green was observed and in nuclei of HeLa cells expressing GFP-histone H2B , suggesting a reticular organization of chromatin. It is tempting to suggest that, at least locally, the chromatin fiber that constitutes the chromosome can be followed in an optical section. Images were subjected to 3-D image restoration to correct for diffraction-induced distortions. The advantage of such image restoration is that it significantly improves the quality of the 3-D images. It is conceivable that the restoration procedure preferentially enhances certain spatial frequencies. To verify this, we compared the unprocessed individual consecutive sections through a chromosome territory to the same sections after 3-D image restoration . Careful comparison shows that essentially all details of a chromosome territory in the processed image are precisely matched by features that can be found in the preprocessed image. Apparent slight differences are most likely due to the fact that the 3-D restored images contain information from optical sections above and below those that are shown, whereas the unprocessed images do not. Very low intensity signal may be lost by the image restoration procedure, probably because it cannot be discriminated from noise in the restoration process. These results show that no artificial structures in chromosome territories are created by the 3-D restoration algorithm. To investigate whether the less intensely labeled areas in chromosome territories contained repetitive DNA, which is suppressed during FISH labeling, we performed hybridization with a probe specific for highly repetitive human Cot-1 DNA, in dual labeling with an X chromosome probe (data not shown). Hybridization of metaphase chromosomes with the Cot-1 probe revealed intense labeling predominantly in the centromeric region, which is known to contain a high concentration of repetitive DNA. Simultaneous in situ hybridization with the X chromosome probe and the probe recognizing highly repetitive DNA showed that repetitive DNA does not occur concentrated in the areas in the territory that are less intensely labeled with the chromosome-specific probe in the FISH procedure. We conclude that the less intensely labeled areas in chromosome territories constitute a compartment that contains little or no DNA. To establish the 3-D distribution of transcription sites in relation to chromosome territories, chromosome painting was combined with the visualization of transcription sites. Fluorescent labeling of nascent RNA shows that transcription occurs scattered throughout the nucleoplasm , often with exception of nucleoli, due to inaccessibility of the anti-BrU antibody . The 3-D images show that transcription sites occur throughout the territory of one of two X chromosomes , whereas almost no transcription sites are found in the other X chromosome . Most likely, the X chromosome territory containing transcription sites represents the Xa and the territory devoid of transcription is the Xi. We also analyzed the distribution of transcription sites with respect to the autosomal chromosome 19. Chromosome 19 replicates early in S phase and is relatively gene rich, as indicated by the number of CpG islands . Transcription sites were found throughout the chromosome 19 territories , similar to that observed for Xa. These results show that transcription sites are present throughout chromosome territories with exception of the Xi chromosome territory. Importantly, no striking differences in the substructure of the territories of the Xa, Xi, or chromosome 19 can be seen. This implies that the substructure does not depend on differences in overall transcriptional activity, or on differences between sex chromosomes and autosomal chromosomes. We have analyzed the relationship between the spatial distribution of transcription sites and the substructure of chromosome territories. Strikingly, transcription sites in Xa, as well as in chromosome 19 , were mainly found between the intensely labeled chromosomal subdomains and almost never overlapped with them. Line scans were made to obtain more quantitative information about the spatial relationship between transcription sites and chromosome substructure. Fig. 4 E shows typical examples of line scans through Xa and Xi and chromosome 19 territories. The position of the scans are marked in Fig. 4B III and C IV. The line scans through the territory of putative Xa and of chromosome 19 territories confirm that transcription sites (indicated by arrowheads) are often localized near the surface of the intensely labeled chromosomal subdomains . The transcription sites marked with an asterisk have their 3-D center of gravity exactly on the scanned line. These transcription sites clearly show that nascent and newly synthesized RNA accumulates near a chromosomal subdomain, generally not overlapping with it. Despite our efforts (see Structural Preservation during In Situ Hybridization) we cannot fully rule out that the FISH procedure induces local rearrangement of chromatin and/or nascent RNA. Therefore, we have used an alternative procedure to label chromatin, using cells that stably express GFP-tagged histone H2B . Since essentially all histone protein is incorporated in chromatin, the distribution of GFP–H2B faithfully represents that of chromatin . Obviously, this procedure does not allow visualization of individual chromosome territories. However, it does allow analysis of the chromatin distribution in nuclei under in vivo conditions. Intense GFP-labeling is seen particularly at the nuclear periphery and around nucleoli, suggesting a compact, heterochromatin-like local structure. In addition, an irregular, apparently reticular labeling of chromatin is seen throughout the nucleoplasm, with exception of the nucleoli. It is important to note that, like after FISH labeling, intensely labeled areas are observed with a diameter in the range of 300–450 nm . Visualizing transcription sites in cells expressing GFP–H2B showed strikingly little overlap between transcription sites and chromatin . Line scans confirm these observations . This observation is fully in agreement with the results after FISH labeling of chromatin in chromosome territories (see Relationship between Transcription Sites and Chromosomal Substructure). These results confirm that FISH does not affect nuclear structure at the light microscopy level and underscore our observations showing that chromosome territories have a distinct substructure and that transcription sites are located near the surface of intensely labeled subchromosomal domains. Two major principles of organization of the interphase nucleus can be distinguished. First, many nuclear components and processes occur compartmentalized in well-defined domains in the interphase nucleus . Second, individual chromosomes occupy discrete chromosome territories that are apparently not intruded by chromatin from other chromosomes . The dynamic structure of the interphase nucleus in general, and that of chromosomes and higher-order chromatin in particular, are important elements in the control of gene expression. Here, we investigate the spatial relationship between transcription sites and chromosome territories in the interphase nucleus. Chromosome territories were labeled by FISH, using chromosome-specific DNA whole library probes . We have analyzed in female primary fibroblasts the localization of transcription sites in relation to the territories of chromosomes X and 19. In addition, we have examined the spatial relationship between total chromatin in nuclei of HeLa cells that express GFP-tagged histone H2B and transcription sites. Sites of transcription were visualized by fluorescent labeling of nascent RNA . Our results show that transcription sites are present throughout chromosome 19 (a gene-rich chromosome) and, as expected, in only one of the two X chromosome territories (most likely the Xa). The other X chromosome is devoid of transcription sites (most likely the Xi) and acts as an internal control. FISH labeling of chromosomes reveals a distinct substructure of interphase chromosome territories. Territories consist of areas that are intensely labeled, probably reflecting compact chromatin, and domains that are unlabeled, containing little or no DNA. Analysis of HeLa cells that express GFP–H2B supports this conclusion. Although no individual chromosomes can be distinguished, a similar pattern of intensely labeled domains and areas with little or no GFP signal is observed. These results show that chromosome territories are relatively open structures, rather than compact lumps of chromatin. Strikingly, newly synthesized and nascent RNA accumulates specifically in the interchromatin domains, indicating a distinct compartmentalization of transcriptionally active and inactive chromatin within territories. Each of these conclusions is discussed in detail below. The relatively harsh FISH procedure may alter the structure of the nucleus and of chromosomes, even after formaldehyde fixation. Therefore, we have thoroughly analyzed the degree of structural preservation during the chromosome painting procedure. Recently, the spatial distribution of acetylated histones in interphase nuclei has been used as a sensitive marker to visualize possible alterations in chromatin structures that are induced by FISH labeling at the light microscopy resolution level . We show that our FISH procedure does not alter the spatial distribution of acetylated histone H4 in fibroblast nuclei. The same is true for the distribution of DNA, stained with Sytox green. These results indicate that the FISH protocol has no major effect on the structure of interphase chromosome territories at the light microscopy resolution level. This is in agreement with observations of Robinett et al. 1996 , who demonstrated that the interphase chromosome structure is well-preserved after in situ hybridization in formaldehyde-fixed cells. In further support of this conclusion, we found that the chromosome painting procedure does not affect the spatial distribution of transcription sites. In addition, the 3-D distributions of centromeres and of PML bodies, compared in one and the same nucleus before and after carrying out the FISH protocol, was hardly affected by the FISH procedure. This is in agreement with other studies demonstrating that the distribution of other subnuclear structures, i.e., nuclear speckles and kinetochores, are not affected by the FISH procedure . Finally, in cells expressing histone H2B–GFP, we observe that chromatin is organized in compact domains surrounded by nonchromatin material, very similar to the substructure of chromosome territories observed after FISH labeling. We conclude that the chromosome painting procedure does not significantly alter the structure of the nucleus and of interphase chromosomes at the light microscopical resolution level. Our results show that chromosome territories have a well-defined substructure and consist of distinct subchromosomal chromatin domains. In optical sections, chromosomes appear as clusters of such domains with a diameter in the range of 300–450 nm. Domains are sometimes interconnected, forming thread-like, folded structures. Consistently, a similar substructure is observed in nuclei after DNA staining with Sytox green. Similar types of spots and threads have been shown by Brakenhoff et al. 1985 in optical sections of mithramycin-labeled mouse neuroblastoma 2A cell nuclei. Chromosomal substructure is not only found in territories of chromosome 19 and Xa, but also in Xi territories, indicating that the presence of chromosomal subdomains does not depend on transcriptional activity. It may be argued that the compartmentalization in subchromosomal domains that are intensely labeled and domains that appear to contain little or no DNA is an artifact. It cannot be ruled out that the chromosome-specific whole library probe that we used gives incomplete labeling. However, the probe libraries label metaphase chromosomes homogeneously over their complete length . Alternatively, nonlabeled areas may be rich in repetitive sequences, the labeling of which is suppressed in the FISH procedure. However, dual label in situ hybridization with a fluorescently labeled repetitive Cot-1 DNA probe and the X chromosome-specific probe library shows that highly repetitive sequences do not occur in the less intensely labeled domains in chromosome territories (data not shown). Finally, the presumed chromosomal substructure could be an artifact due to the 3-D image restoration protocol. However, careful comparison of optical sections before and after image restoration shows that essentially all intensity modulations in the restored image are also present in the unrestored optical section, albeit in a more blurred state. If chromatin is visualized in a different manner, i.e., in cells expressing GFP-histone H2B, a very similar distribution of strongly labeled domains and areas containing little or no chromatin is observed, compared with the substructure in chromosome territories after FISH labeling. From this, we conclude that chromosome territories have a distinct substructure and are only partially filled with compact chromatin. In earlier studies in which chromosome painting is used, territories are described as compact objects without a well-defined substructure . However, close visual inspection of published images in these publications does show distinct intensity modulations in labeled territories, supporting the idea that chromosome territories do have a substructure. Understanding higher-order levels of chromatin structures above that of nucleosomes and 30-nm wide solenoid fibers is limited . It is evident that additional levels of organization exist between the 30-nm chromatin fiber and the 700-μm wide dense sister chromatids of metaphase chromosomes . Higher-order structures, such as 100–130-nm chromonema fibers, have been described by Belmont and Bruce 1994 and Robinett et al. 1996 , and 240-nm fibers have been observed by Manuelidis 1990 . Recently, Zink et al. 1998 showed that chromosome territories in living cells have a similar substructure. Analysis of cell cycle dynamics of a heterochromatic chromosomal region in vivo reveals that various levels of large-scale chromatin organization can be visualized by light microscopy and EM . EM studies have shown that there are two major, intermingled compartments in the nucleus, one containing compact chromatin, whereas the other is mainly filled with proteins and RNA . Our observations are in line with these studies, showing chromatin domains with comparable dimension. It is tempting to speculate that the tightly packed, irregularly shaped substructures represent the chromosome fiber that is folded in a highly convoluted way inside the territory. We find that with the use of FISH labeling, newly synthesized and nascent RNA accumulates in the interchromatin space inside and around chromosome territories. The same result was obtained using cells expressing GFP-tagged histone H2B. This indicates that the observed distribution is not due to labeling artifacts. It may be argued that antibodies recognizing BrU-containing RNA cannot penetrate into the chromosomal subdomains and, therefore, bind only to peripheral RNA. However, this is unlikely since, for instance, anti-DNA antibodies readily label DNA in interphase nuclei and antihistone antibodies can be used to visualize chromatin in situ . Our results indicate that transcription sites are located predominantly at the surface of chromosomal subdomains representing compact chromatin. These results are in agreement with those of Abranches et al. 1998 , who showed that transcription occurs throughout wheat nuclei chromosomes in rapidly dividing root cells. Earlier studies, using tritiated RNA precursors and high resolution autoradiography, have shown that perichromatin fibrils, the ultrastructural in situ form of nascent transcripts, are located in the interchromatin space near the surface of compact chromatin domains . This has been confirmed in more recent studies, showing that BrU-immunogold–labeled nascent RNA coincides with perichromatin fibrils . Nascent or newly synthesized RNA is not found inside compact chromatin domains, confirming that transcriptionally active chromatin is exclusively located at or near the surface of compact chromatin domains. Our interpretation is in line with the model for the haploid interphase chromatid structure proposed by Manuelidis et al. , stating that transcriptional active chromatin occurs on locally decondensed chromatin fibers that extend from a compact chromatin fiber. It has been postulated that the interphase nucleus contains an interchromosomal domain (ICD) space, i.e., the space between the chromosome territories . The ICD space is thought to be an interconnected reticular nuclear compartment in which transcription, RNA processing, and RNA transport take place. Our results extend this model by showing that the interchromosomal compartment between chromosome territories is continuous with the interchromatin space inside the territories. In fact, there is no sharp distinction between the surface of a territory and that of compact subchromosomal domains. This reconciles the at-first-sight conflicting results of Kurz et al. 1996 and our observations. They showed that coding DNA is found preferentially at the periphery of chromosome territories, whereas a noncoding genomic locus was found predominantly in the interior of the territory. The following picture is emerging. The chromosome fiber, which may be compacted or locally unfolded to some degree, follows an irregular, convoluted path inside a chromosome territory, very much as suggested by Belmont et al. 1989 , Belmont and Bruce 1994 , and Robinett et al. 1996 . Large-scale chromatin folding is strictly organized in such a way that transcriptionally active DNA is at the surface of the chromosomal fiber . It is not known whether this compartmentalization of active chromatin is static or dynamic, i.e., whether all actively transcribed genes and those poised for transcription are in the active compartment, or whether a gene, if activated, is moved from the interior of a subchromosomal domain to its surface. Also, whether transcriptionally active loci loop out into the interchromatin space remains to be established. If looping out occurs, it will be only locally and over relatively short distances, since perichromatin fibrils are localized close to the surface of compact chromatin domains. This compartmentalization of active and inactive chromatin allows direct deposition of newly synthesized RNA into the interchromatin space, which contains the molecular machineries for packaging, processing, and transport of RNA. Since the interchromatin space inside territories and the interchromosomal domain between chromosome territories is continuous, such nuclear organization would allow transport of RNA molecules directly to the nuclear pores or to other parts of the nucleus. This model leads to a number of important questions: what is the molecular basis for the strict compartmentalization of active and inactive chromatin; do proteins that are involved in, e.g., epigenetic silencing and activation, induce large scale remodeling of chromatin in terms of this compartmentalization; does constitutive heterochromatin play a structural role in this higher-order chromatin organization; or is transcriptional activity itself responsible for the observed compartmentalization? Presently, we are using EM approaches to obtain insight in this matter. | Study | biomedical | en | 0.999996 |
10508852 | All translational fusions were generated by standard cloning protocols using the previously described plasmid pEMT that expresses the shorter, oocyte-specific Dnmt1 isoform . Dnmt1 sequences present in the fusions are indicated in the figures. In addition, amino acids 361–1061 of the Escherichia coli β-galactosidase were added in-frame at the COOH terminus. In two cases, a short nuclear localization signal (NLS) derived from SV-40 large T antigen (PKKKRKV) was added at the NH 2 terminus of some deletion constructs. Plasmid DNA was purified using Qiagen columns according to the manufacturer's instructions. Monkey COS1 cells and mouse fibroblast cells (C3H10T1/2 and NIH3T3) were grown in a humidified incubator at 37°C and 5% CO 2 in DME supplemented with 10% fetal calf serum. Mouse C2C12 myoblast cells were grown as above except that the media was supplemented with 20% fetal calf serum. COS1 cells were transfected with the DEAE-dextran pretreatment method and 2 d later, cells were scraped, extracted, and the fusion proteins analyzed by Western blotting as described in Leonhardt et al. 1992 . Mouse myoblast and fibroblast cells were transfected by the calcium phosphate-DNA coprecipitation method followed by glycerol shock treatment ∼8–12 h later. 36–48 h after DNA addition, cells were fixed and stained for the localization of the fusion protein. Mouse embryos were obtained from FVB superovulated female mice mated with C57BL/6 males. Female mice (∼3–4 wk old) were injected intraperitoneally with 5 IU of pregnant mare's serum (PMS) followed by an intraperitoneal injection of 5 IU of human chorionic gonadotropin (HGG) 46–48 h later to induce superovulation, which is necessary to obtain an adequate amount of fertilized eggs upon mating. Matings were set up with fertile stud males after the HGG injection. Zygotes were collected from the oviduct of 0.5 d post-coitum (p.c.) donors into flushing-holding medium (FHM) containing hyaluronidase (0.65 mg/ml), to remove the cumulus cells. After several washes in FHM, the eggs were transferred to kSOM medium in microdrop cultures and incubated in a humidified CO 2 chamber at 37°C. Culture conditions and media compositions have been described in detail . For injection, DNA fragments containing all the regulatory sequences but lacking the prokaryotic vector part were used. DNA was purified with standard glassmilk absorption techniques (Qiagen), resuspended in TE buffer at a final concentration of 1–10 μg/ml. On average 1–2 pl were injected into one of the pronuclei. Tissue culture fibroblast or myoblast cells were washed once with PBS, fixed for 10 min in 3.7% formalin in PBS, and permeabilized with 0.25% Triton X-100 in PBS for 10 min. After blocking 30 min in 3% BSA or 0.2% gelatin in PBS, cells were incubated for 60 min with mouse anti–β-galactosidase antibody (Promega) diluted 1:1,000. After extensive washes with 0.1% NP-40 in PBS, cells were incubated for another 60 min in FITC-conjugated goat anti–mouse (Boehringer Mannheim) or in biotinylated goat anti–mouse IgG antibodies (Amersham), the latter followed by washing and 30 min incubation in streptavidin–Texas red (Amersham) as described before . DNA was counterstained with Hoechst 33258 and cells were mounted in mowiol with 2.5% DABCO . All dilutions were made in 3% BSA or 0.2% gelatin in PBS, and all incubations were carried out at room temperature. Specimens were examined and photographed on Zeiss Axiophot and Axiovert microscopes equipped with phase-contrast and epifluorescence optics, using 63× and 100× plan-apochromat and plan-neofluor lenses. Pictures were taken with Kodak Ektar 100 film. Mouse embryos were fixed and stained in microtiter plate wells and moved from one solution to the other with handmade capillaries under a stereo microscope. The staining procedure was as above using mouse anti–β-galactosidase antibody or rabbit antisera to Dnmt1 , the latter followed by FITC-conjugated goat anti–rabbit IgG antibody (Boehringer Mannheim). Embryos were mounted in mowiol with 2.5% DABCO on 8-well multitest slides. Stained embryos were analyzed on a Zeiss Axiophot microscope equipped with differential interference contrast (DIC), epifluorescence, and confocal laser scanning system using 40× and 63× plan-neofluor lenses. Epifluorescence and DIC images were recorded on film as described above. Optical sections were obtained using the Bio-Rad MRC-600 confocal imaging system and micrographs were taken on Kodak Tmax 400 film. Images were scanned, assembled, and annotated with Adobe Photoshop on a Power Macintosh computer and printed on a Phaser 440 dye sublimation printer (Tektronix). To study the mechanism responsible for the cytoplasmic localization of Dnmt1 during preimplantation development, we first tested whether this phenomenon can be reproduced in embryos that are cultured in vitro. Fertilized mouse eggs were collected at the one-cell stage and were cultured in vitro for up to 4 d. Under optimal conditions embryos develop until the blastocyst stage. Embryos were collected and fixed at different stages of preimplantation development and then stained for Dnmt1 with a specific polyclonal antiserum raised against the NH 2 -terminal domain of Dnmt1. The localization of Dnmt1 was then determined by confocal analyses . Dnmt1 was localized in the cytoplasm of one-, two-, and four-cell embryos as well as in blastocysts. Only at the eight-cell stage was the enzyme found in the nucleus. These results are in good agreement with previous studies on naturally developed embryos , and show that the developmentally controlled subcellular localization of Dnmt1 can be reproduced with in vitro cultured embryos. In somatic cells, Dnmt1 is strictly localized in the nucleus and is targeted to replication foci during S-phase . This raises the question how Dnmt1 enters the nucleus of somatic cells but remains in the cytoplasm during most of preimplantation development. One possible explanation for this different localization could be the fact that different isoforms are expressed in these cells. Indeed, the Dnmt1 gene is transcribed from different promoters and a 118–amino acid shorter isoform of Dnmt1 was identified in oocytes and preimplantation embryos . Therefore, we expressed this shorter isoform in different somatic cell lines including COS1, mouse fibroblasts, and myoblast cells. For visualization by immunofluorescence, the oocyte-specific isoform was fused with an unrelated β-galactosidase epitope. In all tested somatic cell lines the fusion protein showed a clear nuclear localization like the longer somatic isoform (data not shown). To further study the regulation of the nuclear uptake of Dnmt1, we generated a set of deletion mutants and determined their subcellular localization . A series of deletions showed that the first 139 amino acids of the oocyte-specific form are sufficient for nuclear uptake of the β-galactosidase fusion in somatic cells. Deletion of one candidate NLS (KKRR from position 72–92) prevented nuclear uptake. This region clearly contains a functional NLS since this sequence alone allows nuclear uptake of the fusion protein . The regulatory domain of Dnmt1, however, contains further stretches of basic amino acids that could serve as NLS. Further deletion constructs identified at least two independent additional NLS (positions 140–259 and 511–638). These results clearly show that the oocyte-specific Dnmt1 form enters the nucleus of somatic cells and contains at least three independent NLS. Though these three regions function as NLS, a set of internal deletions (from positions 310–637, 310–511, 511–637, and 636–972) that retain all three NLS or at least the two first NLS show cytoplasmic localization of the fusion protein in somatic cells . Similar deletions constructs (not fused to β-galactosidase) expressed in mammalian cells are enzymatically inactive (Margot, J.B., A.M. Aguirre-Arteta, V. Di Giacco, S. Pradhan, R. Roberts, M.C. Cardoso, and H. Leonhardt, manuscript submitted for publication) suggesting an important role of this region in the proper folding of Dnmt1, rather than a specific effect on nuclear localization. The fact that Dnmt1 contains at least three independently functional NLS raises the question of how the oocyte-specific form is maintained in the cytoplasm during early development. This cytoplasmic localization of Dnmt1 could be caused by alternative splicing leading to the expression of an isoform without functional NLS. Alternatively, trans-acting factors could be present in the oocyte and early embryo that prevent nuclear localization of Dnmt1. To test these hypotheses, we established an experimental approach to analyze deletion constructs of Dnmt1 in early embryos . To identify sequences controlling subcellular localization in early embryos and to visualize truncated proteins by immunofluorescence, we fused the shorter, oocyte-specific open reading frame of Dnmt1 with the unrelated β-galactosidase gene. These fusion constructs were cloned into a mammalian expression vector and injected into fertilized mouse eggs. These injected eggs were cultured in microdrops and fixed and stained at different time points. Fusion proteins were detected with β-galactosidase-specific antibodies and their localization analyzed by confocal microscopy. Fig. 3 b shows a two-cell embryo expressing an almost full-length Dnmt1 (amino acids 1–1,490) fused to β-galactosidase. This fusion protein is clearly localized in the cytoplasm just like the endogenous Dnmt1 protein at this stage. The same fusion protein has also been tested in somatic cells and was found in the nucleus ruling out possible artifacts caused by the fusion (data not shown). In other words, this experimental system reproduces the subcellular localization of Dnmt1: the fusion protein is localized in the nucleus of somatic cells and in the cytoplasm of early embryos. Since embryos are known to have a high level of autofluorescence control embryos were injected with TE buffer alone and analyzed in parallel . The comparison with expressing embryos shows that the obtained signals are clearly distinguishable from the autofluorescence. Moreover, a truncated fusion protein comprising amino acids 1–259 including the first two NLS of Dnmt1 was clearly localized in the nucleus of two-cell embryos ruling out unspecific retention of the fusion protein caused by the β-galactosidase part. Finally, a fusion protein containing amino acids 1–638 exhibited an intermediate phenotype with less efficient cytoplasmic retention . These results clearly show that this experimental system is suitable for the identification of sequences that control subcellular localization during early development. The comparison of the full-length and the truncated fusion protein in Fig. 3 suggests that Dnmt1 contains oocyte-specific retention sequences, since both fusions contain functional NLS. Therefore, we generated a set of deletions to map that putative retention sequence . All constructs shown in this figure were first tested in somatic cells and showed a clear nuclear localization (data not shown). A deletion series coming from the COOH-terminal end indicated that the first 638 amino acids of the regulatory domain of Dnmt1 are sufficient for retention in the cytoplasm and that the catalytic domain is not required . A similar deletion series starting from the NH 2 terminus is not possible since the major NLS is located between amino acids 72 and 92. Therefore, internal deletions were done to further narrow down the region involved in cytoplasmic retention. These deletions showed that fusions containing the region from amino acid 308–854 are efficiently retained in the cytoplasm and constructs containing the region from amino acid 427–638 were still retained but less efficiently so that some signal could also be detected in the nucleus . We propose that the binding interface mediating the cytoplasmic retention is complex and may involve several stretches of amino acids from different parts of the primary sequence. Deletion of some of these interacting parts may reduce but not totally abolish the affinity for the target(s). Similarly, deletions may affect the three-dimensional structure of the interface and thereby reduce the binding affinity and thus cause some leakage into the nucleus. In the center of this retention sequence lies the cysteine-rich region that had been shown to bind zinc ions . To test whether this region is involved in the cytoplasmic retention we deleted this region in the full-length fusion construct. However, all deletions in this region as well as in neighboring regions caused an aberrant cytoplasmic localization even in somatic cells despite the fact that they all contained several functional NLS . One possible explanation could be that the remaining functional NLS were sequestered by an aberrant globular folding of the deletion construct. In fact, similar results were obtained in deletion studies to map the catalytic center of Dnmt1 . Several deletions in the NH 2 -terminal domain affected the activity of the COOH-terminal, catalytic domain. These results suggest that the NH 2 -terminal domain has a complex folding that is required for efficient cytoplasmic retention as well as for protein functions located in other parts of the protein. To test whether the retention sequence is dominant over heterologous NLS that are derived from an unrelated protein we added the SV-40 NLS at the NH 2 terminus of the full-length fusion construct. As summarized in Fig. 4 , the addition of the SV-40 NLS could not affect nuclear uptake suggesting that the regulatory domain of Dnmt1 in fact contains a dominant cytoplasmic retention sequence. The fact that this retention sequence is dominant over a heterologous NLS suggests that it does not act through a specific modification or masking of the endogenous Dnmt1 NLS but through active retention by a retention factor and binding to some immobile cytoplasmic structures. Confocal images of endogenous Dnmt1 staining had shown stronger signal in the peripheral cytoplasm and were taken as indication that Dnmt1 binds to some structures at the cell membrane . Similar results were also obtained in this study and were also reproduced with the fusion proteins . However, very different results were obtained using regular fluorescence microscopy showing an uniform distribution throughout the cytoplasm . Interestingly, the same embryos viewed by confocal microscopy showed again the peripheral signals . These results suggest that the extreme peripheral staining signals seen in the confocal are most likely an optical artifact. Due to light absorption by the sample, the intensity of the excitation light decreases as it penetrates the specimen and the inner fluorophores are not excited to the same extent as the outer ones . This inner filter effect is particularly evident in thick specimens like mouse embryos and results in stronger peripheral signal. However, in fluorescence microscopy, out of focus light from layers above and below is also detected compensating this absorption effect. In other words, a detailed localization of Dnmt1 is not possible with either method and further ultrastructural analyses by, e.g., electron microscopy, are needed to clarify this issue and to provide clues as to what structures Dnmt1 binds to in the cytoplasm of oocytes and early embryos. An interesting possible mechanism could involve an allosteric control through interacting factors. Since the folding of the NH 2 -terminal domain (in particular amino acids 300–1,000) seems to be complex and required for functions residing in other part of the protein, it seems possible that interacting proteins might affect the globular folding of Dnmt1 and thereby inactivate the NLS similar to the internal deletions in somatic cells . One further prediction from these experiments is that if Dnmt1 is actively retained rather than modified in an enzymatic process, then retention should be saturable. In fact, injection of a four times higher amount of plasmid DNA resulted in cytoplasmic and nuclear localization of the fusion protein (data not shown). Also longer incubation of injected embryos lead to an accumulation of the fusion protein and gradual uptake into the nucleus (data not shown). Although these results were clear, one has to keep in mind that injecting these high amounts of plasmid DNA disturbs normal development of mouse embryos. With this cautionary note, these results suggest that the retention of Dnmt1 is indeed saturable. All tested Dnmt1 fusion constructs are relatively large, ranging in size from 100 kD to ∼250 kD. Although this size range does not make any difference for nuclear uptake in somatic cells, we generated a similar fusion construct of equal size using the DNA ligase I, which is involved in DNA replication and is like Dnmt1 targeted to nuclear replication foci . Fig. 5 shows the side-by-side comparison of both the Dnmt1 and the DNA ligase I construct. In somatic cells, both fusion constructs are targeted to nuclear replication foci, but only the DNA ligase fusion protein enters the nucleus of early mouse embryos. These results clearly show that the observed cytoplasmic retention of fusion proteins is specific for the Dnmt1 protein and is developmentally controlled. Moreover, the retention mechanism is active also in early preimplantation embryos and not only in oocytes, where the maternal stock of Dnmt1 is accumulated. The data presented in this work suggest that Dnmt1 is actively retained in the cytoplasm, but cannot rule out the formal possibility that Dnmt1 is enriched in the cytoplasm by a nuclear export mechanism. However, the mapping of a cis-acting sequence allows now a specific search for interacting factors and the generation of dominant-negative mutants to further study the regulation and role of the cytoplasmic localization of Dnmt1 during early development. The active retention of Dnmt1 in the cytoplasm from the oocyte to the blastocyst stage correlates well with the overall decrease in the genomic methylation level and thus is a likely mechanism to regulate DNA methylation by separating Dnmt1 from chromosomal DNA in the nucleus. Demethylation could occur by preventing maintenance methylation of newly synthesized DNA strands and/or by preventing remethylation of actively demethylated sites. Recently, a cDNA coding for a protein with demethylase activity was isolated from HeLa cells and shown to be ubiquitously expressed at the mRNA level in somatic cells . It is still unknown whether it is active during early development and the fact that this demethylase is ubiquitous means that it cannot by itself cause the specific demethylation during preimplantation development. Cytoplasmic accumulation of maternal nuclear gene products has been extensively observed during oocyte maturation and early development in Xenopus . Some transcription factors are retained in the cytoplasm until the mid-blastula stage when transcriptional activity is restarted. Nuclear exclusion in this developmental system has been proposed as a mechanism to control the function of maternal proteins until the time during development when they are required. These results parallel the regulation of Dnmt1 nuclear uptake during mammalian preimplantation development. Until the blastocyst stage, Dnmt1 is retained in the cytoplasm with the exception of the eight-cell stage when it briefly enters the nucleus . Also, in growing oocytes, Dnmt1 enters the nucleus at a time when parasitic sequences are heavily methylated . Analogously, it is conceivable that specific sequences are methylated at the eight-cell stage. In that regard, it is interesting to note that modifications occur in eight-cell stage embryos that make them unable to recapitulate the normal program of gene expression when transplanted into one-cell embryos . This different behavior of transplanted nuclei from two- and eight-cell embryos might in part be caused by methylation of specific genomic DNA sequences around the eight-cell stage. In fact, the Igf2r gene locus is methylated exactly at the eight-cell stage and could thus be one such candidate genes . The high level of the enzyme in oocytes possibly represents a maternal stock to be used for maintenance and/or de novo methylation in later cell cycles. This large amount would last through subsequent cell divisions and might thus also allow development of Dnmt1-deficient mice till mid-gestation. In this context, it is noteworthy that the oocyte-specific Dnmt1 isoform is fully active, can restore DNA methylation in Dnmt1 -null embryonic stem cells, and can thus rescue their capacity to differentiate in vivo . In addition, one cannot exclude that Dnmt1 might also have a function in the cytoplasm during early development, in particular since this cytoplasmic store is enzymatically active. Interestingly, the bacterial restriction-modification system is also localized in the periplasmic space and serves as a first line of defense against foreign DNA. One possible function of the high concentration of Dnmt1 in the cytoplasm could be to protect the developing embryo and the future germ line by methylating intruding DNA and thus rendering it transcriptionally inactive. The identification and characterization of a cytoplasmic retention sequence makes it now possible to directly investigate functions of Dnmt1 during early development and to screen for interacting factors. | Study | biomedical | en | 0.999996 |
10508853 | Precursors labeled with [ 35 S]methionine or [ 35 S]cysteine were generated by translation in a rabbit reticulocyte lysate (Promega Corp.) using template RNA synthesized by either SP6 or T7 RNA polymerase . During the course of the experiments, a nonspecific protease activity was found to be present in the reticulocyte lysate. In a typical control experiment, [ 35 S]methionine-labeled preHSP21 was incubated with immobilized SPP for 30 min at 24°C. The supernatant containing the transit peptide subfragment was separated from the immobilized SPP fraction and incubated for 1 h at 24°C either without inhibitor or in the presence of 2 mM PMSF. Without inhibitor, the transit peptide subfragment was fully degraded. In contrast, in the presence of PMSF, ∼90% of the subfragment remained. Consequently, 2 mM PMSF was used in the experiments to minimize the nonspecific protease activity of the reticulocyte lysate. An extract of E . coli cells expressing recombinant SPP with a biotinylated peptide tag , immobilized SPP, or a chloroplast extract were used as sources of SPP. To ensure reproducible results, cultures of the E . coli strain carrying the expression construct for recombinant SPP were grown and prepared under identical conditions. Typically, an expression culture (120 ml) was started from an overnight culture (dilution 1:100), cultured at 30°C, and induced by 2 mM IPTG at OD 560 nm 0.22–0.24 after 2 h and 15 min. The culture was continued for 3 h and 30 min (final OD 560 nm 1.2–1.3) and cell extracts were prepared as described previously . For processing reactions with either E . coli cell extract containing recombinant SPP or chloroplast extract, 10 μl extract was incubated in a total volume of 20 μl with 25 mM Hepes-KOH, pH 7.5, 2 mM PMSF, at 24°C. If immobilized SPP was used, SPP protein from 100 μl E . coli extract was bound to 50 μg streptavidin-coated magnetic beads and incubated in a total volume of 20 μl . The amount of radioactive-labeled substrate per reaction was optimized for detection of small products, transit peptides and their subfragments, upon electrophoresis (preFD, 1 μl; preHSP21, 4 μl; preLHCP, 5 μl; preRBCA, 4 μl; preRBCS, 2 μl). Reactions were stopped by mixing with gel-loading buffer and boiling. However, using immobilized SPP, magnetic beads were removed by magnetic separation after boiling. If binding and release of processing products was studied, immobilized SPP was separated from supernatant, washed with 50 μl 25 mM Hepes-KOH, mixed with gel-loading buffer, and boiled. Tricine SDS-PAGE was employed for detection of transit peptides and their subfragments. Standard SDS-PAGE was used to monitor the generation of mature FD and RBCA. All gels were analyzed by autoradiography. Recombinant SPP protein from 1 ml E . coli extract was immobilized onto 500 μg of streptavidin-coated magnetic beads. [ 35 S]methionine-labeled preFD (50 μl) was diluted in a total of 150 μl buffer (25 mM Hepes-KOH, pH 7.5, 2 mM PMSF), mixed with the immobilized SPP and incubated at 24°C. Separation of the supernatant from immobilized SPP was performed either after 3 min to obtain the FD transit peptide or after 120 min to obtain the subfragment. The supernatants were frozen and stored overnight at −70°C. During thawing of the supernatants, a protein precipitate formed, which was removed by centrifugation (5 min, 10,000 g ). The supernatants were subsequently dialyzed (at 4°C for 5 h in 1 liter of 25 mM Hepes-KOH, pH 7.5) using a cellulose ester dialysis membrane with a cut off at mol wt 500 (Spectrum Medical Industries). Around 120 μl of sample was usually recovered from one preparation. To test for nonspecific protease activity, 10 μl of sample was incubated for 1 h at 24°C and compared with a nonincubated control sample upon tricine SDS-PAGE. No degradation was observed for either substrate. Immobilized SPP was incubated with 10 μl of substrate (preparations of FD transit peptide or its subfragment) in a 20-μl vol for 5 min using 25 mM Hepes-KOH, pH 7.5, at 24°C. The immobilized SPP fraction was separated from the supernatant, washed twice with 50 μl 25 mM Hepes-KOH, and once with 20 μl of solution of NaCl at different concentrations to elute bound substrate. The immobilized SPP fraction was finally resuspended in gel-loading buffer and liberated from magnetic beads by boiling before tricine SDS-PAGE. Gels were analyzed by autoradiography and scanned by a PhosphorImager (Molecular Dynamics) for quantification using ImageQuant software (Molecular Dynamics). Typically, 10 μl chloroplast extract was incubated with 10 μl substrate (preparations of FD transit peptide or its subfragment) using 25 mM Hepes-KOH, pH 7.5, at 24°C. Reactions were stopped by mixing with gel-loading buffer and boiling before analysis by tricine SDS-PAGE for autoradiography and quantification. Our previous studies showed that processing of preFD by SPP generates two products: mature FD and its intact transit peptide . The nature of each processing product was established using different amino acids for radioactive labeling of the precursor and by size estimates upon tricine SDS-PAGE. PreFD has four methionines that are found only in the transit peptide, and hence, only the transit peptide with an estimated size of ∼5 kD was detectable upon processing of [ 35 S]methionine-labeled preFD. The five cysteines of preFD are present only in the mature protein. Consequently, using [ 35 S]cysteine-labeled substrate in a processing reaction with SPP, only mature FD was detected. To determine if an intact transit peptide is usually an initial cleavage product, other chloroplast protein precursors were tested in processing reactions with recombinant SPP. In addition to preFD, the precursors of heat shock protein 21 (HSP21), LHCP, ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RBCA), and RBCS, which contain two or more methionines in the transit peptide , were synthesized by in vitro translation as [ 35 S]methionine-labeled polypeptides. Each substrate was incubated with recombinant SPP for five minutes. A fragment that was similar in mobility to the FD transit peptide was observed for each reaction upon tricine SDS-PAGE . Thus, in all cases, SPP initially generated what appeared to be an intact transit peptide by a single endoproteolytic step. Despite the large mass of diverse transit peptides that enter the organelle, nothing is known about their turnover. To investigate if SPP is involved, time courses of processing reactions were carried out using the precursors shown in Fig. 1 a and recombinant SPP, which was immobilized onto magnetic beads (see Materials and Methods). After 2 min, the mature protein and the transit peptide appeared simultaneously in each reaction, although the processing of each precursor progressed at a different rate . Interestingly, using the precursors for FD, HSP21 and LHCP, we found that the initial generation of intact transit peptide was followed by its conversion to one detectable subfragment. The transit peptide of RBCA gave rise to two subfragments . Conversion of the RBCS transit peptide could not be directly monitored. Instead, it nearly disappeared after one hour, probably because the conversion products containing the labeled methionines were too small to be seen upon tricine SDS-PAGE. Progression of this trimming reaction differed considerably between the transit peptides examined. At one extreme, the conversion of the RBCA transit peptide to subfragment 1 was completed in <10 min , whereas an incubation time of 90 min was needed for full conversion of the LHCP transit peptide to a smaller form . Nevertheless, based on our results with four substrates, trimming of transit peptides as another function of SPP is apparently a common step upon precursor processing. The chelator 1,10-phenanthroline inhibits precursor processing by SPP, which is a metallopeptidase . We found that 1,10-phenanthroline also inhibited the conversion of the FD and HSP21 transit peptides to their respective subfragment forms . The transit peptides were stable for one hour in the presence of the chelator. This observation shows that both functions of SPP, precursor processing and transit peptide trimming, depend on metal ions. To investigate the specificity of the proteolytic conversion of a transit peptide to a subfragment form, we took advantage of a preLHCP mutant that was previously used to study cleavage events upon in vitro import and in a chloroplast extract . The transit peptide of this precursor mutant has an altered COOH terminus, where a valine was changed to a glycine at position −9, and three amino acids were deleted from −8 to −6, relative to the site recognized by SPP in the chloroplast extract . This alteration was sufficient to block the conversion of the preLHCP transit peptide to the subfragment, an indication that specific features of the transit peptide serve as determinants for the trimming reaction. When these features are disrupted, in this case by the COOH-terminal mutation, the transit peptide was not further processed by SPP. Using immobilized SPP, two steps were observed during trimming of the RBCA transit peptide. The transit peptide was first converted into a slightly smaller subfragment 1 that was subsequently trimmed to subfragment 2, which was only detected under modified PAGE conditions . However, neither fragment was detected after one hour incubation, which indicated that either inhibition of the intrinsic protease activities in the reticulocyte lysate by 2 mM PMSF (see Materials and Methods) was not sufficient to preserve these barely detectable amounts of peptide, or SPP converted them to smaller subfragments, which were not detectable upon tricine SDS-PAGE. Alternatively, SPP performed complete degradation of the transit peptide. The latter interpretation, however, is not supported by the results for the three other transit peptides, which suggested instead that SPP performed limited proteolysis of the transit peptide. Nevertheless, the fate of the RBCA transit peptide demonstrates that trimming of transit peptides by SPP can occur in more than one cleavage step that may depend on features specific to each transit peptide. The results presented in Fig. 2 demonstrate that the mature protein and the transit peptide appear simultaneously in the course of a processing reaction, supporting the idea that transit peptide removal from a chloroplast precursor polypeptide initially occurs in one endoproteolytic step. Whereas the mature protein is generated by this single cleavage, the transit peptide is further trimmed by SPP. We investigated if SPP specifically binds the intact transit peptide and its subfragment using an in vitro binding assay . A protocol was established for preparation of [ 35 S]methionine-labeled FD transit peptide and its subfragment (see Materials and Methods). Aliquots of a FD transit peptide preparation were incubated with immobilized SPP for five minutes. Each immobilized SPP fraction was separated from the supernatant and individually washed with a solution of NaCl at different concentrations for elution of bound transit peptide. To determine the amount of transit peptide that remained bound to immobilized SPP at 1,000 mM NaCl, one SPP fraction was washed in 1,000 mM NaCl and the bound material was liberated for analysis by boiling in the presence of gel-loading buffer. A representative supernatant, all eluates, and the material released by boiling were subjected to tricine SDS-PAGE to analyze by both autoradiography and PhosphorImager scanning for quantification. The supernatant was separated from the immobilized SPP fraction and contained unbound transit peptide . Washing of the immobilized SPP fraction without NaCl did not elute detectable amounts of transit peptide . Washing with low NaCl (50 mM) released 3% of the substrate originally added to one reaction . Using a higher NaCl concentration (100 mM) for washing of another immobilized fraction, a substantially greater amount of transit peptide, 13%, was released . However, if parallel fractions were washed with even higher concentrations of NaCl, the amount of eluted transit peptide did not increase, not even at 1,000 mM NaCl . Surprisingly, however, boiling of the SPP fraction after washing with 1,000 mM NaCl released an additional 7% of the original substrate added to the reaction . Together, the eluate at 1,000 mM NaCl and its corresponding SPP fraction contained in total 20%, i.e. 13% + 7%, of the original substrate, and thus was equivalent to the portion of transit peptide initially bound by immobilized SPP in one binding reaction. Two-thirds of the bound transit peptide was released from SPP at high NaCl concentration, but, importantly, one third remained bound. This observation suggested that SPP may have two states of transit peptide binding, and one of these has an especially high affinity for the intact transit peptide. SPP activity depends on metal ions. To investigate the importance of metal ions on the interactions between SPP and the transit peptide, binding assays were carried out in the presence of 1,10-phenanthroline. We found that neither eluates with different NaCl concentrations nor a SPP fraction after washing at 1,000 mM NaCl contained significant amounts of transit peptide . Apparently, treatment by 1,10-phenanthroline causes conformational changes in SPP, preventing a stable interaction with the transit peptide. In addition, it was tested whether SPP contains cysteines that are necessary for transit peptide binding by adding of the sulfhydryl group inhibitor N -ethylmaleimide (NEM) into a binding assay. Interactions of this inhibitor with the transit peptide could be excluded since the FD transit peptide does not have cysteines. Analysis of this experiment revealed that 10 mM NEM disrupted the interaction between SPP and FD transit peptide (not shown). Only unbound transit peptide, present in the supernatant, was detected. No transit peptide was found in eluates with different NaCl concentrations (200, 500, 1,000 mM) or in an immobilized SPP fraction after washing with 1,000 mM NaCl. Therefore, the sulfhydryl group of one or more cysteines is required for SPP's ability to bind a transit peptide. To determine if the magnetic beads matrix used for immobilization of SPP nonspecifically binds the FD transit peptide that would interfere with our binding studies, magnetic beads were incubated with a control extract of E . coli cells not expressing SPP . The binding assay was carried out as described above. Elution at 500 mM NaCl did not release detectable amounts of transit peptide from the matrix incubated with control extract . An aliquot of this matrix was boiled to release strongly bound transit peptide, but none was detected (not shown). Hence, the matrix alone did not bind the FD transit peptide establishing that the binding of the intact transit peptide to the matrix fraction was due to the presence of SPP. The FD transit peptide is converted by SPP to a subfragment that remains relatively stable for at least 30 min . We examined if SPP has an affinity for this subfragment. A subfragment preparation was incubated with immobilized SPP in a binding assay. Only unbound subfragment was found in the supernatant, whereas nothing was detected in a NaCl eluate . We conclude that SPP did not recognize the subfragment. Apparently, conversion of the transit peptide into its subfragment causes the loss of features necessary for binding by SPP. The results presented in Fig. 3 demonstrate that there is a stable interaction between SPP and the intact transit peptide, which can be disturbed by metal ion chelator or sulfhydryl group inhibitor. In contrast, binding between SPP and the subfragment was not observed. The finding that SPP does not bind the FD transit peptide subfragment showed that trimming of the transit peptide alters SPP's affinity for the transit peptide, and suggested this may serve as a specific step to trigger its release from SPP. To understand the temporal relationship between transit peptide conversion to the subfragment form and release from SPP, time courses of precursor processing by immobilized SPP were carried out. Binding of the processing products to SPP was monitored by separate analysis of the supernatant and the immobilized SPP fraction. Using preFD and preHSP21 as substrates, during the first 10 min of processing, both mature proteins, FD and HSP21, and their respective transit peptides were produced simultaneously, but they were found in different fractions . The mature proteins were immediately released into the supernatant . In contrast, the intact transit peptides remained bound to immobilized SPP . Beginning at 10 min, proteolytic conversion of the transit peptide coincided with the release of its subfragment into the supernatant . This demonstrates that generation and release of the mature protein is accompanied by an initial accumulation of the intact transit peptide bound to SPP. Subsequently, limited proteolysis by SPP releases the subfragment, perhaps, to clear a binding site for new substrate. The intact transit peptide was identified as an initial product of precursor processing by SPP. However, the fate of the transit peptides in vivo is unknown. They are not stable upon in vitro import into the chloroplast using preFD as a substrate . SPP has the capability to trim transit peptides, but to complete their turnover in the chloroplast, one predicts that other degradative activities are needed. Precursor processing by a chloroplast extract was analyzed to explore this prediction. Using preFD and preHSP21 as substrates, within five minutes both the mature protein and the transit peptide appeared at the same time . After ten minutes, conversion of the transit peptide to a subfragment was observed . The initial appearance and trimming of the transit peptide within the first 20 min of the reaction using the chloroplast extract resembled the pattern found for processing using immobilized SPP . In sharp contrast to the results using immobilized SPP in a processing reaction, however, the subfragment was then quickly degraded by the chloroplast extract . These results indicate that transit peptides are most likely trimmed in the chloroplast by SPP in a discrete step before their final degradation. Furthermore, degradation of both the FD and HSP21 transit peptides and their respective subfragments was sensitive to metal ion chelators. If 1,10-phenanthroline or EDTA was added during the processing reaction, they were stable in the chloroplast extract . However, interestingly, the two reactions, i.e. proteolytic conversion versus subfragment degradation, showed differential sensitivity to 1,10-phenanthroline. Conversion of the transit peptide to the subfragment needed as much as 10 mM 1,10-phenanthroline to be fully inhibited . Subsequent degradation, in contrast, was efficiently inhibited at only 1 mM 1,10-phenanthroline . These results support the idea that two distinct activities are involved in transit peptide turnover after precursor cleavage, the first dependent on SPP. We analyzed the fate of the FD transit peptide and its subfragment separately. [ 35 S]methionine-labeled FD transit peptide and its subfragment were prepared (see Materials and Methods) and used as substrates in separate reactions with chloroplast extract. We observed rapid conversion of the transit peptide and complete degradation of the subfragment within 30 min . Mature FD was prepared by incubation (30 min) of [ 35 S]cysteine-labeled preFD with immobilized SPP. The supernatant of this processing reaction was added directly to a chloroplast extract. Mature FD was relatively stable over 30 min ; we observed only a 15% loss of fragment within the first 2 min. The distinct stability of mature FD compared with its transit peptide in the chloroplast extract demonstrates that selective degradation of the latter occurred. Transit peptide conversion to a subfragment and its subsequent degradation showed differential sensitivity to 1,10-phenanthroline in the course of a processing reaction . To refine this analysis using separate reactions, preparations of FD transit peptide and its subfragment were incubated in a chloroplast extract at different 1,10-phenanthroline concentrations ranging from 0.1 mM to 10 mM. Transit peptide conversion was less sensitive to 1,10-phenanthroline than subfragment degradation . At 1 mM 1,10-phenanthroline, only 16% of transit peptide was left after 30 min, whereas 86% of subfragment remained after the same incubation time. At 10 mM 1,10-phenanthroline, 80% of the transit peptide remained and no degradation was found for the subfragment. Hence, in the chloroplast, transit peptide turnover is metal-dependent and can be dissected into two different steps, initial conversion and subsequent degradation, each sensitive to a different concentration of the chelator 1,10-phenanthroline. To characterize the nature of the activity that carries out the subsequent degradation step, the subfragment of FD transit peptide was used as a substrate to test group specific protease inhibitors for their ability to block degradation in the chloroplast extract. Divalent metal ion chelators other than 1,10-phenanthroline also inhibited the degradation activity, although to a lower extent. Using 10 mM EDTA, 73% of the originally added subfragment was detected in the sample after 30 min and using 10 mM EGTA, 46% of the original amount remained. The divalent metal ion requirement was directly tested. Degradation of the subfragment, which was inhibited at 5 mM 1,10-phenanthroline, was assayed in the presence of zinc ions . At a concentration of 300 μM Zn 2+ , the degradation activity was restored. Other group specific inhibitors were tested as well, including NEM (10 mM), PMSF (10 mM), which inhibits serine proteases, E64 (100 μM), specific for cysteine proteases, and pepstatin (100 μM), which inhibits some aspartic proteases. None of these inhibitors significantly decreased subfragment degradation within 30 min incubation (not shown). However, the degradation activity and SPP showed a different sensitivity to NEM. NEM did not significantly decrease degradation activity, but it inhibited SPP cleavage of precursors (not shown). The inhibitor profile found for the degradation of transit peptide subfragments suggested that this reaction is carried out by a metallopeptidase that is most probably distinct from SPP. We tested whether degradation activity is ATP-dependent. A chloroplast extract was preincubated at different concentrations of apyrase to hydrolyze ATP. The subfragment of the FD transit peptide was subsequently added to assay degradation. We found a decrease of degradation with increasing concentration of apyrase . At the highest apyrase concentration, 100 units/ml, 86% of the originally added substrate remained after 30 min incubation. If ATP (20 mM) was added back to this reaction, 49% of the original substrate was detected, demonstrating that ATP partially restored degradation activity. No significant inhibition of degradation was observed when the extract was preincubated with apyrase inactivated by boiling. These results indicate that an ATP-dependent protease is involved in transit peptide turnover. Other properties of the degradation activity were established. Using a chloroplast extract clarified by ultracentrifugation (1 h at 139,000 g ), all activity was found in the supernatant and no significant degradation was observed using the pellet containing the membrane vesicle fraction (not shown). Thus, the degradation activity is soluble and does not reside in the membrane. Furthermore, this activity showed a broad pH optimum (between pH 5.0 and 9.0, using 20 mM Hepes-KOH buffer). Complete substrate degradation occurred within 30 min at temperatures between 24°C and 42°C. It was slightly reduced at 16°C, whereas 43% of the added subfragment remained at 4°C. Taken together, these results show that SPP trims transit peptides to subfragments that are specifically degraded by an ATP-dependent, soluble metallopeptidase activity with broad pH and temperature optima. The experiments presented in this study provide evidence that transit peptide removal and turnover are regulated processes, and significantly, degradation of the transit peptide is an ATP-dependent step not previously recognized in the general import pathway. Our observations have been incorporated into a model that makes a number of important predictions about how SPP carries out its function during precursor import. First, we propose that SPP initially recognizes, binds, and cleaves the transit peptide from the precursor as it is translocated across the inner membrane. This is based on our finding that SPP has a strong affinity for the transit peptide as a separate domain of the precursor, as demonstrated in both binding assays and time courses of precursor processing. It is also supported by studies showing that translocation intermediates can be cleaved before complete transport into the stroma, a stage when the precursor is likely to be in an unfolded conformation while still spanning the envelope membranes . Once the precursor is cleaved and the mature protein released, SPP continues to interact with the intact transit peptide, although the duration and equilibrium of this interaction remains to be fully explored. Second, SPP then carries out a second processing reaction and converts the transit peptide to a subfragment form which it no longer recognizes. Specific, yet uncharacterized, features of the transit peptide are necessary to promote its conversion, which was shown by the failure of SPP to trim a mutated LHCP transit peptide with a three amino acid deletion near its COOH terminus. The dissociation of the subfragment also frees SPP, which becomes available for additional rounds of precursor processing at the inner membrane. Thus, transit peptide trimming may serve as a recycling step for SPP. In fact, immobilized SPP can be used after a processing reaction again in another reaction (Richter, S., and G. Lamppa, unpublished result). Third, we predict that the subfragment, upon being released from SPP, becomes a target for rapid ATP-dependent degradation by a stromal metallopeptidase. That the reaction depends on ATP suggests an energy requirement. Either the subfragment is degraded to oligopeptides no longer detectable in our assays, or it is fully hydrolyzed to free amino acids. Overall, we propose a regulated sequence of events that is mediated by SPP and results in transit peptide turnover by an ATP-dependent proteolytic machinery. Aspects of the model may apply to certain other members of the pitrilysin family that share the His-X-X-Glu-His zinc-binding motif . Representatives include the mitochondrial processing peptidase , the human insulin-degrading enzyme responsible for β-endorphin processing among other proposed functions , and a yeast homologue of insulin-degrading enzyme needed for processing of pro- a -factor . In general, we propose that each metalloendopeptidase recognizes a precursor with a removable NH 2 - or COOH-terminal extension peptide because of the enzyme's high affinity for this domain. This interaction determines the substrate specificity of the peptidase. A single endoproteolytic cleavage occurs that immediately releases the mature product. However, to disrupt the interaction with the cleaved extension peptide, the enzyme carries out one or more internal cleavages of the extension, which may yield a subfragment for complete degradation. The observation that SPP specifically binds the transit peptide, but does not interact with its subfragment, suggested that another proteolytic activity exists in the chloroplast for transit peptide turnover. Indeed, the degradation activity identified in our work can be distinguished from SPP. SPP activity is ATP-independent in vitro and is blocked by the sulfhydryl group inhibitor NEM. Furthermore, SPP was found to be about tenfold less sensitive to the metal ion chelator 1,10-phenanthroline than the degradation activity. Little is known about the pathways employed by the chloroplast for protein degradation in general. Two ATP-dependent proteolytic systems have been described, but their natural substrates in the organelle remain to be established. One system is comprised of ClpP and ClpC, which are homologues of E . coli serine protease ClpP and a chaperone-like ClpA activity . The other system is the homologue of E . coli FtsH, a membrane anchored metallopeptidase . However, neither proteolytic system fully resembles the activity responsible for transit peptide subfragment degradation. On the one hand, we found that the degradation activity is insensitive to the serine protease inhibitor PMSF. On the other, the degradation activity was found in the soluble fraction and not the thylakoid membranes where FtsH is localized in chloroplasts. At present, it appears that a novel activity separate from SPP is needed for transit peptide turnover. SPP cleaves a large diversity of precursors targeted to the chloroplast, all with transit peptides of different length and primary sequence (see introduction). Despite this variability, our results indicate that the transit peptide alone contains sufficient information to mediate the interaction of the precursor with SPP. What does SPP recognize that determines a highly specific association with the intact transit peptide? There are some indications that critical determinants for processing are located near the COOH terminus of the transit peptide . As a corollary to this question, where in SPP is the binding site for the transit peptide? One can speculate where the binding domain of SPP will be found based on a comparison with the structure of MPP. Unlike SPP, which is a single polypeptide of ∼125 kD (based on its primary sequence), MPP is comprised of α and β subunits . Only subunit α is necessary for the binding step of the processing reaction . Subunit β contains the catalytic His-X-X-Glu-His zinc-binding motif within a domain that shows sequence conservation with the NH 2 terminus of SPP . Thus, it is reasonable to predict that the transit peptide binding site of SPP will be found in a downstream COOH-terminal region. The binding site of SPP, as well as that of MPP, remains to be mapped, and its structural features characterized in order to elucidate the molecular mechanism of precursor recognition and cleavage. What is the nature of the proteolytic machinery that is responsible for transit peptide degradation? As discussed above, our current results suggest that a novel activity, separate from SPP, functions after transit peptide conversion to a subfragment form. However, an answer to this question depends on knowing more about the identity of the degradation activity. Another important goal is to establish which step during transit peptide turnover exhibits an ATP requirement. Given the enormous amount of transit peptide that must be digested as proteins are imported, the degradation process is likely to be essential for normal chloroplast biogenesis. | Study | biomedical | en | 0.999997 |
10508854 | Control and US11-expressing U373-MG astrocytoma cells were cultured as described previously . Cells were detached from tissue culture flasks with trypsin and incubated in suspension in methionine- and cysteine-free DME for 1 h at 37°C. Cells at 1 × 10 7 /ml were pulse-labeled for 10 min at 37°C in 290 μCi/ml [ 35 S]methionine and cysteine ( 35 S-Protein Express Labeling Mix; New England Nuclear). At the beginning of the chase period, 5 mM nonradioactive methionine and 1 mM cysteine were added. Samples were taken at various timepoints and lysates were made as described below. In all pulse–chase experiments, whether on intact or permeabilized cells (see below), when present, the proteasome inhibitor ZL 3 VS was at 50 μM throughout. Cells were labeled as described above, but for only 3 min, placed on ice, and washed once with PBS. They were then resuspended at 1.6 × 10 7 cells/ml in PB (25 mM Hepes 7.3, 115 mM potassium acetate, 5 mM sodium acetate, 2.5 mM MgCl 2 , 0.5 mM EGTA) containing 0.025% digitonin , an ATP-regenerating system , and protease inhibitors (10 μg/ml leupeptin, 5 μg/ml chymostatin, 3 μg/ml elastatinal, and 1 μg/ml pepstatin). After a chase period at 37°C, lysates were made from samples taken at various time points and immunoprecipitations were carried out as described below. 5 min into the chase period, >95% of cells were permeable to Trypan blue and >90% of their lactate dehydrogenase activity was released . Permeabilized cells were resuspended at 6 × 10 6 cell equivalents/ml in homogenization buffer (PB with 250 mM sucrose, PMSF, aprotinin, and leupeptin). Homogenization was carried out using a ball bearing device . The homogenates were fractionated by centrifuging sequentially at 1,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 1 h, and the resulting pellets were resuspended in homogenization buffer. The resuspended pellets and the 100,000 g supernatant were diluted with NP-40 lysis buffer and immunoprecipitations were carried out as described below. In the experiments shown in Fig. 12 , the intact cells were resuspended at a concentration of 1.2 × 10 7 cells/ml in homogenization buffer that contained an ATP regenerating system . Soluble, cytosolic proteins were squeezed out of permeabilized cells by centrifugation. At the indicated chase times, two samples were taken from each permeabilization reaction. Both were centrifuged in a microfuge at 14,000 rpm at 4°C for 10 min. The supernatant and pellet of one sample were remixed to represent the total starting material. The supernatant of the other sample was removed and saved. The pellet fraction was resuspended in PB containing digitonin and the ATP regenerating system. Lysates of each fraction (total, supernatant, and pellet) were made and immunoprecipitations were carried out, as described below. Samples from the permeabilization reactions were added to ice-cold trypsin in PB, such that the final trypsin concentration in each reaction was as indicated . After 30 min on ice, trypsin digestion was stopped by the addition of the protease inhibitors PMSF and Nα-tosyl-lys chloromethyl ketone, hydrochloride (TLCK; Calbiochem-Novabiochem). Denaturing SDS lysates were made and heavy chains were immunoprecipitated as described below. US11 astrocytomas were harvested, washed once in DME containing calf serum and glucose at 0.45 g/liter, and resuspended at 2 × 10 6 cells/ml in the same media containing 1 mCi/ml 3 H-mannose (New England Nuclear) with or without 50 μM ZL 3 VS. After labeling at 37°C for 1 h with frequent agitation, denaturing SDS lysates were made and sequential immunoprecipitations with αHC and αUb serum were carried out as described below and in the legend to Fig. 6 B. As noted in the figure legends, lysates were made in three different ways, depending on the experiment. In all cases, 1–2 × 10 6 cells (or cell equivalents) were used to make 1–1.5 ml of lysate for each immunoprecipitation. The type of lysate did not affect the overall outcome of any of the immunoprecipitation experiments (data not shown), although the yield of immunoprecipitated heavy chain was greater when SDS was present. Nondenaturing NP-40 lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions so that the final buffer was 0.5% NP-40 (or Igepal CA-630; Sigma Chemical Co.), 50 mM Tris, pH 8, and 10 mM MgCl 2 . Samples were agitated for 20 min at 4°C and then clarified by centrifuging in a microfuge at full speed for 10 min. The resulting supernatant was used for immunoprecipitation. Denaturing SDS lysates were made by resuspending cell pellets, permeabilized cells, or cell fractions in 100–150 μl of 1% SDS and 2 mM DTT. Samples were heated to 95°C for 5 min, cooled to room temperature, agitated vigorously, and diluted into NP-40 buffer so that the final lysate used for immunoprecipitation was 0.1% SDS, 0.2 mM DTT, 0.5% Igepal, 50 mM Tris, pH 8, and 10 mM MgCl 2 . In some experiments , nondenaturing NP-40 lysates were made and then SDS and DTT were added after the clarifying spin to final concentrations of 0.1% and 0.2 mM, respectively. All immune complexes were recovered by precipitation with fixed Staphylococcus aureus bacteria (Staph A). Anti-heavy chain serum (αHC) was made by injecting rabbits with the luminal domains (amino acids 1-275) of both HLA-A2 and HLA-B27 that had been expressed in bacteria (kindly provided by D. Garboczi, Harvard University). This serum recognizes free MHC class I heavy chains, as did a similar antibody which was described previously . Antibodies were raised against purified bovine ubiquitin exactly as described . The quality of the αUb serum that was obtained was evaluated in Western blots by its ability to bind Cdc34p-linked multi-ubiquitin conjugates . US11 antiserum was raised in rabbits against a mixture of three US11 peptides (MPELSLTLFDEPPPLVETE, ESLVAKRYWLRDYRVPQRT, and FWGLYVKGWLHRHFPWMF) that were coupled to keyhole limpet hemocyanin. The monoclonal antibody 12CA5, which recognizes the influenza hemagglutinin (HA) epitope, was purified from tissue culture cell supernatants by standard methods . The monoclonal antibody 66Ig10 was used to immunoprecipitate transferrin receptor . Immunoprecipitated heavy chains were eluted from Staph A by heating to 95°C in 2% SDS and then diluted into Con A precipitation buffer (final conditions: 0.5 M NaCl, 50 mM Tris, pH 7.5, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM MnCl 2 , 0.5% Igepal, 0.1% SDS, and 0.1% BSA). Con A–Sepharose (Pharmacia Biotech) was added and samples were mixed gently for 15 h at 4°C. The precipitates were washed four times in Con A precipitation buffer before being analyzed by SDS PAGE. One-dimensional isoelectric focusing (IEF), and fluorography were carried out as described previously . Quantitation of proteins labeled with 35 S and 125 I was done with a Fuji PhosphorImager BAS1000. Densitometry on gels of 3 H-mannose–labeled proteins was done with a BioRad Fluor-S MultiImager. HLA-A*0201 heavy chain (HLA-A2), with the mouse H-2 K b signal sequence replacing its own , was cloned into the pcDNA3.1 expression vector (Invitrogen Corp.). An HA epitope tag was inserted in the α2 domain at amino acid 127, where amino acid 1 is the first amino acid of the protein after the signal sequence is removed. For the K→R HA/A2 construct, site-directed mutagenesis and standard cloning techniques were used to change the three lysine residues in the cytosolic tail (positions 311, 316, and 340) to arginines. The HA/A2 constructs were transfected into US11 and control astrocytoma cells using Superfect (Qiagen). Cell lines stably expressing wild-type (wt) HA/A2 or K→R HA/A2 astrocytomas were selected with G418-sulfate (GIBCO BRL). Bovine ubiquitin (Sigma Chemical Co.) was iodinated using chloramine T . Each 310-μl iodination reaction contained 2.6 mg/ml ubiquitin, 3.5 mCi/ml Na 125 I (NEN Life Science Products), and 0.33 mg/ml chloramineT in 0.32 M potassium phosphate, pH 7.6. Iodination was allowed to proceed for 1 min and was stopped by addition of sodium metabisulfite and nonradioactive NaI. Iodinated ubiquitin was separated from unincorporated Na 125 I by running Sephadex G-25 (Pharmacia Biotech) spin columns that had been equilibrated with PB. The final ubiquitin concentration was estimated to be 200 μM, labeled to 10,000–15,000 cpm/pmol. 125 I-ubiquitin was added to US11 cells in permeabilization reactions at a final concentration of 60 μM or 15 μM . To begin to dissect the mechanism of US11-dependent dislocation and degradation of MHC class I heavy chains, a permeabilized cell system was developed. We devised permeabilization conditions, using the mild detergent digitonin, such that cells appear microscopically intact, but are permeable to Trypan blue and ATP as well as to proteins such as hexokinase, trypsin, and lactate dehydrogenase (see below and Materials and Methods). In a typical experiment, human astrocytoma cells expressing US11 or control astrocytoma cells were pulse-labeled briefly at 37°C with [ 35 S]methionine to load the endoplasmic reticulum (ER) with radioactive MHC class I heavy chains. Labeled cells were permeabilized by incubation in a buffer containing a low concentration of digitonin and an ATP-regenerating system, and returned to 37°C for a chase period. Lysates were made from samples taken at various timepoints and class I heavy chains were recovered by immunoprecipitation with rabbit anti-heavy chain serum (αHC). As in intact cells , in the permeabilized cell system, heavy chains were degraded only when US11 was present . The half-life of MHC class I heavy chain in permeabilized US11 cells was ∼10 min , somewhat longer than its 2–3-min half-life in intact US11 cells . In the presence of the proteasome inhibitors ZL 3 VS or lactacystin (data not shown), degradation was largely prevented and a lower molecular mass heavy chain species accumulated in US11 cells. This species is endoglycosidase H (Endo H) resistant (data not shown) and its molecular mass by SDS-PAGE corresponds to that of the deglycosylated heavy chain species that accumulates in the cytosol of intact US11 cells treated with proteasome inhibitors . As was observed in intact cells, the change in molecular mass of the heavy chain in the permeabilized cells is accompanied by a change in isoelectric point upon hydrolysis of the glycoamide bond. This is best seen by comparison with bacterial N -glycanase (PNGase F) treated samples . Thus, deglycosylated heavy chains also accumulate in permeabilized US11 cells in the presence of proteasome inhibitor. As in intact cells, the deglycosylated heavy chains in the permeabilized cells were soluble and cytosolic . The glycosylated heavy chains from both permeabilized US11 and control cells fractionated mostly with the particulate fractions, as did the control membrane proteins transferrin receptor (TfR) and US11. The light chain β 2 m, a soluble secretory protein, fractionated with membrane pellets in permeabilized control cells, as expected from its tight association with the MHC class I heavy chain. In US11 cells, because there is little heavy chain, most of the β 2 m is soluble in the ER lumen . We found β 2 m in both the 10-K pellet and the 100-K supernatant fractions in permeabilized US11 cells , indicating that a portion of the ER content was released during homogenization. Because the mechanical homogenization was a rather harsh procedure, resulting in some disruption of vesicles, a squeeze-out fractionation technique was also applied. At each timepoint after permeabilization, samples were taken, subjected to centrifugation in a microfuge, and separated into pellet and supernatant fractions. Under these conditions, β 2 m fractionated identically in US11 and control cells, >90% of the β 2 m was found in the pellet fractions, confirming that little disruption of vesicles occurred. US11 was found only in pellet fractions (data not shown). The deglycosylated heavy chain was found mostly in the squeezed-out, soluble fractions whereas the glycosylated heavy chain was found in the membrane pellet fractions. The cytosolic localization of the deglycosylated heavy chain in permeabilized cells was confirmed by proteolysis protection experiments. Samples of permeabilized cells were treated with increasing amounts of trypsin. Trypsin at 100 μg/ml degraded nearly all of the deglycosylated heavy chain present, while the glycosylated, membrane-bound heavy chain was largely protected . Further experiments showed that the faster-migrating heavy chain species, seen at 100 μg/ml and 200 μg/ml trypsin, corresponds to glycosylated heavy chain lacking the 30–amino acid cytosolic tail. It was not immunoprecipitable with antibodies specific for the heavy chain cytosolic tail (data not shown) and it was sensitive to treatment with endo H . Thus, deglycosylated heavy chain in permeabilized US11 cells accumulates in the cytosol and not in a membrane-bound compartment. In intact astrocytomas expressing US11 or US2, accumulation of deglycosylated heavy chain requires ATP . To test whether this also applies to the permeabilized cell system, we depleted ATP from permeabilization reactions carried out in the presence of proteasome inhibitor. Simply omitting the ATP-regenerating system from the permeabilization reactions significantly reduced the amount of deglycosylated heavy chain that appeared . When remaining ATP was depleted by the addition of hexokinase and glucose, no deglycosylated heavy chain was detectable . Moreover, ATP could not be substituted with the nonhydrolyzable ATP analogue AMPPNP . Thus, as in intact cells, the US11-dependent accumulation of deglycosylated heavy chain in permeabilized cells requires ATP. Taken together, these data demonstrate that the permeabilized cell system faithfully reproduces the US11-dependent degradation of MHC class I heavy chain seen in intact cells. Although ubiquitin conjugates of ER proteins degraded in the course of quality control have been detected in both mammalian cells and in yeast , ubiquitinated MHC class I heavy chains in cells infected with HCMV or in cells expressing US11 or US2 have not been reported. We used a direct and sensitive method to test for the presence of ubiquitinated heavy chain intermediates in permeabilized US11 cells. Instead of metabolically labeling cells before permeabilization, we permeabilized the cells in the presence of 125 I-ubiquitin. After a 10-min incubation at 37°C, lysates were made and heavy chains were isolated by immunoprecipitation. Iodinated, high molecular mass heavy chain species accumulated in permeabilized US11 cells, but only when proteasome inhibitor was present . Much less of this material was seen in control cells, despite the presence of proteasome inhibitors (lane 5). The high molecular mass of the iodinated products is consistent with their being polyubiquitin-conjugated MHC class I heavy chains. They are not coimmunoprecipitating ubiquitinated proteins, because they are also immunoprecipitable with αHC antibody from permeabilized cells lysed under denaturing conditions (data not shown). Nor are they conjugates formed after lysis, because the appearance of 125 I-ubiquitin heavy chains requires incubation with permeabilized cells (lane 4). To establish in intact cells that similar ubiquitin conjugates occur, anti-ubiquitin antibodies were raised and used in immunoprecipitation experiments. US11 and control cells were pulse-labeled with [ 35 S]methionine for 10 min and chased at 37°C in the absence or presence of proteasome inhibitor. MHC class I heavy chains were immunoprecipitated and the material bound to the αHC antibodies was dissociated and denatured with SDS. One aliquot was analyzed directly , and the rest was diluted into immunoprecipitation buffer and subjected to a second round of immunoprecipitation with anti-ubiquitin serum (αUb). High molecular mass, ubiquitin-conjugated heavy chains were detected in US11 cells but not in control cells . Some nonubiquitinated heavy chains were also recovered, probably due to nonspecific interactions (lanes 17, 21, 25, and 29). Thus, MHC class I heavy chain is ubiquitinated in intact US11 cells just as it is in permeabilized cells. In the absence of proteasome inhibitor, a small amount of heavy chain was found ubiquitinated immediately after labeling , but subsequently disappeared during the chase period, presumably because it was degraded. In the presence of proteasome inhibitor, the amount of ubiquitinated heavy chain observed remained essentially constant throughout the chase period (lanes 22–24). This probably reflects a balance in the action of ubiquitinating and deubiquitinating enzymes in the absence of appreciable degradation of the heavy chain. The majority of heavy chain in US11 cells is not ubiquitinated. Given the short labeling time in these experiments and the stability of ubiquitin in cells, we believe that most of the radioactivity in the ubiquitinated heavy chains is incorporated into the heavy chains themselves and not the ubiquitin molecules. With this assumption, we estimate that, at most, 10–20% of the MHC class I heavy chains are ubiquitinated in intact US11 cells treated with proteasome inhibitor. In the absence of proteasome inhibitor, at most 5% of the heavy chains were ubiquitinated. Consistent with the observation that dislocation is slower in permeabilized cells than in intact cells, we found that accumulation of ubiquitinated heavy chain is also slower under those conditions . Interestingly, both deglycosylated heavy chains and ubiquitinated heavy chains accumulate at approximately the same rate , suggesting that a common, preceding step is rate-limiting. Having demonstrated the occurrence of ubiquitinated heavy chains, we wished to know whether ubiquitination precedes deglycosylation or vice versa. Two independent approaches were taken to determine the fraction of ubiquitinated heavy chains that are glycosylated. First, intact US11 cells were treated with proteasome inhibitors and labeled with 3 H-mannose. Very little 3 H-mannose-labeled, ubiquitinated heavy chain was detected. On very long (2 mo) exposures of gels from three independent experiments, the amount of 3 H-mannose–labeled heavy chain precipitated with αUb serum was barely above background levels (data not shown). Furthermore, in αHC immunoprecipitations, 3 H-mannose–labeled, nonubiquitinated heavy chains were efficiently recovered, but very little mannose label was detectable in the area of the high molecular mass, ubiquitinated HC species . In contrast, such species were readily detectable in 35 S-labeled cells . Ubiquitinated glycosylated heavy chains account, at most, for only 1–2% of all glycosylated heavy chains in these experiments. Thus, it appears that the majority of the ubiquitinated heavy chains in US11 cells are deglycosylated. This finding is supported by a second set of experiments, in which glycosylated heavy chains were isolated by their ability to bind the lectin Con A. 35 S-labeled ubiquitinated heavy chains were isolated by immunoprecipitation with αHC serum, followed by reimmunoprecipitation with αUb serum. Heavy chains were eluted from the αUb antibodies with SDS and each sample was divided in two. Half was analyzed directly by SDS-PAGE and the other half was precipitated with Con A–Sepharose. Only a very small amount of ubiquitinated HC precipitated with con A . The precipitation was specific for glycosylated material because, in control experiments carried out in parallel, very little deglycosylated, nonubiquitinated HC was precipitated with Con A . No heavy chain was recovered in samples incubated with Con A in the presence of the competitor methyl α- d -mannopyranoside or in samples incubated only with Sepharose beads (data not shown). We calculated that only ∼10% of the ubiquitinated heavy chains bound to Con A. Taken together, these data suggest that either deglycosylation precedes ubiquitination or that ubiquitinated heavy chains are rapidly deglycosylated. Does ubiquitination occur while heavy chains are still associated with membranes? We first examined permeabilized US11 cells that had been treated with proteasome inhibitors because ubiquitinated heavy chains are most abundant under those conditions. In fractionation experiments, after mechanical homogenization of permeabilized cells that had been incubated in the presence of 125 I-ubiquitin, 50% of the ubiquitinated heavy chain was found in the 100-K supernatant and 30% in the 100-K pellet . In squeeze-out fractionation experiments on permeabilized US11 cells labeled with 125 I-ubiquitin in the same way, ∼75% of 125 I-ubiquitinated heavy chains fractionated with the cytosol (data not shown). A similar result was obtained in 35 S-labeled permeabilized US11 cells, where ∼80% of ubiquitinated heavy chains fractionated with the cytosol after 20 min of chase . Thus, most of the ubiquitinated heavy chains in US11 cells treated with proteasome inhibitors are released from the ER, although a small percentage seems to be membrane-associated. We next examined the localization of ubiquitinated heavy chain in cells not treated with proteasome inhibitors. To follow the small amount of ubiquitinated heavy chain that accumulates in intact cells under these conditions, we permeabilized the cells after the chase period and fractionated their contents using the squeeze-out technique (see Materials and Methods). We found that almost all of the ubiquitinated heavy chain fractionated with the membrane pellets in US11 cells not treated with proteasome inhibitor . This result was not simply due to the failure to achieve permeabilization because, under the same conditions, proteasomes and other cytosolic proteins can be squeezed out of these cells (data not shown). Moreover, in an experiment carried out in parallel using cells treated with proteasome inhibitor, deglycosylated heavy chain and ubiquitinated heavy chains (lanes 8 and 11) were squeezed into the cytosolic supernatant. Note that in the presence of proteasome inhibitor, the amount of soluble, ubiquitinated heavy chain increases with time . Thus, these results are consistent with those shown in Fig. 9A and Fig. B . They suggest that, both in the presence and absence of proteasome inhibitor, heavy chain is ubiquitinated while it is still associated with the ER membrane. Over time, in the absence of proteasome inhibitor, the ubiquitinated heavy chain is degraded, while, in the presence of proteasome inhibitor, it is released into the cytosol. With heavy chain ubiquitination occurring at the ER membrane, it seemed possible that ubiquitination might be the initiating signal for heavy chain dislocation. To determine whether ubiquitination of the heavy chain is required for its dislocation, we prevented ubiquitination of the heavy chain cytosolic tail by removing all lysines from the tail domain. Two different HA epitope-tagged heavy chain constructs, based on the HLA allele A2, were made, one with a wild-type (wt) cytosolic tail and one with the three cytosolic tail lysines mutated to arginine (K→R). Cell lines stably expressing the HA-tagged heavy chains (HA/A2) were selected. We found that K→R HA/A2 is degraded at approximately the same rate as wt HA/A2 in US11 cell lines, while both HA/A2 proteins are stable in control cells . When pulse–chase experiments were conducted in the presence of proteasome inhibitor, deglycosylated HA-tagged wt and K→R heavy chain intermediates accumulated at roughly the same rate . In all cases, the HA/A2 heavy chains were dislocated and degraded more slowly than endogenous heavy chain in cells not expressing HA/A2 constructs. We attribute this to the overexpression of the HA/A2 degradation substrates, which may saturate the degradation machinery . These results were reproducible in multiple, independently derived cell lines. Ubiquitination of the heavy chain cytosolic tail, therefore, is not required to initiate US11-dependent destruction of the protein. Next, we asked whether the K→R heavy chain is ubiquitinated. We isolated ubiquitinated HA/A2 from [ 35 S]methionine-labeled cell lines by sequential immunoprecipitation with αHA and αUb antibodies. Ubiquitin-conjugated K→R HA/A2 was detected in US11 cells both in the absence or presence of proteasome inhibitor. In fact, K→R HA/A2 was ubiquitinated to approximately the same extent as wt HA/A2. The finding that dislocated heavy chains are not ubiquitinated exclusively on residues in the cytosolic tail implies that dislocation must start before ubiquitination of the heavy chain. In squeeze-out fractionation experiments, the majority of ubiquitinated K→R HA/A2 and wt HA/A2 fractionated with membrane pellets in the absence of proteasome inhibitors . In the presence of proteasome inhibitors, slightly more than half of the ubiquitinated HA/A2 in each cell line fractionated with the cytosol . These results were confirmed in experiments in which the cell lines expressing the two different HA/A2 alleles were homogenized mechanically and fractionated by differential centrifugation. Both ubiquitinated HA/A2 species showed identical fractionation behavior. In the absence of proteasome inhibitors, they were clearly membrane-associated, with most found in the 1-K pellet . In the presence of proteasome inhibitors, the majority of the ubiquitinated HA/A2 was in the 100-K pellet and supernatant . Although we have not characterized the heavy chain in the 100-K pellet fraction further, we have found proteasome subunits in this fraction as well (data not shown), raising the possibility that some ubiquitinated heavy chain is tightly associated with proteasomes in the presence of proteasome inhibitor. Taken together, these data support the idea that the ER luminal domain of heavy chain is ubiquitinated while the protein is still associated with the membrane. We have developed a permeabilized cell system that recapitulates many of the important aspects of US11-dependent dislocation and degradation of MHC class I heavy chains that have been observed in intact cells. In the presence of proteasome inhibitors, deglycosylated heavy chain accumulates in the cytosolic fraction of permeabilized cells. As in intact cells, appearance of this deglycosylated species is ATP-dependent. We have used this permeabilized cell system, in combination with experiments carried out in intact cells, to identify and order intermediates in US11-dependent dislocation. In so doing, we have provided strong evidence that US11-dependent degradation of class I heavy chain follows a sequence of events similar to that described for other proteins that exit the ER for degradation in the cytosol. First, using two independent methods, we have shown that ubiquitinated MHC class I heavy chains accumulate in US11 cells. Experiments involving the addition of 125 I-ubiquitin to permeabilized cells followed by immunoprecipitation with αHC serum provide direct evidence that heavy chain is ubiquitinated. Double immunoprecipitation experiments on lysates of 35 S-labeled cells, using antibodies against MHC class I heavy chain and against ubiquitin, confirmed the identity of the ubiquitinated heavy chain. Importantly, ubiquitinated heavy chains were detected in US11 cells regardless of whether they had been treated with proteasome inhibitors, indicating that they are not an artifact of proteasome inhibitor addition. The majority of the ubiquitinated heavy chain that accumulates in US11 cells treated with proteasome inhibitor is not glycosylated, suggesting that deglycosylation may precede ubiquitin conjugation. However, the presence of a small population of glycosylated, ubiquitinated heavy chains and the observation that, at least in permeabilized US11 cells, ubiquitinated heavy chains seem to accumulate simultaneously with deglycosylated heavy chains , suggest that there might be no obligatory order to the deglycosylation and ubiquitination steps. We addressed the role of heavy chain ubiquitination in US11-dependent export from the ER by mutating all of the lysine residues in the heavy chain cytosolic tail to arginine, creating a K→R heavy chain mutant that lacks potential ubiquitination sites in its cytosolic domain. This mutant is still dislocated and degraded at approximately the same rate as wt heavy chain. It is also ubiquitinated. Thus, ubiquitination of the cytosolic tail of MHC class I heavy chain is not required to initiate heavy chain dislocation and degradation. Our data lead to the following model for US11-dependent degradation of MHC class I heavy chain . The first step is dislocation, in which all or part of the luminal domain of heavy chain enters the cytosol. We do not know the source of the force that pulls or pushes the heavy chain from the ER, nor do we know which segment of the protein exits the ER first. However, once the luminal domain of the heavy chain has been dislocated, the protein seems to undergo ubiquitination while still associated with the ER membrane. We find ubiquitinated heavy chains, including the K→R heavy chain mutants, in cell membrane fractions. Thus, at this stage, the ubiquitinated heavy chains are either completely dislocated from the ER but still tightly associated with the membrane on the cytosolic side , or they are partially dislocated from the ER, with part of the protein integrated in the ER membrane or held in the dislocation channel. Partially dislocated heavy chain could be in two different orientations: with a portion of the luminal domain situated in the ER membrane , or, more likely for energetic reasons, with the transmembrane domain in the ER membrane and with both NH 2 and COOH termini in the cytosol . ER membrane-associated ubiquitin-conjugating enzymes have been identified in yeast and they are required for ER-associated degradation . Our results suggest that similar enzymes may play a role in US11-dependent heavy chain degradation. Ultimately, the heavy chain is degraded. It is not clear how the heavy chain is brought into contact with the proteasome, nor where degradation takes place. If we assume that the proteasome inhibitor simply causes a backup of intermediates of the normal degradation pathway, then both soluble deglycosylated and soluble ubiquitinated heavy chains must also be present in the absence of inhibitor, but as very short-lived species. Thus, degradation would occur on cytosolic proteasomes. Alternatively, it is possible that the proteasome inhibitor actually alters the degradation pathway and that the soluble heavy chain species are an artifactual consequence of proteasome inhibition. If this is the case, then heavy chain degradation would occur at the ER membrane, carried out by a population of proteasomes that localizes there . The work presented here suggests that US11 accelerates a process that normally occurs with misfolded proteins in uninfected cells. By inference, US2 probably operates similarly. In fact, MHC class I heavy chains are also ubiquitinated in cells expressing US2 (Shamu, C.E., unpublished observation). Thus, understanding the exact mechanism by which US11 and US2 induce rapid MHC class I heavy chain degradation will be important not only for understanding viral immune evasion, but also for understanding ER quality control. The speed and specificity with which heavy chain is degraded in the presence of US11 will be particularly useful for further characterization of the dislocation pathway using our permeabilized system. It should be possible to dissect the cytosolic requirements of the pathway and to carry out more detailed mechanistic studies. | Study | biomedical | en | 0.999997 |
10508855 | Hippocampal or cortical cultures from embryonic rat brain have been previously used as a model system to study mRNA and protein localization as these cells differentiate distinct axonal and dendritic processes and have demonstrated similar molecular distributions to that observed in vivo . We have described the use of cerebrocortical cultures in studying the localization of β-actin mRNA to neuronal processes and growth cones . In this study, we used a chick forebrain neuronal culture system, which also differentiates axon and dendrite-like neurites , as an alternative to rat cortical neurons. We observed that neurons within these cultures frequently have larger growth cones that have a flattened, lamellar morphology that is characteristic of the type that localize β-actin mRNA . The general method of neuronal culture that we use has been described in detail and modified for use with chick forebrain neurons . Cerebral hemispheres were dissected from 8-d-old chick embryos and trypsinized (0.15% in HBSS) at 37°C for 7 min, washed in HBSS, and placed in MEM with 10% FBS. Cells were mechanically dissociated by pipetting, washed three times in MEM, and plated at low density (6,000 cells/cm 2 ) on poly- l -lysine (0.2 mg/ml, 16 h) and laminin (0.02 mg/ml, 12 min) coated coverslips or plates in MEM with 10% FBS for 2 h. Cells were inverted onto a monolayer of rat astrocytes in N 2 -conditioned medium with serum (2% FBS) and cultured for 4 d at 37°C in 5% CO 2 . N 2 -conditioned medium contained glutamate-free MEM supplemented with transferrin (100 μg/ml), insulin (5 μg/ml), progesterone (20 nM), putrescine (100 μM), selenium dioxide (30 nM), glucose (6 mg/ml), sodium pyruvate (1 mM), and ovalbumin (0.1%). The cells were then fixed in paraformaldehyde (4% in PBS) at room temperature for 15 min and washed in PBS containing 5 mM MgCl 2 three times. Neurons were cultured in N 2 -conditioned medium with 2% FBS for 4 d and then starved in minimal essential medium (MEM) without N 2 supplements or serum for a period of 6 h at 37°C. Before fixation, cells were treated at various time points (10 min through 2 h) with drugs or growth factors as follows: FBS (8%), forskolin (5 μM; Calbiochem-Novabiochem), neurotrophin-3 (NT-3) (25 ng/ml; Austral Biologicals), db-cAMP (N 6 , O 2 -dibutyryl-adenosine 3′,5′-cyclic monophosphate) (25 μM; Calbiochem-Novabiochem), brain derived neurotrophic factor (BDNF) (25 ng/ml; Sigma), nerve growth factor (25 ng/ml; GIBCO-BRL). In some experiments, the cells were treated with NT-3 in a series of time points, i.e., 1, 2, 5, and 10 min, after starvation in MEM to observe the changes of β-actin mRNA and protein localization in growth cones. K252a (200 nM; BioMol), an inhibitor for a broad range of serine and threonine protein kinases including Trk receptor tyrosine kinases and Rp-cAMP (50 μM; Calbiochem-Novabiochem), a nonhydrolyzable analogue competitor of cAMP , were both used to inhibit the NT-3 induction of β-actin mRNA localization. After 5 h of starvation in MEM, K252a or Rp-cAMP was added for 1 h followed by treatment with NT-3 (in the continued presence of the inhibitor). The cells were fixed in 4% paraformaldehyde in PBS after treatment with drugs or growth factors. To disrupt microfilaments, cells were treated with 5 μg/ml of cytochalasin-D (Sigma Chemical Co.) in culture media at 37°C for 30 min before NT-3 stimulation. To depolymerize microtubules, cells were treated with colchicine (10 μg/ml, Sigma Chemical Co.) in culture media for 30 min before NT-3 stimulation. In each case, the cells were first starved in MEM for 5.5 h before addition of cytochalasin-D or colchicine. Stock solutions of cytochalasin-D and colchicine were made up in DMSO and ethanol, respectively, and the concentration of these solvents was diluted below 0.1% in the culture media, so as not to be toxic to neurons. Neurons were fixed in paraformaldehyde (4% in PBS with 5 mM MgCl 2 ) after drug treatment and NT-3 stimulation. Six amino group modified oligonucleotides (50 bases each) complementary to 3′ untranslated sequences (3′-UTR) of chick β-actin mRNA were synthesized on a DNA synthesizer . Each oligonucleotide was modified at five positions within the sequence and chemically labeled using digoxigenin succinamide ester (Boehringer Mannheim). To ensure isoform specificity, probes were selected from unique regions within the 3′-UTR and were of identical length, guanine/cytosine (GC) content and hapten incorporation. Oligonucleotide probes complementary to β-galactosidase mRNA or Mu phage DNA were used as controls. In situ hybridization for β-actin mRNA was completed as previously described . Cells were equilibrated in 1× SSC with 40% formamide for 5 min. Each coverslip was incubated at 37°C overnight in hybridization reactions containing 20 ng of oligonucleotide probe, 1× SSC, 40% formamide, 10% dextran sulfate, 0.4% BSA, 20 mM ribonucleotide vanadyl complex, salmon testes DNA (10 mg/ml), E. coli tRNA (10 mg/ml), and 10 mM sodium phosphate. Cells were washed twice with 4× SSC/40% formamide and then twice with 2× SSC/40% formamide, both at 37°C, and then with 2× SSC three times at room temperature. The hybridized probes labeled with digoxigenin were detected using Cy3-conjugated monoclonal antibody (mAb) to digoxigenin and anti–mouse mAb-Cy3 (from Jackson ImmunoResearch Labs.). After blocking in TBS with BSA (2%) and FBS (2%) at 37°C for 1 h, the coverslips were incubated with Cy3-mAb to digoxigenin in TBS (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) with 1% BSA at 37°C for 1 h. After washes in TBS with 1% BSA, cells were mounted with n-propyl gallate (anti-fading agent). β-actin protein was detected with a mouse monoclonal antibody (Sigma) and secondary antibodies were conjugated with Cy3 (Jackson ImmunoResearch Labs.). Immunofluorescence signal was viewed using an Olympus-IX70 microscope equipped with a 60× Plan-Neofluar objective and Nomarski (DIC) optics. Cells were viewed using a 100 watt mercury arc lamp and light was filtered using HiQ bandpass filters (ChromaTech). The images were captured with a cooled CCD camera (Photometrics) using a 35-mm shutter and processed using IP Lab Spectrum (Scanalytics) running on a Macintosh G3. After identification of growth cones using DIC optics, a fluorescence image was immediately acquired. All exposure times with the CCD camera were kept constant (1 s for β-actin mRNA, 0.5 s for β-actin protein) and below grey scale saturation to permit a linear response to light intensity and quantitative analysis of differences in fluorescence intensities. The perimeter of each growth cone was traced using the DIC image and IP Lab software to identify a region of interest (ROI) and measure total fluorescence intensity. For quantitative image analysis of β-actin mRNA and protein localization using this method , 20 cells were imaged for each cell culture condition. For quantitative analysis using a visual scoring method, 100 cells per coverslip were analyzed for each cell culture condition. Experiments were done with duplicate coverslips for each variable and each experiment was repeated at least three times. The scoring method involved visualization of the presence or absence of β-actin mRNA granules in the axon-like growth cone from each cell. Cells were scored as localized if several granules were observed, and scored as nonlocalized if the signal was not distinguishable from background levels (hybridization with control probe). Localized cells would be expected to have a higher amount of fluorescent signal in growth cones compared with nonlocalized cells. Examples of localized and nonlocalized cells are shown in Fig. 1 . This scoring method was used to show that NT-3 promotes an increase in localization and that the results were comparable to the quantitation of fluorescence intensity using the CCD camera . The value for each bar within the histogram reports the mean and standard deviation between independent samples . The Student's t test was used to compare the percentage of localized cells under a number of experimental conditions. This scoring method produced similar results to that of quantitation of fluorescence intensity within each growth cone using digital imaging analysis (see above). The advantage of the scoring method is that one can rapidly score hundreds of cells within a population and evaluate multiple variables. SignaTECT cAMP-Dependent Protein Kinase Assay (Promega) was used to monitor neurotrophin-3 stimulation of PKA activity in primary neuronal cultures . This assay uses a biotinylated Kemptide substrate, derived from pyruvate kinase , which is highly specific for cAMP-dependent PKA (Km = 5–10 μM). The biotinylated peptide substrate is phosphorylated by cellular PKA using γ-[ 32 P]ATP. The 32 P-labeled biotin substrate is then recovered from the reaction mix and captured by a streptavidin-linked matrix (SAM TM Membrane). In brief, cultured neurons were treated with NT-3 as described, washed with HBSS, and then lysed in 0.3 ml cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM PMSF). Extracts were centrifuged for 5 min at 4°C at 14,000 g and 5 μl of the supernatant was incubated with 20 μl of the reaction mixture containing biotinylated peptide substrate and γ-[ 32 P]ATP for 5 min at 30°C. The reaction was terminated and 10 μl was spotted onto a biotin capture membrane. After washing and drying of the membrane, radioactivity was measured using a liquid scintillation counter. This assay detects 0.012 casein units (2.5 Kemptide units) or less of purified cAMP-dependent PKA. A linear regression analysis, using calibrated amounts of casein units, produced an R2 value >0.95. Triplicate samples were used for each time point and the PKA activities and standard deviation is shown in Fig. 8 (expressed relative to starved cells not exposed to NT-3). Neurons (8 × 10 5 cells) were cultured in N 2 -conditioned medium supplemented with 2% fetal bovine serum on poly- l -lysine and laminin-coated plastic dishes for 4 d. Total cellular RNA was isolated using Tri Reagent following the product instructions (Molecular Research Center, Inc.). Cellular RNA was dissolved in DEPC-treated, distilled water. RNA (8 μg) was run in each lane in 0.8% formaldehyde-denatured agarose gel and transferred to Zeta-probe GT Genomic Tested Blotting Membrane (Bio-Rad Lab). The cDNA fragments (372 bp) complementary to the β-actin mRNA reading frame sequence were labeled with 32 P-dCTP (Amersham Corp.) by using the Random Primers DNA Labeling System (GIBCO-BRL) and purified by Quick Spin Columns (Boehringer Mannheim). The RNA binding membrane was prehybridized in 5× SSPE supplemented with 5× Denhardt's, 0.2% SDS, 5% dextran sulfate, 300 μg/ml salmon testes DNA, and 50% formamide at 45°C for 4 h, and hybridized with 32 P-labeled cDNA probes at 45°C overnight. After washes, the membrane was exposed to x-ray film (Kodak). Bands on the exposure film were scanned using a densitometer (Molecular Dynamics) and the optical densities were analyzed quantitatively using ImageQuant software. After 4 d of culturing in N 2 -conditioned medium with 2% FBS and 6 h of starvation in MEM, the cells were incubated in MEM with NT-3 (25 ng/ml) for 1, 2, 5, and 10 min. Cells were extracted in buffer (20 mM Hepes, pH 7.4, 138 mM KCl, 4 mM MgCl 2 , 3 mM EGTA, 0.1 mg/ml saponin, and 1 mM ATP) with 0.45 μM of rhodamine-labeled actin (rho-actin) at room temperature for 1 min and fixed for immunofluorescence microscopy . Rho-actin was rabbit G-actin labeled with rhodamine (provided by J. Condeelis, Albert Einstein College of Medicine). Rho-actin was thawed on ice and diluted in buffer (1 mM Hepes, pH 7.4, 0.2 mM MgCl 2 , and 0.2 mM ATP) to a final concentration of 12 μM. Rho-actin (75 μl) was added into 2 ml of extraction buffer just before application to the cells. To investigate the distribution of actin mRNA isoforms within neuronal processes and/or growth cones of chick forebrain neurons, chemically modified oligonucleotide probes were synthesized to specific β-actin mRNA sequences and conjugated for use with fluorescence in situ hybridization. With this approach it is possible to detect a signal that may not be apparent using conventional bright-field or epifluorescence microscopy. Also, the control of probe length, GC content, and hapten labeling efficiency using oligonucleotide probes has eliminated variability encountered previously with enzymatic labeling. Using these probes we can compare the signal obtained for specific mRNAs with that of control probe sequences such as lacZ which have identical probe complexity (not shown). We have previously used this method to show that specific mRNAs have distinct intracellular distributions in primary cultures and that β-actin mRNAs localize within axonal growth cones of cultured cortical neurons . In chick forebrain neurons, hybridization signal to β-actin mRNA was observed in the cell body and extended into processes and growth cones in a highly punctate or granular pattern . To visualize β-actin within these forebrain neurons, an isoform-specific monoclonal antibody was used for immunofluorescence localization. In the somatodendritic compartment, β-actin labeling was highly enriched in the distal tips of minor neurites and no staining was observed within the cell body and proximal segments . β-actin protein was highly enriched within the peripheral margin of the axon-like growth cone and filopodia . In contrast to β-actin, γ-actin was distributed throughout the cell body, all processes and filopodia (not shown). The enrichment of the β-actin isoform within growth cones in these chick forebrain neurons was identical to that observed previously in rat cortical neurons . To investigate whether β-actin mRNA localization to growth cones is regulated by signal transduction mechanisms, we used an assay to study the effects of signaling molecules in the absence of the astrocyte coculture, which secretes neurotrophins, and other potential signaling molecules within the culture medium, e.g., insulin. This approach involved removing the neurons from their normal defined media with N2 supplements and placing them in MEM for 6 h, before challenging with specific growth factors. This brief deprivation of N2 supplements did not result in adverse effects on neuronal morphology or cytoskeletal integrity as judged by DIC microscopy or immunofluorescence staining with anti-tubulin antibody (not shown). The objective was to have the cells in a quiescent state but still sensitive to signaling mechanisms. It has been shown that starvation of nonneuronal cells in MEM causes them to enter a quiescent phase with reduced β-actin gene expression and mRNA levels . Serum stimulation promotes β-actin mRNA localization and enhances fibroblast motility . This previous work provides a useful methodology to study the effects of neurotrophins on β-actin mRNA localization in neurons. We investigated whether treatment of starved cells with neurotrophins could stimulate β-actin mRNA localization. Neurotrophins are a family of proteins that include NGF, BDNF, and the neurotrophins, NT3, NT4, and NT6. These proteins bind the tyrosine receptor kinases, TrkA, TrkB, and TrkC with selective affinity and activate several signaling pathways . Both NT-3 and BDNF bind trk B receptors, whereas NT-3 also binds trk C . NGF binds TrK A receptors and shows little affinity for Trk B/C receptors . We observed that addition of NT-3 to the medium of starved cells could promote the rapid localization of β-actin mRNA within growth cones. Cells were scored as either localized or nonlocalized for β-actin mRNA (see Materials and Methods), as has been done previously, as a means to quantitate mRNA localization . Cells were scored as localized if several granules were observed, and scored as nonlocalized if the signal was not distinguishable from background levels (hybridization with control probe). Localized cells would be expected to have a higher amount of fluorescent signal in growth cones compared with nonlocalized cells. After a 10-min stimulation with NT-3, >80% of the cells exhibited β-actin mRNA localization within growth cones, compared with only 45% of the starved cells . BDNF, but not NGF, was also shown to promote localization of β-actin mRNA within >80% of the cells . These results suggest involvement of specific neurotrophin-receptor interactions in the localization of β-actin mRNAs within growth cones. The localization of β-actin protein within growth cones was also stimulated by NT-3. Starved cells showed little accumulation of β-actin protein within growth cones . After 10 min in NT-3, a dramatic increase in the signal for β-actin protein within growth cone filopodia was observed . This signal for β-actin protein colocalized with F-actin, as assayed using phalloidin, suggesting that there is the stimulation of new F-actin filaments (not shown). To further quantitate the observations that NT-3 treatment results in an increase in the amount of detectable β-actin mRNA and protein within growth cones, images were acquired of both starved and NT-3–treated cells, using identical exposure times with a cooled CCD camera (see Materials and Methods). Fluorescence intensity values can be either expressed as a total amount or after dividing by growth cone area to get a measure of fluorescence density. Since growth cone area varies naturally from neuron to neuron, data expressed as fluorescence density was a more relevant parameter (total intensity/area). The perimeter of each growth cone was traced using DIC optics and the total fluorescence intensity was measured for each growth cone and divided by its area to obtain a measure of the density of mRNA and protein per pixel. NT-3 treatment resulted in a 3.3× increase in the mean pixel intensity for β-actin mRNA after 10 min in NT-3, and a 2.2× increase after 2 h in NT-3 . The increase in the mean pixel intensity for β-actin protein was increased by 1.75× after 10 min in NT-3, and 3.25× after 2 h in NT-3 . We also analyzed total fluorescence intensity, independent of growth cone area, and similarly observed an increase in β-actin mRNA and protein levels (not shown). To determine whether the localization of β-actin mRNA particles to growth cones was dependent on the integrity of cytoskeletal filament systems, cells were pretreated with cytochalasin-D or colchicine to depolymerize microfilaments and microtubules, respectively. After treatment with cytochalasin-D, localization of β-actin mRNA by NT-3 was still observed within neuronal processes and growth cones . Neurons treated with cytochalasin-D showed a disruption of filamentous actin in the peripheral margin of growth cones; labeling of F-actin was primarily localized to cytoplasmic aggregates . The cytochalasin dose was tenfold above that used to delocalize β-actin mRNA from fibroblast lamellae . Interestingly, treatment with cytochalasin-D actually produced an increase in the density of β-actin mRNA in growth cones after NT-3, as measured by quantitative digital imaging microscopy . Since cytochalasin-D can induce actin gene expression , it was possible that new β-actin mRNA transcripts were also transported and localized in the presence of cytochalasin. An alternative explanation could be that cytochalasin-D may decrease growth cone area that would result in a higher fluorescence density even if mRNA levels were unaffected. However, we saw no evidence that growth cone area was affected by either cyotchalsin-D or colchicine treatment . Consistent with these images, quantitative analysis of growth cone area did not reveal any evidence for decreased growth cone area after 30 min in either cytochalasin or colchicine (not shown). The preservation of growth cone structure after treatment with cytoskeletal disrupting agents can largely be attributed to their brief exposure to the cells (only 30 min). These brief treatments were also insufficient to perturb the steady state distribution of β-actin mRNA in control cultures (not shown). We have previously shown that longer treatments with colchicine (1.5 h) can delocalize mRNA form processes, whereas cytochalasin-D has no effect . These previous results are consistent with a hypothesis that mRNAs are anchored to microtubules. The current study focused instead on the dynamic aspects of mRNA localization in response to NT-3 stimulation. Here we show that perturbation of microtubules and not microfilaments can block the dynamic process of mRNA localization. These results suggest that β-actin mRNA transcripts can be localized into processes and growth cones by NT-3 signals despite the presence of disorganized actin filaments. To confirm the role of microtubules in the localization of β-actin mRNA to processes, microtubules were depolymerized with colchicine before fixation and hybridization. A filamentous distribution for microtubules was not observed after colchicine treatment . Also, minor alterations in neuronal morphology were evident, as most neurites were thinner with varicosities; however, the growth cones did not collapse . Depolymerization of microtubules inhibited the NT-3 signaling of β-actin mRNA localization within processes and growth cones . These results indicate that the localization of β-actin mRNA within processes and the central domain of growth cones was dependent on drug-labile microtubules. The above results using cytochalasin suggest a specific mechanism to localize β-actin mRNA that is independent of any signals that NT-3 may directly impart on new actin synthesis and polymerization. We propose that the first neuronal response to NT-3 is to rapidly promote actin polymerization within the peripheral margin of the growth cone that is then followed by the microtubule-dependent targeting of β-actin mRNAs to the growth cone. We performed a time course experiment to determine whether β-actin protein localization may occur before and perhaps independent of mRNA localization. After starvation in MEM, 62% of the cells were scored as localized for β-actin protein . NT-3 treatment for 2 min resulted in >85% of the cells being scored as localized for β-actin protein . β-actin protein localization in response to NT-3 was initially concentrated in one or two foci within the peripheral margin, suggesting local accumulation and/or polymerization . After several additional minutes, β-actin protein staining was observed throughout the entire peripheral margin . An increase in β-actin mRNA localization was also observed after 2 min, although it took longer than β-actin protein to reach maximal levels, peaking at 10 min . Therefore, the relocalization of β-actin mRNA was delayed relative to the protein localization. These results support the model that NT-3 signals may also promote the localization of β-actin protein by a mechanism independent of mRNA localization. We hypothesized that NT-3 may signal the polymerization of monomeric actin within growth cones that may be diffuse within the cytoplasm. The de novo formation of F-actin within growth cones would create very high levels of actin within a given pixel consistent with our quantitative observations . To address this issue, we used an actin polymerization assay developed to visualize actin polymerization at the leading edge of motile cells in response to EGF treatment . The assay involves extraction of live cells in the presence of rhodamine-labeled actin, which adds on to the growing barbed ends of actin filaments within the cell, allowing direct visualization of the location of NT-stimulated nucleation sites. Cells were treated with NT-3 and then extracted for 1 min with saponin in the presence of rhodamine-labeled G-actin. Cells were briefly washed, fixed, and examined by fluorescence microscopy. There was no incorporation of rhodamine-actin in control cells that were not treated with NT-3 . After a 5-min treatment, incorporation of rhodamine-actin was observed within the cortex of the cell body as well within growth cones . Staining levels increased progressively over the next several minutes. After a 10-min treatment with NT-3, rhodamine-actin incorporation was observed throughout the peripheral margin of the growth cone . There was no appreciable level of rhodamine-actin incorporation within the neurite shaft. Peripheral cytoplasm was the preferred locus for continued actin polymerization. These results indicate that actin polymerization, β-actin protein localization, and β-actin mRNA localization are coordinately regulated by neurotrophin signals. Neurotrophin stimulation of growth cone motility may involve activation of a cAMP-dependent signaling pathway . We sought to determine whether NT-3 signaling of β-actin mRNA localization could be mimicked by activation of the cAMP signaling pathway, and whether inhibitors of intracellular kinases could block the NT-3 response. Treatment of starved forebrain cells with either db-cAMP, a membrane permeable analogue of cAMP, or forskolin, an activator of adenylate cyclase, was observed to rapidly stimulate a 40% increase in β-actin mRNA localization within growth cones after 10 min . This stimulation in β-actin mRNA localization persisted throughout the various time points studied (up to 2 h). NT-3 signaling of β-actin mRNA localization could be blocked by prior incubation of the cells with K252a , an inhibitor for a broad range of serine, and threonine protein kinases including Trk receptor tyrosine kinases . A reduction in mRNA localization was also observed with Rp-cAMP , a nonhydrolyzable analogue competitor of cAMP , which has been previously used to affect growth cone motility in response to neurotrophin treatment . The reduction in mRNA localization observed with the specific PKA inhibitor, Rp-cAMP was less than that observed with K252a, suggesting the possibility that other signaling pathways may also be involved. To determine whether NT-3 signaling can specifically activate a cAMP signaling pathway, we measured protein kinase A (PKA) activity in cell lysates after cultures were treated with NT-3 at various time points. This assay used a biotinylated Kemptide substrate that is highly specific for cAMP-dependent PKA (see Materials and Methods). PKA activity was observed to peak at 2 min (345% increase over control levels) and then declined to baseline levels . We conclude from this assay that there is a rapid (within 2 min) increase in PKA activity that precedes our observations of β-actin mRNA localization. The localization of β-actin mRNA to growth cones in response to neurotrophin signaling is suggestive of a mechanism that promotes a redistribution in the preexisting mRNA population. In addition, neurotrophins may also enhance β-actin gene transcription that would result in increased levels of mRNA in the cytoplasm. We evaluated β-actin mRNA levels by Northern blot analysis in normal, starved, and stimulated cultures . Starvation of cells for 6 h resulted in a 52% decrease in β-actin mRNA levels. Treatment of starved cells with NT-3, forskolin, or fetal bovine serum (also promotes mRNA localization, data not shown) did not result in an increase in β-actin mRNA levels within the 10-min time period during which mRNA localization occurred. These results suggest that mRNA localization can be due to a rapid redistribution of preexisting mRNAs. A 30-min treatment with NT-3 did result in an increase in β-actin mRNA levels which then declined at 2 h (not shown). These results indicate that neurotrophin signals can also upregulate β-actin mRNA levels, and that this response may be one of the last events in a signal transduction cascade that collectively affects many aspects of actin gene expression and localization which include actin polymerization, localization of β-actin mRNA and protein, and perhaps transcriptional activation. The elucidation of regulatory mechanisms for mRNA localization within neuronal growth cones is important as it would provide support for the hypothesis that external cues encountered by the growth cone during pathfinding could locally control protein synthetic activities. The regulated synthesis of cytoskeletal proteins could influence growth cone structure and affect the rate and/or direction of process outgrowth. In nonneuronal cells, the active transport of β-actin mRNA to the cell's leading edge was induced by serum and PDGF. This induction was, in fact, required to obtain maximal rates of cell motility . The localization of β-actin mRNA within neurons could similarly influence local actin polymerization and process outgrowth. In this study, we identify a new function for the neurotrophins: the localization of β-actin mRNA granules within growth cones. We suggest that β-actin mRNA localization into neuronal processes provides a local mechanism to control G-actin concentration and facilitates de novo nucleation of actin polymerization. Our results suggest that Trk receptor activation leads to the anterograde transport of β-actin mRNA granules from the cell body into growth cones. We speculate that this mechanism might involve Trk receptors within the cell body, which would signal the microtubule-dependent transport of mRNA through activation of a cAMP-dependent pathway. In this study, we have also observed that NT-3 stimulated the rapid polymerization of actin within growth cones (within 2 min) which is consistent with previous observations . This could be due to the binding of NT-3 or BDNF to tyrosine kinase receptors (Trk C and/or B) which are localized within growth cones. Receptor activation could stimulate the rapid and local polymerization of G-actin into F-actin within the growth cone and filopodia. We suggest that this initial stimulus for polymerization would be independent of mRNA localization and local protein synthesis. We propose that after the initial signal to polymerize actin, active transport of β-actin mRNA into the growth cone occurs, which further supports actin polymerization and long-term process outgrowth. An alternative hypothesis is that the anchoring of β-actin mRNA within the growth cone may be secondary to the polymerization of actin which is induced by NT-3. However, NT-3 treatment was observed to induce the localization of mRNA in cells that cannot polymerize new actin filaments. These data imply the presence of a specific signaling mechanism for mRNA localization. Evidence in support of regulation of mRNA localization to growth cones was also obtained from a previous study using a micropipet to remove cytoplasm from growth cones and amplify a heterogeneous population of mRNAs, which included MAP2 and intermediate filament proteins . The amount of mRNA within growth cones was dependent on the stage of neuronal development, and was varied for each mRNA species . Translation of these mRNAs within growth cones was demonstrated by transfection of mRNA encoding an epitope tag and immunofluorescence localization. Collapse of growth cones with the calcium ionophore A23187 promoted transport of mRNAs encoding intermediate filaments into growth cones, further suggesting that local synthesis may be regulated . Our observations, along with the results of these recent studies, suggest a novel type of developmentally regulated sorting mechanism. Further work should provide new insight into the local control of cytoskeletal organization and growth dynamics. It is of fundamental interest to identify how the cytoskeletal composition of the growth cone differs from that of the perikarya and to identify mechanisms involved in cytoskeletal sorting and assembly. Not only must the neuron target cytoskeletal proteins to growth cones, it must also target a distinct set of proteins whose identity, stoichiometry, and structural organization differs from that of the perikarya. There is an increasing consensus that actin isoforms in several cell types are sorted to different intracellular compartments, and that β-actin has a specific role in regions of motile cytoplasm . β-actin may be the predominate isoform at submembranous sites where it is involved in local de novo nucleation of actin polymerization in response to extracellular signals . Our evidence here is that β-actin is highly concentrated within growth cones and filopodia. We propose that β-actin protein is the preferred isoform for de novo nucleation within growth cones and filopodia. The paucity of β-actin protein within the cell body and neurite shaft is consistent with a model in which β-actin mRNA is first transported to the growth cone and then translated locally to stimulate or bias the process of actin nucleation and polymerization near the peripheral margin. Evidence indicates that RNA granules or particles are found in many different cell types and may represent a transported form for mRNAs and translational machinery . These RNA particles may be translocated along cytoskeletal filaments via interactions of mRNA localization elements, motor molecules, and accessory proteins . The microtubule-dependent transport of RNA granules into neuronal processes may provide a rapid mechanism for localized protein synthesis in distal compartments. Previous studies have demonstrated the presence of RNA granules within neuronal and oligodendrocyte processes . These RNA granules were shown to contain specific mRNAs and translational components . Granules containing myelin basic protein mRNA (MBP) moved into oligodendrocyte processes at 0.2 μm/s , and microtubule depolymerizing drugs and antisense oligonucleotides to kinesin inhibited granule translocation into processes . RNA granules labeled with the nucleic acid binding dye, SYTO14, have been shown to move into minor neurites at an anterograde rate of 0.1 μm/s . Translocation of SYTO labeled granules was blocked by prior depolymerization of microtubules with colchicine . Our present work indicates that colchicine treatment inhibited the NT-3 stimulation of β-actin mRNA localization within growth cones, suggesting that microtubules were required for the active transport of RNA from the soma to the growth cone. Further work is needed to define the transport rates for granules containing β-actin mRNA which localize to neuronal processes and growth cones. Previous work in live cells using the fluorescent nucleic acid binding dye, SYTO14, has shown that the anterograde translocation of RNA granules was induced by NT-3 . It was proposed that Trk receptor signaling through receptors within the cell body could stimulate the movement of preexisting mRNA granules from the cell body into the process. It was not known previously whether the RNA granules induced by NT-3 contained specific mRNAs or whether they localized to growth cones. Our work here has shown that granules localized to growth cones in response to NT-3 contain β-actin mRNA. These new findings suggest an important function for neurotrophin-mediated mRNA localization: local synthesis of cytoskeletal precursors that are essential for actin polymerization and filopodial-directed outgrowth. The continued study of the dynamic regulation of mRNA localization should provide new insight into active mRNA transport mechanisms and the role of local protein synthesis in growth cone dynamics. | Study | biomedical | en | 0.999998 |
10508856 | Early passage normal human keratinocytes were transduced with retroviral expression vectors for Shh-IRES-GFP as well as GFP and lacZ controls as noted below and seeded on devitalized human dermis and grafted to SCID mice 5 . 4 mice were grafted per vector group; untransduced keratinocytes as well as GFP and lacZ vectors groups were used as additional controls. At 1 and 3 wk, a biopsy was performed and 5-μM-thick tissue cryosections were prepared. The human species origin of regenerated epithelium was confirmed in tissue sections using species-specific antibodies to human involucrin 24 (BTI Inc.). Propidium iodide staining was performed to highlight cellular nuclei in Shh and GFP control human epidermis at 1 and 3 wk using propidium iodide at 40 ng/ml for 1 min. For study of indicators of cellular proliferation in vivo, bromodeoxyuridine (BrdU) (Sigma) 100 mg/kg was administered by intraperitoneal injection to mice bearing genetically engineered human skin grafts. 2 h after injection, human skin was excised and 5-μM cryosections prepared. Antibody staining for BrdU (Becton Dickinson) and Ki-67 (Novocastra) was performed as described 28 . Quantitation of fold increase in BrdU[+] and Ki-67[+] cell numbers per 100 μM unit surface length was performed at 1 and 3 wk, with GFP epidermis at 1 wk assigned the standard value of 1.0. Error bars were calculated as SD between four independent grafts in each vector group. Primary human keratinocytes were freshly isolated from human skin 26 and transduced with retroviral expression vectors for Shh-IRES-GFP as well as GFP and lacZ controls 7 11 . Greater than 99% efficiency of both gene transfer and gene expression maintenance was verified by fluorescence microscopy (or X-gal staining at pH 8.0 for E . coli lacZ), both at 48 h after transduction and at serial passaging to passage 15 as previously described 7 11 . A similar gene transfer approach was used for the human p21 Cip1 retroviral expression vector except that high efficiency gene transfer was confirmed by immunofluorescence staining of cell aliquots using antibodies to p21 Cip1 (Santa Cruz Biotechnology). For p21 Cip1 studies, transduction with p21 Cip1 retroviral expression vectors was performed 48 h after initial transduction with either Shh-IRES-GFP or GFP and lacZ controls. Senescence-associated β-galactosidase (SA-β-gal) expression was determined at pH 6.0 as described 9 before and at day 0, 3, 5, 7, and 9 after transduction with retroviral vector for expression of p21 Cip1 . Keratinocytes were used from the same donor for each series of experiments to control for possible tissue variations between individuals. All experiments were performed using triplicate independent transductions, with data presented as the average of these independent transductions ± SD. Cell cycle distribution was determined in proliferating cells grown in a 1:1 mixture of SFM media (GIBCO BRL) and 154 media (Cascade Biologics) growth media as described 28 . Terminal differentiation-associated growth inhibition was induced by addition of 1.5 mM calcium to this media for 48 h. Shh and control cells were stained with propidium iodide and analyzed for DNA content via flow cytometry 23 . Cumulative cell yield was determined as previously described 17 , with cells grown in a low calcium 1:1 media mixture of SFM:154 media 28 . Cells were passaged at identical densities in triplicate after triplicate independent transductions for each condition. Each independent transduction was passaged separately after initial gene transfer, with the average cell numbers ± SD presented for each condition. Three independent series of these long-term experiments were performed to assess replicative capacity and cumulative cell yield. Analysis of CDK2 and CDK4 kinase activity was performed as previously described 20 . In brief, cells were rinsed with cold PBS and harvested in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, and 0.1% Tween-20 with 1 mM DTT, 0.1 mM PMSF, 0.2 U/ml aprotinin, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, and 1 mM sodium fluoride). After a freeze-thaw cycle, lysates were collected after centrifugation at 12,000 g for 10 min and immunoprecipitated using antibodies to human CDK2 and CDK4 (Santa Cruz). Histone H1 (Boehringer-Mannheim) was used as a substrate for kinase assays in 50 mM Hepes, pH 7.0, 10 mM MgCl 2 , 2.5 mM EGTA, 1 mM DTT, 20 μM ATP, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, and 1 mM sodium fluoride. The amount of CDK2 and CDK4 protein in precipitates was confirmed in parallel by immunoblotting. To determine if Shh impacts epithelial cell cycle regulation, we expressed Shh in human epidermis regenerated on immune deficient mice from normal keratinocytes in an approach known to effect hedgehog pathway activation 11 . Genetically engineered human epidermal grafts were then examined at 1 and 3 wk after grafting. Compared with normal findings seen in GFP[+] and lacZ[+] controls, Shh[+] epidermis displays hyperplasia and an over sixfold increase in cell numbers per unit surface length by 3 wk . Shh[+] epidermis is hyperproliferative, with >10-fold increases in Ki-67 expression and >8-fold increase in BrdU incorporation compared with controls . Even in Shh[+] tissue sections with comparable thickness to control, Ki-67- and BrdU-positive cells are seen well above the basal layer of Shh[+] epidermis , suggesting a failure to undergo the cell cycle arrest that normally occurs before outward migration in stratified epithelium. Such findings raise the possibility that Shh may oppose stimuli for epithelial cell cycle arrest. To determine if Shh confers a resistance to differentiation-associated cell cycle arrest, Shh[+] and control cells were grown to confluence in vitro then media calcium concentration was raised to 1.5 mM in a process that recapitulates features of differentiation seen in vivo, including cell cycle arrest and activation of certain terminal differentiation genes 18 . Cell cycle distribution was analyzed both before differentiation stimuli and 48 h after addition of calcium. Shh[+] and control cells are indistinguishable under low calcium conditions promoting cellular proliferation, with the majority of cells in G2/M and S phase . As anticipated, the addition of calcium induces exit of the majority of normal control cells into G0/G1. Whereas Shh[+] cells alter their cell cycle distribution in response to calcium, instead of redistributing to G0/G1 these cells are disproportionately in G2/M and S phase . These findings indicate Shh opposes cell cycle inhibitory effects of a primary in vitro stimulus for epithelial differentiation. These findings suggest that Shh pathway activation may confer resistance to growth inhibitory mechanisms that are of potential importance in epithelial growth control and neoplasia. To test if Shh augments the long-term growth capacity of nonimmortalized human epithelial cells, we performed serial passaging of Shh[+] cells along with GFP- and lacZ-expressing controls. Nonimmortalized keratinocytes exhaust their replicative capacity after multiple passages in vitro, allowing the total cell yield of a defined number of input cells to be determined 17 19 . Shh[+] cells display more active sustained proliferation, growing to higher densities and demonstrating evidence of more active DNA replication compared with controls . Consistent with this, Shh[+] cells generate a cumulative increased cell yield of 64–150-fold over controls before exhaustion of replicative capacity . Shh[+] cells continue to proliferate until passage 16 whereas controls cease active proliferation after passage 12. Less than 5% of cells were nonviable at each passage for all groups (as judged by trypan blue exclusion), indicating that this increase is not due to alterations in frequency of cell death. These findings indicate that Shh augments the proliferative capacity of normal epithelial cells. Another negative growth regulatory mechanism bypassed in neoplasia are cyclin-dependent kinase inhibitors (CKIs). To determine if Shh could also confer resistance to CKI-induced growth arrest, we studied its impact on the effects of p21 Cip1 , a CKI of known importance in epithelial growth inhibition 8 23 . p21 Cip1 is known to be important in the cell growth arrest that occurs before keratinocyte terminal differentiation 8 23 . Shh[+] and control cells were transduced at high efficiency with a retroviral expression vector for p21 Cip1 and kinetics of cell growth were determined. As anticipated, p21 Cip1 profoundly inhibits proliferation of both untransduced and GFP[+] control cells, however, p21 Cip1 expression fails to inhibit exponential growth by Shh[+] cells . In addition to resisting p21 Cip1 growth arrest, Shh[+] cells fail to induce a biomarker seen in permanent cell cycle arrest, SA-β-gal 9 , in response to p21 Cip1 . Whereas ∼50% of control cells are SA-β-gal[+] by day 5, only 15% of Shh[+] cells express this biomarker . Shh expression in these cells is also associated with augmented levels of CDK2 and CDK4 associated kinase activity, with this activity not fully repressed by p21 Cip1 in the case of CDK2 . These data indicate that Shh opposes p21 Cip1 -induced growth arrest and suggest that Shh leads to activation of key core cell cycle machinery elements CDK2 and CDK4. Cell cycle exit before outward migration and terminal differentiation is a cardinal feature of normal stratified epithelium. Here we report that Shh inhibits this process. We observe that Shh induces dramatic increases in the proportion of actively proliferating cells within stratified epithelium, including cells in normally growth-arrested suprabasal layers. This observation suggests that Shh opposes the normal growth arrest necessary for homeostasis in stratified epithelium. Implicated as important in this growth arrest process in vivo are calcium and the CKI p21 Cip1 . Consistent with this, we found that Shh rendered cells resistant to the exit from S and G2/M that occurs in normal keratinocytes exposed to elevated calcium concentrations in vitro in a process that may recapitulate the local differences in calcium concentration that exist within normal epidermis 10 . This resistance was characterized by an enhanced accumulation of cells in G2/M, raising the possibility that Shh could impact regulators acting at later points in the cell cycle. In addition to calcium, another factor implicated in the promotion of normal epithelial growth arrest is the CKI p21 Cip1 8 . Shh also opposes p21 Cip1 growth inhibitory effects, raising the possibility that Shh may exert a dominant positive impact on core cell cycle machinery capable of bypassing negative signals from CKIs. The possibility that Shh acts dominantly on core cell cycle machinery is supported by the observation that Shh expression is associated with an increase in activity of CDK2 and CDK4 under normal growth conditions. This is most notable in the case of CDK2 whose inhibition by p21 Cip1 is an important control point in the G1-S transition 3 . Shh[+] cells sustain CDK2 activity even in the presence of p21 Cip1 , although the undetectable levels of CDK2 kinase activity in controls make conclusions about relative impact of Shh on p21 Cip1 inhibition of CDK2 difficult. Detailed knowledge of the impact of loss of Shh pathway function on mammalian epithelial cell growth in settings where it is normally active, is not currently available. The failure of hair follicle morphogenesis in mice lacking Shh 4 , however, suggests that such loss may lead to a failure of the epithelial cell expansion required in this process. Consistent with these data, our findings indicate that Shh enhances epithelial proliferation and opposes stimuli for normal cell cycle exit. Evidence of Shh pathway activation is present in the vast majority of human BCCs, with induction of the Shh target Gli1 a consistent finding even in the absence of abnormal Shh or Ptc expression 6 . The fact that misexpression of Gli1 alone can induce tumors in vertebrate embryos in the absence of primary perturbations in Shh expression 6 underscores the fact that Shh pathway downstream effectors may account for the impact of induced Shh expression observed in these studies. Unlike the classical step-wise model of carcinogenesis requiring multiple genetic lesions, hedgehog pathway activation alone appears sufficient to produce cardinal features of human BCC 11 . This observation may account for the fact that BCC arises without precursor lesions, unlike another common cancer of stratified epithelium, cutaneous squamous cell carcinoma 22 . In light of the mechanism of Shh pathway induction, it is possible these effects may occur within normal hair-bearing skin and in BCC in a non-cell autonomous manner. This single hit induction of neoplasia by Shh stands in sharp contrast to the step-wise model of carcinogenesis requiring multiple genetic alterations believed for some time to be operative in other neoplasias, such as colorectal carcinoma 12 . In addition, this proposed requirement for activation of only one pathway to trigger this cancer may offer an explanation as to why BCC is the most common malignancy in the United States, with an estimated incidence of up to 1,000,000 yearly 21 . Recent studies support a need for cells to surmount several major obstacles in order to progress to neoplasia; apoptosis and irreversible growth arrest have emerged as two of the most important of these obstacles 30 . We have found that Shh augments cellular capacity for long-term growth. The fact that Shh opposes p21 Cip1 -induced growth arrest is consistent with this observation as p21 Cip1 loss allows other cell types to resist replicative exhaustion 2 . The Shh effects on growth capacity observed here, however, may likely be due to Shh-enhanced resistance to terminal differentiation-associated cell cycle arrest rather than a direct extension of the Hayflick limit on population doublings. Combined with the ability of Shh to induce bcl-2 11 , and thus potentially resist apoptosis, the ability to bypass growth arrest stimuli shown has been increasingly recognized as an important milestone in carcinogenesis and may explain the potent effects of the Shh pathway in inducing BCC. In spite of these potent effects, Shh-induced neoplasia appears to lack the aggressiveness of other malignancies characterized by multiple genetic lesions. In general, BCCs are slowly growing tumors that do not exhibit markedly invasive features and characteristically fail to metastasize 22 . These biologically mild behavioral characteristics may be due to the fact that BCC lacks the additional genetic lesions necessary for more aggressive behavior. Our findings indicate that Shh action promotes cellular proliferation by opposing cell cycle arrest. This observation may provide a platform for future studies examining Shh impacts on cell growth during development as well as in efforts aimed at defining the mechanistic basis of normal growth control in epithelial tissues. | Study | biomedical | en | 0.999996 |
10508857 | pMMTV-ErbB4ΔIC contains a truncated human ErbB4 cDNA in which sequences encoding ErbB4 up to P705 are fused to sequences encoding tandem Flag epitope tags (Kodak), immediately followed by two stop codons. The coding sequences are under regulatory control of a mouse mammary tumor virus (MMTV) long terminal repeat. The vector pMMTV-SV40-Bssk was first modified to generate the vector pMMTV-Aat using the AatII linker 5′-ACTAGTGACGTCA at the unique SpeI site. pMMTV-ErbB4ΔIC was produced by trimolecular ligation joining the HindIII (site filled with T4 DNA polymerase)-EcoRI digested pMMTV-Aat; the 3′ ∼2.1 Kb SalI (site filled with T4 DNA polymerase)-SpeI fragment from pLXSN-ErbB4 ; and the 285-bp SpeI-EcoRI digested PCR product generated from pLXSN-ErbB4 using the forward primer upstream of the unique SpeI site 5′-CCCACTAGTCATGAC and the reverse primer with an EcoRI linker 5′-CGGAATTCTTATTACTTGTCATCGTCGTCTTGTAGTCCTTGTCATCGTCGTCCTTGTAGTCGGGTG-CTGTGCC-3′ . The riboprobe template pBl-ErbB4ΔIC was produced by subcloning the 285-bp SpeI-EcoRI digested PCR product described above into pBluescript I S/K (Stratagene). Riboprobe template pBl-β-casein was generated by subcloning the 170-bp HindIII-StuI fragment from pFLAG1-β-casein (generously supplied by Dr. Nancy Hynes, Friedrich Miescher Institute, Basel, Switzerland) into HindIII-SmaI digested pBluescript I S/K. The riboprobe template pBl-WAP, for quantifying mouse whey acidic protein (WAP) RNA, was generated by reverse transcriptase PCR (RT-PCR) of mouse mammary gland RNA isolated from a female at 3-d postpartum using the downstream primer 5′-CTATCTGCATTGGGCACGGCCCGG . The cDNA was amplified by PCR using the downstream primer specified above, and the upstream primer 5′-CCTCATCAGCCTTGTTCTTGGCCT . The 295-bp PCR product was made blunt with T4 DNA polymerase and subcloned into the SmaI site of pBluescript I S/K. The riboprobe template pBl-α-lactalbumin, was generated by RT-PCR with the downstream primer 5′-GGGCTTCTCACAACGCCACTGTTC and the upstream primer 5′-CATAGATGGCTATCAAGGCATCTC . The PCR product was digested with HincII-HindIII and the resultant 214-bp fragment was subcloned into pBluescript I K/S. Vector sequences for microinjection were separated from pMMTV-ErbB4ΔIC by digestion at unique AatII-XhoI sites. The ∼6.2-kb fragment containing the MMTV LTR, a 600-bp 5′ untranslated region of c-Ha- ras , the truncated human ErbB4 cDNA with COOH-terminal tandem Flag epitope tags, and simian virus 40 3′ mRNA processing signals, was purified by agarose gel electrophoresis. The purified DNA fragment was microinjected into single-cell B6SJL/F2 zygotes at a concentration of 12 μg/ml in 10 mM Tris, pH 7.5, 0.1 mM EDTA (by Ms. Carole Pelletier under the direction of Dr. David Brownstein at the Transgenic Mouse Shared Resource of the Yale University School of Medicine, New Haven, CT). Transgenic mice were identified by PCR of DNA isolated from tail biopsies. DNA purification, PCR conditions, and controls have been described previously . Primers for the amplification of a 360-bp MMTV-ErbB4ΔIC fragment were 5′-CAAGTATGCTGATCCAGATCGGGA and 5′-GAATTCTTATTACTTGTCATCGTC, which hybridizes to the 3′-terminal Flag epitope tag and unique EcoRI site of MMTV-ErbB4ΔIC. RNA was isolated from the number 4 inguinal mammary gland by TRIzol extraction (GIBCO BRL). Riboprobe synthesis and purification, and RNA analysis using the RPA II ribonuclease protection assay kit (Ambion) were performed as described . For hematoxylin/eosin staining, immunohistochemistry, and in situ hybridization, a portion of the number four inguinal mammary gland was spread onto a glass microscope slide and fixed in freshly prepared 4% paraformaldehyde in PBS (15 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 137 mM NaCl, 3 mM KCl, pH 7.2) overnight at 4°C. The fixed tissue was embedded in paraffin and 6-μm sections were dried onto gelatin-coated slides using standard procedures. Immunohistochemical detection of Flag-tagged ErbB4ΔIC and Stat5 was performed as described elsewhere with the following modifications. For Flag immunohistochemistry, the primary antibody was rabbit anti-Flag probe (Santa Cruz) diluted to 0.67 μg/ml and secondary antibody was biotinylated goat anti–rabbit diluted to 15 μg/ml (Vector Labs, Inc.). Negative controls included similarly treated mammary gland paraffin sections from nontransgenic siblings and affinity-purified rabbit IgG diluted to 0.67 μg/ml as the primary antibody. For Stat5 immunohistochemistry, primary antibody was rabbit anti-Stat5 LH743 diluted 1/600 and secondary antibody was biotinylated goat anti–rabbit diluted to 15 μg/ml (Vector Labs, Inc.). Sections were blocked and all antibodies were diluted in PBS containing 10% goat serum. Similarly treated mammary gland paraffin sections from nontransgenic mice at 3- and 18-d postpartum served as Stat5 positive and negative controls, respectively. An additional Stat5 negative control was rabbit serum (Pierce Chemical Co.) diluted 1/600 as the primary antibody. For immunohistochemical detection of Stat5 phosphorylated at Y694, sections were pretreated to expose phosphorylated Stat5 epitopes. Deparaffinized sections were treated with 1 mg/ml of buffered trypsin (Sigma Chemical Co.) for 20 min at 37°C. Endogenous peroxidase activity was inactivated by incubating the sections in 0.5% H 2 O 2 in PBS for 15 min at room temperature. The sections were incubated in 2 N HCl for 1 h at room temperature followed by two washes in 100 mM borate buffer, pH 8.5, for 5 min per wash. The sections were treated with 0.2% NP-40 for 30 min at room temperature. Between each treatment the sections were washed twice in PBS for 5 min per wash. The remainder of the procedure was performed as described elsewhere with the following modifications. Primary antibody was rabbit antiphospho-Stat5 (Zymed Labs, Inc.) diluted to 10 μg/ml and secondary antibody was biotinylated goat anti–rabbit diluted to 15 μg/ml (Vector Labs, Inc.). PBS containing 10% goat serum was used to block sections and dilute antibodies. For peptide blocking experiments, phospho-Stat5 antibody was diluted to 10 μg/ml in PBS containing 10% goat serum and 400 μg/ml of phospho-Stat5 peptide immunogen (Zymed Labs, Inc.). The antibody/peptide solution was incubated for 15 min at room temperature before application to the sections. Positive and negative controls for phospho-Stat5 immunohistochemistry were similarly treated paraffin sections from mammary glands at 3- and 18-d postpartum, respectively. Affinity-purified rabbit IgG diluted to 10 μg/ml as the primary antibody also served as a negative control. Immunostained sections were lightly counterstained in hematoxylin (Polysciences Inc.) or methyl green (Vector Labs, Inc.) according to the manufacturer's instructions, dehydrated in EtOH, cleared in xylene, and coverslipped with Permount (Fisher Scientific Co.). Buffers used for riboprobe synthesis and transcript purification were generally pretreated with DEPC. For in situ hybridization experiments, DNA template was linearized for sense and antisense riboprobe synthesis and contaminating ribonucleases were inactivated by proteinase treatment at 37°C for 1 h in 10 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA, 0.6% SDS, and 150 μg/ml proteinase K. In vitro transcription and subsequent DNase treatment were performed using a MAXIscript in vitro transcription kit (Ambion) with 1 μg of template DNA and 130 μCi of 35 S-UTP (DuPont-NEN) exactly as described by the manufacturer. Transcripts were suspended to 200 μl in a final concentration of 10 mM DTT (Sigma Chemical Co.), 300 mM NaOAc, and 20 μg of t-RNA, and purified by EtOH precipitation with 2 M NH 4 OAc. The precipitated RNA was washed extensively in 70% EtOH, resuspended into 10 mM DTT, and precipitated and washed a second time. The final RNA pellet was resuspended into 10 mM DTT. In situ hybridization was performed on 6-μm paraffin sections of mammary glands from female mice at 1- and 12-d postpartum using 35 S-UTP labeled riboprobes. Sections were deparaffinized in xylene, washed in 100% EtOH, and defatted by incubating with chloroform for 5 min. The sections were hydrated through a descending EtOH series and washed in PBS for 5 min. The tissue was etched in 2 μg/ml of protease K in PBS for 10 min at 37°C and rinsed in PBS. Tissue sections were postfixed in 4% paraformaldehyde in PBS for 10 min, quenched with 0.2% glycine in PBS for 5 min, and washed in PBS for 5 min. Nonspecific binding sites were blocked by incubating the sections for 10 min in 100 mM triethanolamine (Sigma Chemical Co.), pH 8.0, 0.9% NaCl, containing 0.25% acetic anhydride (Sigma Chemical Co.). The slides were washed in 2× SSC (20× SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.0) for 5 min, dehydrated through an ascending ethanol series, treated with chloroform for 5 min, washed two times with 100% ethanol for 2 min per wash, and air dried. The hybridization mixture contained 10 mM Tris, pH 7.5, 600 mM NaCl, 2 mM EDTA, 10 mM DTT, 1× Denhardt's (Sigma Chemical Co.), 500 μg/ml total yeast RNA (Ambion), 100 μg/ml poly-A (Pharmacia Biotech, Inc.), 100 mg/ml dextran sulfate (Sigma Chemical Co.), 50% deionized formamide (Ambion), and 4 × 10 4 cpm/μl of 35 S-UTP–labeled riboprobe, and was heated at 80°C for 10 min immediately before use. To each section, 50 μl of hybridization mixture was applied, the sections were overlaid with parafilm coverslips, and hybridized at 50°C for 16 h in a humid chamber containing 10 mM Tris, pH 7.5, 600 mM NaCl, 2 mM EDTA, and 50% formamide (Sigma Chemical Co.). After hybridization, the parafilm coverslips were removed and the slides were washed twice at low stringency for 15 min per wash at 50°C in 2× SSC, 50% formamide, and 0.1% β-mercaptoethanol. Nonhybridized probe was digested by placing the slides in 10 mM Tris, pH 8.0, 500 mM NaCl, containing 20 μg/ml of RNase A (Sigma Chemical Co.) for 30 min at 37°C. The low stringency washes were repeated and the slides were washed an additional two times in 0.1× SSC and 1% β-mercaptoethanol at 50°C for 15 min per wash. The slides were dehydrated through an ascending ethanol series, with a final concentration of 600 mM NaCl included in ethanol solutions under 80%, and air dried. Dried slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1:1 with ddH 2 O at 45°C, and were exposed at 4°C in light-tight slide boxes containing silica gel desiccant packets (Sigma Chemical Co.). Before developing, the slides were warmed to room temperature and developed in Kodak D-19 developer for 2.5 min, washed in ddH 2 O for 30 s, fixed in Kodak fixer for 3 min, and washed in running tap water for 15 min. The sections were lightly counterstained with hematoxylin using the same procedure described for immunohistochemistry. 293T cells were transfected using FuGENE6 transfection reagent (Boehringer Mannheim Corp.) according to the manufacturer's instructions. In brief, cells 25% confluent in 100-mm tissue culture dishes were transfected with 500 μl of growth medium without serum, containing 10 μl of FuGENE6 and 2 μg of each plasmid, for a total of 4 μg. The cells were incubated with transfection mixture for 48 h in a humidified incubator at 37°C with 5% CO 2 . Plasmid pLXSN served as a vector control for ErbB4 transfections. pLXSN-ErbB4 expresses human ErbB4 . pEF-neo served as a vector control for Stat5a transfections. pEF-Stat5a encodes mouse Stat5a cDNA. pEF-Stat5/Y694F is pEF-Stat5a with a tyrosine to phenylalanine mutation at residue 694, which abrogates Stat5 nuclear localization and DNA binding . pEF-Stat5/R618V is pEF-Stat5a with an arginine to valine mutation at residue 618, which ablates SH2 function . This mutation was created using QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the oligonucleotide primer 5′-GCGAAAGCAGTCGACGGATTCGTGAAGCCACAG. Transfected 293T cells were lysed in 2.0 ml of ice-cold EBC buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% NP-40, with 1× Complete protease inhibitor cocktail [Boehringer Mannheim Corp.], 1 mM phenylmethylsulfonyl fluoride, and 1 mM pervanadate) on ice for 10 min. The cell lysates were cleared by centrifugation in a SS-34 rotor at 5,000 rpm for 15 min at 4°C. Immunoprecipitation of ErbB4 and Stat5a from 500 μl of lysate was performed by adding 50 μl of preswollen protein A–Sepharose (Pharmacia Biotech, Inc.) and 2 μg of anti-ErbB4 (C-18; Santa Cruz) or 2 μg of anti-Stat5b . Precipitated proteins were resolved by SDS-PAGE on a 7.5% acrylamide gel and the resolving gel was transferred to Trans-Blot 0.45-μm nitrocellulose (Bio-Rad Laboratories) using standard procedures. Western blot analysis was performed as described previously . For detection of phosphotyrosine containing proteins, mAb 4G10 (Upstate Biotechnology Inc.) was diluted to 1 μg/ml, followed by sheep anti–mouse conjugated with HRP (Nycomed Amersham Inc.) diluted 1:3,000. For detection of Stat5a phosphorylated at Y694, rabbit antiphospho-Stat5 (Zymed Labs, Inc.) was diluted to 0.5 μg/ml; for detection of ErbB4, rabbit polyclonal ErbB4 (C-18) was diluted to 0.2 μg/ml; and for detection of Stat5, rabbit polyclonal Stat5b (C-17) was diluted to 0.2 μg/ml. The secondary antibody for rabbit primary antibodies was donkey anti–rabbit conjugated with HRP (Nycomed Amersham Inc.) diluted 1:3,000. Signal was detected using SuperSignal West Pico chemiluminescence substrate (Pierce Chemical Co.) according to the manufacturer's instructions. Since the embryonic lethality of ErbB 4 gene disruption precludes characterization of postnatal mammary development , we instead used a dominant-negative strategy to inactivate ErbB4 in the mammary gland. For this purpose, ErbB4 coding sequences encoding a receptor lacking the cytoplasmic domain were placed under the regulatory control of the MMTV promoter. pMMTV-ErbB4ΔIC encodes a protein of ∼120 kD, as determined by anti-Flag Western blot analysis of stably transfected FR3T3 cells (data not shown). Expression of pMMTV-ErbB4ΔIC inhibits EGF-stimulated tyrosine phosphorylation of the endogenous EGFR, verifying that, like cytoplasmic deletion mutants of other receptor tyrosine kinases, ErbB4ΔIC has dominant-negative activity (data not shown). The in vivo specificity of pMMTV-ErbB4ΔIC dominant-negative activity is described in the Discussion. To determine the effect of dominant-negative ErbB4 activity within the developing mammary gland, transgenic mice were derived by injecting MMTV-ErbB4ΔIC DNA into the pronuclei of fertilized one-cell zygotes from B6SJL/F2 mice. 19 founders with transgene integration (identified by PCR) were crossed into an FVB strain, and transgene expression by the F2 female offspring was determined by RNase protection analysis. Transgene expression was detected in mammary glands of five week-old female offspring from six different founders (data not shown). The highest levels of transgene expression were observed in the offspring of founders 5963 and 5997. Phenotypic analysis of mammary glands from mice expressing ErbB4ΔIC was performed on F3 females derived by crossing founder line 5963 F2 mice with FVB strain mice. The phenotype of line 5963 was confirmed by analysis of the second founder line, 5997. The temporal expression pattern of ErbB4 Δ IC RNA in the mammary gland was determined by RNase protection assay . The riboprobe hybridizes to the extreme 3′ end of the ErbB4 Δ IC transgene, including unique sequences encoding the tandem Flag epitope tags, resulting in a protected fragment of 285 bp . Transgene expression was first detected in prepubescent females at 3 wk and expression levels increased slightly with age, reaching maximal expression in the mature nulliparous mammary gland at 10 wk . The apparent decrease in expression at 19 wk was not observed in other experiments. Expression levels were similar from early to mid-pregnancy , increased at late pregnancy (lane 10), were highest at 1- and 3-d postpartum , and were reduced from 12-d postpartum through weaning . To determine the effects of ErbB4ΔIC expression on female mammary gland development, wholemounts and histological sections were examined from virgin mice at 3, 5, 6, 8, 10, and 19 wk of age; during early (12 d), mid- (16 d), and late (19 d) pregnancy; after parturition at days 3, 6, 9, 12, 15, or 18; and 2–4 d after weaning. At least three mice were analyzed at each time point. Despite the extensive time frame for transgene expression, and the fact that expression was highest shortly after parturition , the only identifiable phenotypes were detected on day 12 postpartum. The fat pad of a nontransgenic mouse at 12-d postpartum is completely invested with engorged lobuloalveoli displacing stromal adipose cells. Secretory activity is demonstrated by lumens lined with protruding secretory epithelium . Engorged active secretory lobuloalveoli were also observed in ErbB4ΔIC-expressing mice at 12-d postpartum . In some transgenic mice (3 out of 5 examined), however, a subpopulation of lobuloalveoli failed to expand and contained an unusually high level of lumenal secretory lipids . Adipose cells were still abundant in this region of the mammary gland fat pad. The condensed lobuloalveoli resembled undifferentiated lobuloalveoli that are normally predominant during late pregnancy. We next used anti-Flag immunohistochemistry to determine if the condensed lobuloalveoli expressed the Flag-tagged ErbB4 Δ IC transgene. Intense cytoplasmic immunostaining of epithelium within condensed lobuloalveoli was observed . Anti-Flag immunostaining was not observed in distended lobuloalveoli in the same tissue sections . The lack of detectable transgene expression in this subpopulation of lobuloalveoli may be a result of variegated transgene expression. Variegated promoter expression within the mouse mammary gland has been reported for several mammary specific promoters, including the MMTV LTR promoter used in this study . Although the alveolar condensation associated with high ErbB4ΔIC expression might be caused by selective growth inhibition or apoptosis, neither BrdU incorporation experiments, nor TUNEL analysis revealed differences between the phenotypically normal and condensed lobuloalveolar populations in ErbB4ΔIC animals (data not shown). These results suggest instead that ErbB4ΔIC expression inhibits normal lobuloalveolar development and function at 12-d postpartum. ErbB4ΔIC expression at 12-d postpartum impaired lobuloalveolar development, resulting in condensed alveolar structures with pronounced lipid secretory activity. These structures resembled normal undifferentiated lobuloalveoli observed at late pregnancy and parturition. To determine if the ErbB4ΔIC-expressing lobuloalveoli were lactationally active, we performed in situ hybridization using antisense riboprobes specific for the milk genes β-casein, WAP, and α-lactalbumin. Serial paraffin sections containing both normal expanded lobuloalveolar structures and condensed lobuloalveoli were examined . ErbB4ΔIC expression within condensed lobuloalveoli was confirmed by anti-Flag immunohistochemistry . The sense probes for β-casein, WAP, and α-lactalbumin yielded similar levels of background hybridization in both expanded and condensed lobuloalveoli . With antisense probe, equivalent high levels of β-casein RNA expression was observed in both the normal and ErbB4ΔIC-expressing lobuloalveoli . However, the ErbB4ΔIC-expressing lobuloalveoli showed a moderate diminution in WAP expression . Strikingly, α-lactalbumin expression was reduced to sense probe background levels in condensed areas, but not in normal areas of the same section . The decrease in WAP and the absence of α-lactalbumin expression suggests that terminal differentiation in ErbB4 Δ IC -expressing lobuloalveolar epithelium has been disrupted. Similar in situ hybridization analysis performed on mammary glands from female mice at 1-d postpartum yielded equivalent levels of expression of these genes in transgenic and nontransgenic sisters (data not shown). The condensed lobuloalveoli and pattern of impaired milk gene expression observed in ErbB4ΔIC-expressing mammary tissue resembles mammary defects observed in mice with Stat5 gene disruptions . Stat5 expression was determined by immunohistochemistry in sections of mammary glands at 12-d postpartum, containing both normal and ErbB4ΔIC-expressing lobuloalveoli . Strong immunostaining was detected in the nuclei of both normal and ErbB4ΔIC-expressing lobuloalveoli . Since functional Stat5 is phosphorylated at Y694 , we used an antibody specific for Stat5 phosphorylated at Y694 to evaluate the phosphorylation state of Stat5 . Strong nuclear staining and moderate cytoplasmic staining of phosphorylated Stat5 was detected within normal lobuloalveolar epithelium at 12-d postpartum . Immunoreactivity was blocked by preadsorption with the peptide immunogen and was undetectable in sections incubated with affinity-purified rabbit IgG control primary antibody (data not shown). Immunoreactive Stat5 and Phospho-Stat5 were detected in both normal mammary glands and phenotypically normal areas of transgenic mammary glands at 1-, 3-, 6-, 9-, and 15-d postpartum, but not at day 18 (data not shown). However, at day 12 postpartum, Stat5 was localized to the nucleus, but not phosphorylated in areas expressing ErbB4ΔIC . The lack of Y694 phosphorylation of nuclear Stat5 in ErbB4ΔIC-expressing lobuloalveolar epithelium suggests that it is functionally inactive. Since expression of ErbB4ΔIC appears to inhibit phosphorylation of Stat5 at the regulatory site Y694, it is possible that ErbB4 normally regulates this effector protein during mammary development. To determine if ErbB4 can induce phosphorylation of Stat5a at this site, the proteins were ectopically expressed at high levels in human embryonic kidney 293T cells . Despite high levels of Stat5a expression in transfected cell lysates , significant Stat5a tyrosine phosphorylation was observed only when Stat5a was coexpressed with ErbB4 . This phosphorylation included Y694, since it was detected by the anti-Stat5 phospho-Y694 antibody . When Stat5a and ErbB4 were coexpressed in 293T cells, they could be cross-coimmunoprecipitated . Stat5a coimmunoprecipitated with anti-ErbB4 and was not phosphorylated at Y694 , suggesting that phosphorylation of Stat5a results in rapid release of Stat5a from an ErbB4/Stat5a complex. To determine the specificity of ErbB4/Stat5a interaction and ErbB4-mediated phosphorylation of Stat5a on Y694, 293T cells were transfected with mutant STAT5 a alleles, with inactivating mutations in the SH2 domain (R618 to V) or at Y694 (Y to F). The two Stat5a mutants were expressed at levels comparable to wild-type Stat5a , but the Stat5a mutants were not phosphorylated at Y694 when coexpressed with ErbB4 . Interestingly, the Stat5a Y694F mutant was tyrosine phosphorylated at sites other than Y694 when coexpressed with ErbB4 . Alternative tyrosine phosphorylation of the Stat5a Y694F mutant also has been observed in 293T cells when cotransfected with a T cell receptor and Lck tyrosine kinase (Welte, T., and X.-Y. Fu, unpublished observations), and with activation of the EGFR . When expressed alone, the Stat5a mutants Y694F and SH2 were not immunoprecipitated by ErbB4-specific antiserum (data not shown). In summary, ErbB4 and Stat5a were coimmunoprecipitated when coexpressed and the Stat5a SH2 domain mutation prevented association of ErbB4 and Stat5a . Hence, the interaction between activated ErbB4 and Stat5a, and subsequent tyrosine phosphorylation of Stat5a at Y694, requires a functional Stat5a SH2 domain. Members of the EGFR family have important functions during several stages of mammary gland development. Stromal expression of EGFR is required for ductal morphogenesis . Epithelial functions for the receptors are suggested by the patterns of expression of ligands and receptors , by the functional effects of hormone implants , and by the phenotypes of MMTV-driven transgenic animals expressing dominant-negative receptor genes . To elucidate the function of ErbB4 during mouse mammary gland development, we inactivated ErbB4 signaling in the developing mouse mammary gland through the directed expression of dominant-negative ErbB4 as a transgene. Despite significant levels of transgene expression throughout pregnancy and even greater levels of expression early postpartum, an ErbB4ΔIC-specific phenotype was not observed until mid-lactation at 12-d postpartum. Lobuloalveoli expressing ErbB4ΔIC at 12-d postpartum were condensed, with lumens predominantly filled with secretory lipids, a phenotype resembling normal tissue at late pregnancy. Furthermore, the ErbB4ΔIC-expressing lobuloalveoli failed to terminally differentiate, as evidenced by a lack of α-lactalbumin expression. ErbB4ΔIC also inhibited Stat5 phosphorylation at Y694, suggesting that Stat5 is an important downstream mediator of ErbB4 signaling during lactation. The ErbB4ΔIC phenotype is significantly different from the phenotypes observed in transgenic mice harboring MMTV-driven dominant-negative EGFR or ErbB2 . Dominant-negative EGFR inhibited ductal morphogenesis in the pubescent virgin mouse. The dominant-negative receptor did not have an effect, however, during pregnancy or lactation, apparently because of high levels of endogenous EGFR expression during these developmental stages. In contrast, the dominant-negative MMTV-ErbB2ΔIC did not affect virgin mammary gland development, but did inhibit lobuloalveolar development at parturition . This phenotype appears earlier than the ErbB4ΔIC phenotype described here, and is not accompanied by suppression of mRNA for WAP, or α-lactalbumin (Jones, F., unpublished data). Although the dominant-negative receptors have some ability to inactivate heterologous dimerization partners in vitro, the nonoverlapping phenotypes obtained with dominant-negative EGFR, ErbB2, and ErbB4 suggests that each of these dominant-negative receptors does not act as a pan–dominant-negative. Corroborative evidence supporting a role for ErbB4 signaling during mid-lactation comes from the timing of ErbB4 activation during mouse mammary gland development, since ErbB4 tyrosine phosphorylation is dramatically enhanced at 14-d postpartum . These results support the conclusion that ErbB4 signaling plays an important role in lobuloalveolar maintenance and lactation during mid-lactation. Additional members of the EGFR family and their ligands have been implicated in lobuloalveolar development and lactation. Our in vivo experiments identified a role for ErbB2 signaling in lobuloalveolar development at parturition . Similarly, activated ErbB2 induces the formation of alveolar-like structures in a mammary epithelial cell culture system . Waved-2 mice, which carry a spontaneous point mutation within the EGFR kinase domain, have reduced EGFR kinase activity and exhibit impaired lobuloalveolar development and decreased lactation . The ErbB3 and ErbB4 ligand, NRG1, is required for lobuloalveolar development in mammary organ cultures and can induce lobuloalveoli formation and lactation when encapsulated and implanted within mammary glands of virgin mice . In addition, mice lacking the EGFR ligand amphiregulin develop immature lobuloalveoli, and the cumulative loss of EGF and TGFα aggravates this defect . Overlapping expression and activation of ErbB4 with EGFR and ErbB2 during lobuloalveolar development suggests that ErbB4 activity at this developmental stage may be regulated by these receptors and the aforementioned ligands with functions during lobuloalveolar development. The ErbB4ΔIC-expressing mammary epithelium resembles the phenotype observed in mice with a disrupted Stat5a gene . ErbB4 signaling during mid-lactation is required for Stat5 activation, since Stat5 expressed in ErbB4ΔIC-expressing lobuloalveoli was not phosphorylated on the regulatory Y694. Phosphorylation of this residue is essential for some Stat5 functions including dimerization and DNA binding . To our knowledge, this is the first in vivo evidence that an EGFR family member can mediate activation of Stat5. Surprisingly, Stat5 lacking phosphorylation at Y694 was localized to the nucleus of ErbB4ΔIC expressing lobuloalveolar epithelium. The current paradigm is that Stat5 phosphorylation at Y694 and subsequent dimerization are essential for Stat5 nuclear localization . However, this may not always be the case. It is conceivable that Stat is imported to the nucleus in a complex with the ligand-activated progesterone receptor . Johnson and coworkers have observed EGF-induced nuclear translocation of nonphosphorylated Stat2 . These recent observations, along with our results, demonstrate that nuclear translocation of Stat proteins, mediated by activation of EGFR family members, can occur in the absence of Stat tyrosine phosphorylation at Y694. Others have detected ErbB4 within the nucleus of breast epithelium , and nuclear translocation of exogenous ErbB4 ligand, NRG1, has been reported . These results raise the possibility that Stat5 may be transported to the nucleus in a complex with ligand-bound receptor. A similar mechanism has been proposed for Stat nuclear translocation mediated by ligand-activated interferon γ receptor . The coupling of ErbB4 to Stat5 regulation is reinforced by a survey of the ability of ErbB family receptor combinations to regulate Stats . NRG1 stimulated Tyr phosphorylation and activation of Stat5b in fibroblasts coexpressing ErbB2 and ErbB4, but not fibroblasts coexpressing EGFR and ErbB4, or ErbB2 and ErbB3. NRG1 is expressed at moderate levels during lactation , suggesting a means for activation of Stat5. In transient transfection assays, ErbB4 induced phosphorylation of Stat5a on Y694, and the two proteins could be coprecipitated in a Stat5 SH2-dependent manner. This suggests that Stat5 is a direct substrate for ErbB4, although we cannot rule out the possible recruitment of a second tyrosine kinase into the complex. Indeed, c-src is an important mediator of Stat5a activation by ErbB family members, and Janus kinases (JAKs) can associate stably with these receptors . The COOH-terminal portion of ErbB4, which contains tyrosine autophosphorylation sites, is required for ErbB4 and Stat5a coimmunoprecipitation (data not shown). The Stat5 consensus docking site , is present at three sites within the COOH terminus of ErbB4, raising the possibility of a direct interaction between Stat5 and ErbB4. Activation of Stat5 during lactation is thought to be mediated by prolactin receptor (PrlR) signaling . In the rodent mammary gland, PrlR is expressed as long (LPrlR) and short (SPrlR) isoforms . Only the LPrlR can activate Stat5 to stimulate β-casein promoter activity . Moreover, SPrlR acts as a dominant-negative and interferes with β-casein promoter induction by LPrlR . Expression of the two PrlR isoforms is differentially regulated during mammary development, resulting in an increase in the ratio between SPrlR and LPrlR as lactation progresses . The formation of inactive SPrlR/LPrlR heterodimers would, therefore, increase during lactation. One possible function of ErbB4 signaling in the mouse mammary gland would be maintenance of Stat5 activation at mid-lactation and compensation for the potential diminution in PrlR signaling activity. Another possibility is based upon the mechanistic differences between EGFR and PrlR regulation of Stat5 . c-Src is required for coupling of EGFR, but not PrlR to Stat5, and, conversely, Jak2 is required for coupling of PrlR, but not EGFR to Stat5. Moreover, EGFR and ErbB4 (see above) induce additional Tyr phosphorylation in Stat5 besides Y694. Hence, the downstream targets and adaptor functions of Stat5 may be different when regulated by receptor kinases or by cytokine receptors, so that Stat regulation by these two afferent systems is not functionally redundant. In contrast to expression of the other EGFR family members, expression of ErbB4 in breast cancer is associated with favorable prognosis and a differentiating tumor phenotype . In this communication, we present in vivo evidence demonstrating a role for ErbB4 signaling in terminal differentiation of mammary epithelial cells. Our results raise the intriguing possibility that ErbB4 activity in breast cancer cells may activate differentiation pathways and thus antagonize the oncogenic properties of other coexpressed EGFR family members. | Study | biomedical | en | 0.999996 |
10508858 | DMSO, Triton X-100, NP-40, sodium orthovanadate, NaFl, Trizma, Hepes, leupeptin, pepstatin, PMSF, BSA, collagenase type 1A, human fibronectin (FN), collagen I, and vitronectin (VN) were purchased from Sigma Chemical Co. Aprotinin and trypsin were from ICN Biomedicals Inc. DME, OPTI-MEM medium, glutamine, antibiotics, and Lipofectin™ were from Gibco Laboratories. FBS was from JRH Biosciences, COFAL-negative embryonated eggs were from SPAFAS, Inc.; protein G–agarose beads were from Boehringer Mannheim Corp. Polyvinylidene difluoride membranes and enhanced chemiluminescence (ECL) detection reagents were from Amersham Life Sciences. Mek1 inhibitor PD98059 was from New England Biolabs Inc.; purified human single chain urokinase type plasminogen activator (scuPA) was provided by Abbott Laboratories. Purified soluble human uPA receptor (uPAR) was provided by Dr. Francesco Blasi (Milan, Italy). mAbs: anti-phospho ERK 1/2 (anti-phospho-Tyr 204; clone E4), anti-NH2-Jun Kinase (JNK) (clone G7, phospho-Thr 183, and phospho-Tyr 185), anti-Shc (clone PG-797) were from Santa Cruz Biotechnology Inc. Anti-ERK1/2 (clone MK12) and anti-HCK (C18) mAbs were from Transduction Laboratories. Antiphosphotyrosine was from Upstate Biotechnology Inc. Anti-Grb2/sem5/ASH, anti-CD29 ( β1 integrin), and anti–CD55/DAF mAbs were from NeoMarkers. Anti-β1 activating mAb TS2/16 was from Endogen, and anti–α5 antibodies, clone P1D6, was from Chemicon International Inc. Mouse IgG 1 (MOPC1) was from Sigma Chemical Co. Anti-human uPAR 3996 mAb was from American Diagnostica. Anti-human uPAR mAb R2 was provided by Dr. Francesco Blasi (Universita Vita-Salute S. Raffaele, Milan, Italy), rat anti-β1 and α5β1 integrin blocking mAbs AIIB2 and BIIG2 , respectively, were provided by Dr. Caroline H. Damsky (University of California San Francisco, San Francisco, CA), currently available from the Developmental Study Hybridoma Bank, at the University of Iowa. Anti–mouse IgG mAb conjugated with HRP was from Vector Laboratories. Anti-rabbit IgG antibody and anti–HA mAb (clone 12CA5) were from Boehringer Mannheim. Human epidermoid carcinoma HEp3 (T-HEp3) that were tumorigenic and metastatic in the chick embryo and in nude mice were serially passaged on CAMs and used as a source of tumorigenic cells. The source of spontaneous dormant tumor cells (D-HEp3) were HEp3 cells passaged in vitro 120–170 times . HEp3 cells transfected with the expression vector LK444 (control clones, designated LK5 and LK25) or HEp3 cells transfected with LK444 vector expressing antisense uPAR mRNA (clones AS24, AS33, and AS48) in which surface uPAR and uPAR mRNA were reduced by up to 70 and 80%, respectively, was described previously . T-HEp3 or D-HEp3 cells were cultured in DME with 10% heat inactivated FBS (HI-FBS), penicillin (500 U/ml), and streptomycin (200 μg/ml). G418 (400 μg/ml) was added to LK25 and AS24 culture medium. Unless otherwise indicated, all cells were routinely passaged using 0.05% trypsin/EDTA. LK5, LK25, AS24, AS33, or AS48 cells were used between passage 1 and 10 in culture. For in vivo experiments, T-Hep3, LK25, D-HEp3, or AS24 cells growing in culture were detached with 2 mM EDTA in PBS, washed, and inoculated on the CAMs of 9–10-d-old chick embryos. In some experiments, before inoculation, the cells were incubated for 40 min at 37°C with appropriate antibodies. To determine growth in vivo at different times postinoculation, the thickened CAMs indicating tumor cell presence were excised, weighed, and after mincing, dissociated into single cell suspensions by incubation with type 1A collagenase for 30 min at 37°C. Tumor cells, recognized by their very large diameter, were counted with a hemocytometer. Cell viability was determined by trypan-blue exclusion. All antibodies used in vivo or in culture were free of azide. The antibodies used in vivo were tested for endotoxin content, using the Pyrogen-Plus test from Biowhittaker, and found to have <24 pg/ml. For serial passage of T-Hep3 cells, 7-d-old CAM tumors were minced, and small amounts of the mince were reapplied to fresh CAMs of 10-d-old chick embryos. For surface expression of integrins, cells were detached with 2 mM EDTA in PBS, resuspended in cold PBS with Ca 2+ , Mg 2+ , and 1% FBS at 10 7 cells/ml. Antibodies (P1D6, anti-α5; AIIB2, anti-β1) were added to 4 × 10 5 cells at 25 μg/ml and incubated at 4°C for 30 min. Controls were incubated with isotype-matched rat or mouse IgG. After two washes, FITC-conjugated goat anti–mouse or goat anti–rat (1:100) IgG were added and the cells incubated for 30 min at 4°C, washed two times, fixed in 5% formaldehyde in PBS, and analyzed in FACS ® scan equipped with a laser (488; Becton Dickinson). For cells in culture, exponentially growing cells were detached with 2 mM EDTA as described above. For cells in vivo, CAMs were inoculated with 1–2 × 10 6 cells, and at indicated times, tumor cells were isolated as described above, except that cells suspensions were obtained by incubation with collagenase for 20 min. To remove red blood cells, cell suspensions were layered on a 50% cushion of Percoll in 0.15 M NaCl, centrifuged for 15 min at 3,000 rpm, and the cells were collected from the top of the Percoll cushion, washed once by centrifugation, fixed in suspension with 70% ice-cold methanol, incubated with 10 mg/ml RNAse for 30 min at 37°C, washed, and incubated with 50 μg/ml propidium iodide in 0.1% Triton X-100 and 0.1% sodium citrate. The cells were kept in the dark before being analyzed by FACS ® scan, Profile II from Coulter Corp. Dormant and tumorigenic cells were plated in 100-mm dishes and transfected with 5–10 μg vector DNA expressing HA-tagged ERK2 using Lipofectin according to the manufacturer's instructions. After 24 and 48 h, cells were lysed using a buffer with 300 mM NaCl, 10% glycerol, 20 mM Hepes, pH 7.6, 5 mM MgCl 2 , 1 mM EGTA, 1 mM DTT, 1 mM sodium orthovanadate, 10 mM NaFl, and a cocktail of protease inhibitors. For in vivo experiments, 24 h posttransfection with the HA-ERK–expressing vector, the cells were inoculated (2.5 × 10 6 cell/CAM) into 8-mm-diam Teflon rings placed on CAMs. After 24 h of growth in vivo, the CAMs delineated by the rings were excised, slipped into tubes containing lysis buffer, and snap-frozen in liquid N 2 . The tissues were homogenized, centrifuged for 14 min at 14,000 rpm at 4°C, and the supernatants were analyzed for HA-ERK and phospho-HA-ERK as follows: 250 μg of cell protein was incubated with 1 μg of anti–HA antibody (12CA5) for 1 h at 4°C and for 1 h with protein G–agarose beads. The G beads were washed three times, resuspended in 2× sample buffer, heated for 10 min at 95°C, electrophoresed on SDS-PAGE, transferred to PVDF membranes (see below), and blotted with antibodies to HA or to phospho-ERK. The signal was developed using the ECL method. Matrix protein or polylysine plates, (96-, 24-, or 6-well) were coated with matrix proteins at 0.5 to 10 μg/ml or as stated in individual experiments, or with 4–8 μg/ml of polylysine (PL), incubated overnight at 4°C in PBS, and blocked for 1 h at 37°C with 1 mg/ml BSA (BSA or PL was used as a negative control). Cells were detached with 2 mM EDTA, resuspended in DME at 5 × 10 5 /ml, and added (50 μl per 96-well tray) to wells coated with FN and preincubated at 37°C with 50 μl of DME. After 30 min incubation at 37°C, the wells were washed gently, fixed with methanol, stained with 0.5% of crystal violet in water for 10 min, and washed extensively with water. After microscopic inspection, 60 μl of 10% methanol and 5% acetic acid solution was added to each well and, after 10 min, the OD at 570 nm was measured in a microplate reader (Dynatech Laboratory Inc.). Adhesion to other extracellular matrix proteins was done in a similar way except that VN, laminin (LN), or type I collagen were used instead of FN. Cells used in testing the effect of AIIB2 (20 μg/ml), BIIG2 (20 μg/ml), or TS2/16 (10 μg/ml) antibodies, and MnCl 2 (1.5 mM) were prepared as above, but the DME contained 0.2 mg/ml BSA and 10 mM Hepes. The cells were resuspended at 10 6 cells/ml with or without antibodies and incubated on a rocking platform for 30 min at room temperature. The medium was diluted to yield 2 × 10 4 cells/100 μl and 100-μl aliquots were inoculated into wells of a 96-well plate, four wells per sample. The effect of MnCl 2 on cell adhesion was examined without preincubation. All cells were allowed to adhere to FN for 20 min at 37°C and processed as above. LK25 or AS24 cells (0.8 × 10 5 ) were plated into wells of 24-well plates and cultured overnight with 10% serum. The medium was replaced in DME with 10 mM Hepes and 1 mg/ml BSA with or without 10 μM PD98059 (stock prepared in 100% DMSO); and the control medium contained 0.05% DMSO. Cells in three wells per sample were counted every 24 h in a Coulter counter, (Particle Counter, Model Z1; Coulter Corp.). To study the effect of immobilized FN on the growth of T-Hep3, LK25, D-Hep3, or AS24 cells in culture, cells detached from monolayers with EDTA were seeded in 24-well plates coated with FN or BSA in DME with 10 mM Hepes and 1 mg/ml BSA. Every 24 h the cells (three wells per sample) were detached and counted using a Coulter counter. To determine the basal level of ERK and JNK activation, subconfluent monolayers of cells were either kept overnight in DME with serum or in DME with 10 mM Hepes and 1 mg/ml BSA, scraped in PBS, centrifuged, and the pellets were lysed with RIPA buffer (1% Triton X-100, 140 mM NaCl, 10 mM Tris, 0.02% sodium azide, 0.1% SDS, 0.5% deoxycholate, 1 mM orthovanadate, 1 mM NaFl, 200 KIU/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM PMSF) and extracted on ice for 20 min. The lysates were centrifuged for 14 min at 14,000 rpm and the supernatants were saved. Equal amounts of proteins of each cell lysate were electrophoresed on an SDS-PAGE and Western blotted using either anti–phospho-ERK (p42/p44) or anti–phospho-JNK (p46/p54) antibodies. The p42/p44 ERK protein levels were determined using an anti–ERK1 antibody. To test the effect of scuPA on ERK activation, subconfluent cell monolayers were starved overnight in DME with Hepes and BSA, washed and acid-stripped for 3 min using cold 0.05 M glycine-HCl in 0.1 M NaCl buffer, pH 3, to remove uPAR bound uPA, and neutralized using 0.5 M Tris-HCl, pH 7.8. The cells were incubated for 1–60 min with 1–80 nM scuPA in the presence of 200 KIU/ml aprotinin to avoid protease-dependent effects of uPA. In some experiments, acid-stripped cells without or with added scuPA (10 nM) were incubated in presence of 10 μM PD98059 for 10 min. In another set of experiments, serum-starved but nonacid-stripped cells were incubated for the indicated time points with 0.1–10 ng/ml (<0.2 nM) of soluble uPAR in the presence of 200 KIU/ml aprotinin. To study the effect of integrin ligation on ERK and JNK activation, 24 h serum-starved cells were plated at 10 6 on PL- (4 μg/ml), FN- (0.4–4 μg/ml) or CL-I– (4 μg/ml) coated dishes (cells plated on PL attached, but did not spread even after 90 min). At the indicated times, the cells were processed as above. To test whether the antibody would interfere with FN activation of ERK, 10 6 cells were preincubated for 35 min at 37°C with 7 μg/ml of R2 anti–uPAR antibody (recognizes domain 3 of uPAR), or 5 μg/ml of anti–uPAR 3996 antibody (recognizes domain 1, and blocks uPA binding), or 7 μg/ml of isotype matched IgG, or 10 μg/ml of β1-integrin blocking antibodies (AIIB2), or with isotype-matched IgG, plated on surfaces coated with a mixture of PL and FN and, after 20 min incubation at 37°C, were analyzed for ERK activation. To test the effect of uPAR interaction with β1 integrin on ERK activation, T-HEp3, LK25, or AS24 cells were incubated for 5–10 min with increasing doses (0.1–50 μM) of peptide 25 (P25; AESTYHHLSLGYMYTLN-NH 2 ) dissolved in DMSO, that was shown to inhibit such interactions, but not to interfere with uPA or VN binding to uPAR . Controls were treated either with 0.05% DMSO alone or with a scrambled version of peptide 25 (NYHYLESSMTALYTLGH-NH 2 ). Regardless of treatment, to test for ERK activation, the cells were placed on ice, lysed, and the lysates were analyzed by Western blots as described above for scuPA treatment. Subconfluent monolayers were washed three times with cold PBS, and the cells were incubated on ice for 20 min with 5 ml of 0.5 mg/ml sulfo-NHS-biotin (Pierce Chemical Co.). The reaction was stopped by aspirating and washing the cells twice with 10 ml of ice-cold PBS. The cells were scraped in 1 ml of PBS-containing protease inhibitors, spun at 4°C, and the pellets were lysed with a lysis buffer containing 1% Triton X-100, 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and protease inhibitors as described for RIPA buffer. Immunoprecipitation and biotinylated proteins' detection were performed as indicated below. Cells were extracted for 1 h with a lysis buffer containing 1% Triton X-100, 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM orthovanadate, 1 mM NaFl, and protease inhibitors as described for RIPA buffer. Triton X-100 soluble and insoluble fractions of biotinylated or nonbiotinylated surface proteins (400 μg protein) were incubated with 4 μg of TS2/16 anti–β1, anti–α5β1 (BIIG2), anti–α5 (P1D6), anti–uPAR (R2) antibodies, matched isotype IgG or no IgG overnight at 4°C, precipitated with protein G–agarose beads, and washed three times. The beads were resuspended in 2× Laemmli sample buffer, heated to 95°C for 10 min, and analyzed by Western blotting using anti–β1 integrin (anti–CD29) antibody or anti–uPAR R2 antibody. For Western blotting analyses, after SDS-PAGE, the proteins were electrotransferred to PVDF membranes, the membranes probed with the primary and secondary antibodies, and the signal was detected using enhanced chemiluminescence with ECL (Amersham Life Sciences) and X-OMAT films (Eastman Kodak). When indicated, the bands were quantitated by laser densitometry using GelScan XL (Pharmacia Biotech Sverige). To detect surface biotinylated proteins, after immunoprecipitation with appropriate antibodies, the immunoprecipitates were separated in SDS-PAGE (nonreducing) and transferred to PVDF membranes. After blocking 1 h at room temperature with 5% skim milk, the membranes were washed with Tris buffered saline Tween 20 and incubated 1 h at room temperature with 1:2,000 dilution of streptavidin conjugated with HRP (Boehringer Mannheim) in TBS 0.3% BSA. The membranes were washed three times with TBS and the signal was developed using the ECL method. To study the mechanism responsible for dormancy induced by downregulation of uPAR, we used T-HEp3 (a mass culture prepared weekly from CAM tumors and maintained in culture for up to 1 wk), LK5, and LK25 as cell lines with a full complement of uPAR (uPAR-rich), and compared them to dormant cell lines with low uPAR levels, which included D-HEp3 obtained by prolonged passage of T-HEp3 cells in culture, , AS24, AS33, and AS48 clones of T-HEp3 transfected with a vector expressing uPAR antisense . Compared with T-HEp3, LK5, and LK25 cells, the level of uPAR protein in D-HEp3 and AS24, AS33, and AS48 cell lysates was reduced by ∼80% . A similar reduction in uPAR mRNA and surface uPAR number was found previously . We also showed that while all cell types with high uPAR (a total of five) grew rapidly on CAMs, all cells in the group with low uPAR (five total) remained dormant for months, suggesting that these differences were not due to clonal selection. The dormancy was linked to a ∼70% reduction in bromodeoxyuridine incorporation into the DNA of tumor cells maintained in vivo, and not to enhanced cell death , indicating reduced proliferation. To further analyze the proliferative failure in vivo, we inoculated D-HEp3 and T-HEp3 cells on CAMs, excised, and dissociated the CAMs, and either counted tumor cells daily or subjected them to cell cycle analysis . The T-HEp3 cells, which formed exponentially growing tumors, divided rapidly (six divisions in 6 d) on CAMs, whereas the number of D-HEp3, low uPAR cells, which formed very small nodules, did not increase . Cell cycle analysis revealed that in comparison to T-HEp3 cells in culture (day 0), T-HEp3 cells in vivo had a statistically significant larger percentage of cells in S phase, a matching decline in the percentage of cells in G 0 /G 1 and a matching fraction of cells in G 2 /M . This change was noticeable as early as 24 h postinoculation and was maintained throughout the 6 d of observation. In contrast, D-HEp3 uPAR-deficient cells in vivo underwent a rapid increase in the percentage of G 0 /G 1 cells, a rapid decline in the proportion of cells in G 2 /M, and a slower decline in the percentage of S phase cells . There was no significant difference in the proportion of cells in the different cell cycle phases between T and D-HEp3 cells in culture, whereas already after 1 d on the CAMs, the percentage of dormant cells in G 0 /G 1 was significantly larger than that of uPAR-rich cells, ( P = 0.005), and on day 3, the percentage of cells in both G 0 /G 1 and S phases was significantly different ( P = 0.000 and 0.001, respectively). Exit from G 0 /G 1 and entry into S phase is promoted by growth factors that signal predominantly through the ERK pathway. Thus, we examined whether this pathway is altered in uPAR-deficient cells by comparing the basal state of activation of the ERK1/2 in uPAR-rich and low uPAR cells. Cells incubated in serum-free medium for 24 h were tested for levels of ERK and active phosphorylated ERK (ERK1-p44/ERK2-p42) proteins by Western blots. Compared with the level of phospho-ERK in T-HEp3, LK5, or LK25 cells, the level in D-HEp3, AS24, AS33, or AS48 cells was very low (approximately four to sixfold reduction) , suggesting that the signal leading to ERK activation is impaired in uPAR-deficient cells. However, it should be noted, that despite the low level of active ERK, D-HEp3, AS24, AS33, or AS48 cells are capable of rapid proliferation in culture, possibly because a lesser level of activated ERK may be sufficient to initiate cell cycle progression in culture, or because parallel mitogenic pathways may be active. To distinguish between these possibilities, we interrupted the ERK pathway by blocking the activation of its immediate upstream activator MEK-1 with a specific inhibitor . After 1 h of treatment with 10 μM PD98059, basal ERK phosphorylation was strongly reduced in LK25 cells and almost completely blocked in AS24 cells . Increasing concentrations of the compound induced a dose-dependent inhibition of cell proliferation in both cell types , indicating that even at this low basal level, activation of ERK contributed to a mitogenic signal in uPAR-deficient AS24 cells in culture. Together, these results suggest that uPAR may be involved in triggering or coordinating a signaling mechanism that produces a powerful activation of the MEK-ERK pathway that may be crucial for mitogenic stimulus in vivo. Since in tumorigenic HEp3 cells 80–90% of uPAR is occupied by endogenously produced uPA , and since binding of uPA, scuPA, or amino terminal fragment of uPA to uPAR is known to initiate signal transduction , we wished to test whether removal of the endogenous ligand interrupts the signal leading to ERK activation and whether its addition restores it. LK25 and AS24 cells stripped of endogenous uPA were incubated for 5 min with increasing concentrations of scuPA and tested for ERK phosphorylation. To avoid scuPA activation and protease-dependent effects, the reaction was carried out in the presence of 200 KIU/ml aprotinin. In LK25 cells, scuPA binding induced a strong, dose-dependent increase in phospho-ERK levels, with a maximal effect at ∼10 nM . Neither the basal nor the stimulated levels of phospho-ERK were detectable in AS24 cells at the same time of exposure (data not shown). However, after a 10 times longer exposure of the blot both basal and scuPA-induced active ERK became detectable. Densitometric analysis revealed that 1 nM concentration of scuPA (approximately its K d value) caused a maximal (approximately fourfold) stimulation of active ERK in LK25 cells, whereas causing no modulation in AS24 cells. The highest concentration of scuPA (80 nM) added to AS24 cells produced a large stimulation over the basal level , but it still represented only <4% of active ERK in LK25 cells . This result suggests that it is not the fold increase, but the absolute level of active ERK that needs to be high enough to surpass the required threshold for proliferation in vivo. Similar results were observed when T-HEp3 was compared with D-HEp3 cells (data not shown). Cells stripped of endogenous uPA had a very low level of active ERK that could be restored by incubation with exogenous scuPA, but not in the presence of the MEK-1 inhibitor PD98059 , indicating that as shown before the uPA-induced signal was propagated through the MEK1-ERK pathway. Also, time dependence of ERK activation by scuPA was different: whereas 10 nM scuPA induced a large increase in the level of phospho-ERK in LK25 cells, which at 10 min reached a plateau sustainable for at least 60 min, AS24 cells showed a less pronounced and much delayed activation of ERK by scuPA, peaking at 30 min and decreasing at 60 min , suggesting that the ∼80% lower uPAR level in AS24 cells is incapable of evoking an optimal signal through this pathway. Interactions of uPAR with integrins are known to result in integrin activation in leukocytes during in vivo transendothelial migration and in modulating integrin function as adhesion receptors . Therefore, we tested whether the approximately fivefold difference in uPAR expression between tumorigenic and dormant cells influences integrin activation and function. As a test of function, adhesion of cells was measured on immobilized extracellular matrix proteins, including FN, LN, collagen type I (CLI), and VN. Adhesion of uPAR-rich tumorigenic (T-HEp3 or LK25) cells to FN was always three to sevenfold greater than that of uPAR-deficient, dormant cells (D-HEp3, AS24, or AS33 cells), and this difference was maintained over a range of FN concentrations . Adhesion to 0.5 μg/ml of FN revealed even greater differences (data not shown), but cells adhering to 0.5 μg were easily detached by washes, making the experiments more difficult. The differences (four to fivefold) in adhesion was maintained when tumorigenic and dormant cells were plated on 5 μg/ml of the 120-kD fragment of FN that lacks the α4β1 binding site (data not shown), confirming the involvement of the classical FN receptor. The uPAR-rich and low uPAR cells showed no difference in adhesion to other matrix proteins; both types of cells adhered well to CLI, but poorly to VN and LN (results not shown). The difference in adhesion to FN could not be explained by a difference in surface expression of FN-binding integrins, since their surface expression examined by FACS ® analysis using antibodies to α5 and β1 , α3 and αV (results not shown), and isotype-matched IgG as negative controls, showed that in every instance, the percentage of positive cells and the mean fluorescence intensity were similar or greater in the uPAR-deficient, dormant cells , suggesting that integrin function and not its surface level regulate adhesion to FN. Although, in addition to α5, the HEp3 cells express other FN-binding integrins, such as α3 and αV (data not shown), the fact that no difference in adhesion of tumorigenic and dormant cells to LN and VN was noted, suggests that these two integrins, although able to pair with β1, do not play a major role in the differential adhesion to FN, thus pointing to α5β1 as the likely candidate. To test this possibility directly, we examined the effect of β1-activating antibodies (TS2/16) and β 1 (AIIB2) or α5β1 (BIIG2) function–blocking antibodies on adhesion to FN of dormant and tumorigenic cells. We found that adhesion to FN of D-HEp3 or AS24 cells was increased by 67 and 85%, respectively, by TS2/16 antibody, whereas the adhesion of T-HEp3 or LK25 cells was unaffected . Treatment of cells with 1.5 mM MnCl 2 , which substantially increased the adhesion of dormant D-HEp3 or AS24 cells to FN, did not affect the adhesion of tumorigenic T-HEp3, or LK25 cells nor did it affect the adhesion of dormant cells to CLI (data not shown). Further support for a principal role for α5β1 integrin in mediating adhesion of HEp3 cells to FN is found in the observation that β1 and, more importantly, α5β1 function blocking antibodies (AIIB2 or BIIG2, respectively) reduced the adhesion to FN of all tested cells by ∼70–90%, indicating that regardless of the adhesion level (high in uPAR-rich and greatly reduced in low uPAR cells), the FN adhesion is predominantly mediated by α5β1 integrins and that the contribution to adhesion of other FN-binding integrins is marginal. Together, these results indicate that, a large proportion of α5β1 integrins in dormant uPAR-poor cells, although capable of being activated by Mn 2+ or an activating antibody, are intrinsically inactive. In contrast, in malignant, uPAR-rich cells exposed to similar conditions, the α5β1 integrins are maintained in a state that allows for an optimal adhesion to FN. Is the state of α5β1 integrin activation reflected in its function as a signaling receptor? We found that T-HEp3 cells plated for 20 min on dishes coated with increasing concentrations of immobilized FN, showed a dose-dependent increase in the levels of active ERK as compared with cells on PL-coated dishes . In contrast, D-HEp3 cells showed only marginal activation of ERK at 20 min on FN-coated dishes . In LK25 or T-HEp3 cells, but not in dormant cells, FN activated ERK at similar concentrations at which it stimulated adhesion . ERK activation by FN in LK25 cells was maximal at 20 min, and remained at almost peak level for up to 90 min . In contrast, in AS24 cells, not only the magnitude of response was greatly reduced, but also the activation of ERK did not persist beyond 20 min . Some activation of ERK was observed in cells plated on PL for 45 and 90 min . This was most likely caused by integrin-independent cell adhesion or because of deposition of endogenous FN matrix. It also has been reported that integrin engagement may activate the JNK pathway to promote cell cycle progression , but we found a similar basal level of active JNK in dormant and tumorigenic cells that was activated neither by FN nor by CLI (results not shown). We next tested whether FN/α5β1–dependent ERK activation leads to increased cell proliferation. In serum-free medium, immobilized FN stimulated the growth of uPAR-rich LK25 cells in a dose-dependent fashion , with doses even as low as 0.04 and 0.4 μg/ml producing significant stimulation of growth. In contrast, the growth of uPAR-deficient, dormant AS24 cells was only very marginally modulated, even by the highest FN concentration used . This difference in response to FN was also observed with T-HEp3 and D-HEp3 cells (results not shown). These same doses of FN differentially stimulate ERK activation and adhesion in tumorigenic and dormant cells . These results strongly suggest that uPAR-deficient cells may have an impairment in the activation pathway of MEK1/ERK by an FN/α5β1/uPAR signaling mechanism. To answer this question, we first examined whether uPAR and β1, α5, or α5β1 integrins were physically associated by testing the ability of anti-β1, α5, or α5β1 antibodies to coimmunoprecipitate (co-IP) uPAR and of uPAR antibodies to co-IP α5β1 integrins from cell lysates. Cell lysates were IP-ed and Western blotted. IP with anti–β1 antibody revealed a complex with uPAR, which was vastly reduced in the D-HEp3 and AS24 cells as compared with T-HEp3 cells, reflecting, most likely, the low level of uPAR in these cells . All three cell type lysates contained similar amounts of β1 protein . In a second approach, lysates of surface biotinylated cells were IP-ed with anti–β1, α5, α5β1, and uPAR antibodies. Anti–α5β1 (BIIG2) or α5 (P1D6) antibodies coimmunoprecipitated 55-, 116-, and 150-kD bands, which correspond to uPAR, β1, and α5 integrins, respectively (two additional bands of ∼130 and 80 kD were also present, but not yet identified). As shown before for β1 association with uPAR, in spite of similar amounts of β1 and α5 integrins present on the surface of all cells tested, the amount of uPAR in complex with α5β1 was 3.3–7-fold less in AS24 cells than in LK25 or T-HEp3 cells . The identity of uPAR and β1 integrin was verified by a parallel IP with anti–uPAR antibodies and anti–β1 antibodies . The ability of the different antiintegrin antibodies to co-IP uPAR from biotinylated cells suggests a plasma membrane association for these proteins. It also shows that biotinylation does not prevent the antibodies from recognizing their specific antigens. Taken together these results, along with the results showing inhibition of adhesion to FN by anti–β1 or α5β1 antibodies, strongly support the presence of a functional surface adhesion and signaling complex formed by uPAR and α5β1 integrins, which is more prevalent on uPAR-rich cells. Thus, the severe deficiency of dormant cells in integrin α5β1 adhesive and signaling properties is most likely due to the reduced number of active α5β1–uPAR complexes on the surface of these cells. We next examined whether the low level of ERK activation in uPAR-deficient cells can be corrected by exogenously added, soluble uPAR (suPAR). Such an effect would suggest that β1 integrin or other extracellular domains of transmembrane proteins may serve as uPAR adapter molecules mediating its effect on ERK activation. LK25 or AS24 cells were incubated for 5 min with increasing concentrations of suPAR and tested for ERK phosphorylation. While not affecting ERK in LK25 cells, suPAR induced a dose-dependent phosphorylation of ERK in AS24 cells, with maximal stimulation occurring at 5 ng/ml (∼0.5 nM) . The addition of suPAR did not fully restore the level of ERK activation found in uPAR-rich cells, suggesting that GPI-anchored uPAR may be more effective in integrin activation. Taken together, these results strongly suggest that a full complement of uPA/uPAR, through an interaction with α5β1 integrin, may be responsible for the high level of ERK activation in LK25 and T-HEp3 cells. The observation that binding of scuPA to uPAR increases the uPAR–integrin β1-mediated ERK activation signal suggests that a change in uPAR conformation may mediate this interaction. Therefore, we examined whether incubation of uPAR-rich cells with anti–uPAR antibodies affects ERK activation by FN. Preincubation of T-HEp3 or LK25 cells with mAbs (R2), which recognize domain 3 of uPAR, strongly inhibited FN-dependent activation of ERK, whereas irrelevant, isotype-matched IgG had little or no effect . Antibody to domain 1 of uPAR also blocked ERK activation but to a somewhat lesser degree (results not shown). To further examine the notion that uPAR–β1 interaction may be important for ERK activation, we treated T-HEp3 and LK25 cells with a peptide (peptide 25) that has been shown previously to interfere with the physical and functional interaction of uPAR and β1 integrins . Peptide 25, but not its scrambled version, when added to adherent cells, was capable of reducing ERK activation in a dose-dependent manner . Preincubation of suspended T-HEp3 cells with peptide 25, followed by their plating on FN, produced a similar reduction in ERK activation without diminishing the ability of cells to adhere to FN. A recently published report showed a similar lack of effect of peptide 25 on adhesion of uPAR and caveolin transfected 293 kidney cells or smooth muscle cells to FN. ERK activation could also be diminished by preincubation of T-HEp3 cells with a blocking anti–β1 antibody. In this experiment the cells remained adherent, as they were plated on a mixture of FN and PL . Therefore, interfering with uPAR/β1/FN interaction or β1–FN interaction both lead to a similar block in ERK activation. Collectively, our experiments show that binding of uPA to uPAR, by associating predominantly with α5β1 integrin, forms a complex that signals through MEK1-ERK1/2 activation, and that high density of uPAR increases this effect. In an effort to identify additional downstream members of this cascade, we tested whether Shc and Grb2, previously described to coimmunoprecipitate with β1 integrin , were in a complex with β1/uPAR immunoprecipitates. Although the 52- and 46-kD forms of Shc and Grb2 could be readily immunoprecipitated from tumorigenic or dormant cells (data not shown), we have been unable so far to find these proteins in complex with β1 integrins, either in the soluble or insoluble fractions of Triton X-100 lysates. Similarly, an Src-like tyrosine kinase, Hck, could be immunoprecipitated from both types of cells, but was not found to be differentially phosphorylated or in complex with β1 integrin (data not shown). We were also unable to show that, as described previously , binding of HEp3 cells to FN induced Shc phosphorylation (data not shown). In a recent report, active ERK level was shown to be higher in human renal carcinomas as compared with the surrounding normal tissue , suggesting that the findings in our model system may reflect the situation in human cancer. Thus, it was of major importance to test whether the high level of active ERK is present in the uPAR-rich, tumorigenic cells in vivo. Since the available anti–phospho-ERK antibodies recognize equally well chicken and human phospho-ERK (results not shown), to detect the ERK originating specifically in tumor cells, we transiently transfected the tumor cells with a construct expressing HA-tagged ERK2. By immunoprecipitating HA-ERK2 with anti–HA antibodies and analyzing the precipitate by Western blotting using either anti–HA antibodies (loading control) or anti–phospho-ERK antibodies, we showed that HA-ERK expression was maximal at 48 h after transfection and, as with the native ERK, the level of phosphorylation of HA-ERK was much greater in LK25 as compared with AS24 cells . To examine the state of ERK phosphorylation in vivo, 24 h after transfection with HA-ERK, the two tumorigenic and two dormant cell types were inoculated on CAMs and incubated in vivo for an additional 24 h. The areas of CAM containing tumor cells were excised, lysed, subjected to IP with anti–HA antibodies, and analyzed by Western blotting for HA-ERK2 and phospho-ERK content. Fig. 8 B shows that the level of phosphorylated ERK2 was three to sixfold greater in T-HEp3 and LK 25 cells than in D-HEp3 and AS24 dormant cells, respectively, indicating that the difference in the signal leading to ERK activation was maintained in vivo. Our results indicate that a functional association between α5β1 integrins and uPAR in tumorigenic cells with a full complement of uPAR is necessary for their optimal adhesion to FN and for transducing the FN-dependent activation of ERK. Therefore, we asked whether inhibition of ERK-activation by β1 integrin function–blocking antibodies (AIIB2) and/or antibody to uPAR (R2) , will reduce cell proliferation in vivo. Cells were pretreated with the appropriate antibodies, inoculated on the CAMs, and the number of tumor cells per CAM was determined on day 1, 3, and 7 postinoculation. Regardless of treatment, on day 1, only slightly more than a third of the inoculum was recoverable from the CAMs. On day 3, while the T-HEp3 cells, either untreated, or pretreated either with nonimmune IgG or irrelevant antibody (anti-CD55, CD55 is expressed in HEp3 cells, results not shown) underwent at least two divisions, cells pretreated either with anti–uPAR (R2), anti–β1 (AIIB2), or both antibodies, underwent only ∼0.5 divisions . The combination of antibodies exerted a slightly stronger inhibitory effect than each antibody individually. The number of cells on day 7 of in vivo incubation was similar in all groups indicating that the effect was transient. These results show that by interfering with the uPAR/β1/FN signaling complex that activates ERK, we can mimic the effect of uPAR deficiency, and that disruption and reduced abundance of this signaling complex may be responsible for the dormancy of uPAR-deficient cells. We have previously shown that downregulation of surface uPAR expression in human carcinoma cells, brings about a state of tumor dormancy characterized by cancer cell survival unaccompanied by an increase in tumor mass. This outcome is the result of a reduced proliferation rate and not of increased apoptosis . We set out to explore the mechanism through which a reduction in uPAR can evoke such dramatic changes. The proposed hypothesis stated that only levels of uPAR above a given threshold will, when saturated by uPA and laterally interacting with a putative transmembrane partner(s), initiate an outside-in signaling cascade capable of maintaining cancer cells in a proliferative state in vivo. A reduction in the surface expression of uPAR should restrict the magnitude of this signal, leading to dormancy. We were able to show that uPAR is physically associated with an FN-binding integrin, α5β1. Since low uPAR cells express similar or greater levels, of α5β1 than uPAR-rich cells, it could not be their mere physical presence on the cell surface that affects the behavior of uPAR-rich and low uPAR cells. Therefore, we concluded that in uPAR-rich cells α5β1 integrin, through its interaction with uPAR, must be held in an active state. The high level of uPAR leads to an in increase in the proportion of integrins sequestered into these interactions exceeding a threshold necessary for effective adhesion to FN, FN-dependent activation of ERK, and cell proliferation in vivo. Cell cycle progression analysis of the uPAR-rich (T-HEp3 cells) and low uPAR (D-HEp3) cells in culture and in vivo indicated that a reduction in uPAR expression renders the cells either incapable to respond to, or unable to generate a sufficient signal to propel them through G 0 /G 1 in vivo. This was underscored by the fact that in culture, the proportion of cells in the different cell cycle phases was similar, whereas only after 24–48 h of in vivo exposure, the proportion of cells in G 0 /G 1 in D-HEp3 cells rapidly increased and the G 2 /M and S phases declined. In contrast, the S phase fraction of T-HEp3 cells significantly increased and the G 0 /G 1 consistently declined . Our findings strongly suggest that the in vivo growth arrest of the uPAR-deficient cells is due to an interruption of the pathway to ERK activation. However, as indicated by the inhibition of ERK activation and growth of uPAR-deficient cells by a specific MEK1 inhibitor , the low level of active ERK in these cells is sufficient to promote their growth in culture. This is not unexpected as most normal cell lines proliferate rapidly in culture, but do not develop tumors in nude mice, syngeneic animals, or the CAM of chick embryos. These cells become tumorigenic when transformed by oncogenic viruses known to hyperactivate, among others, the ERK pathway. It is also possible that other MAP kinases, such as JNK, which we showed to be equally active in both cell types, may contribute to cell cycle progression in culture. The observed difference in ERK activation in vitro and in vivo correlates with the findings showing that primary lesions of human renal cell carcinoma display hyperactivated ERK1/2 . uPAR, a GPI-linked protein has been previously shown to associate with, and modulate the function of integrins from three different families (β1, 2, and 3) . We confirmed the physical association of uPAR and α5β1 integrin in HEp3 cells by co-IP experiments with anti–α5, β1, or α5β1 antibodies . By comparing the effect of MnCl 2 and anti-β1 activating antibodies (TS2/16), known inducers of integrin activation, we determined that, as measured by cell binding to FN, the α5β1 integrins in uPAR-rich cells were constitutively active and resistant to further stimulation. In low uPAR cells the integrins were inactive but responsive to stimulation. This difference in integrin avidity had an important consequence for intracellular signal transduction: the attachment of uPAR-rich cells to FN was accompanied by a persistent (up to 90 min) increase in the level of phospho-ERK, followed by increased cell proliferation in serum-free medium . In low uPAR cells, the amount of uPAR–α5β1 integrin complex was greatly reduced, the adhesion to FN was low, the maximal ERK stimulation by FN was greatly diminished in scope and duration , and no effect on cell proliferation was noted . We tentatively concluded that since both cell types have similar levels of α5β1 , that it is the high density of uPAR in T-HEp3 and LK25 cells that, through lateral interactions with α5β1, is responsible for the initiation of signal transduction events leading to ERK activation and cell proliferation. This response appears to be specific for FN integrins, since no differential response with regard to effect of attachment on ERK activation was found between uPAR-rich and low uPAR cells adherent to CLI. . Our findings are in agreement with published evidence showing a proximity by resonance energy transfer technique of uPAR and β1 integrin in HT1080 fibrosarcoma cells , which like T-HEp3 and LK25 cells form large, progressively growing tumors on CAMs . The growth of other cancer cells, such as primary and metastatic melanomas and colon carcinoma HT29 , showed an α5β1-mediated stimulation of cell growth on FN but the level of uPAR in these cells was not tested. Also, growth inhibition in nude mice was observed in transformed bronchial epithelium treated with anti–α5β1 antibodies . If ERK activation is dependent on the abundance of uPAR, it should theoretically be possible, by adding suPAR to uPAR-deficient cells, to increase the frequency of lateral interactions between uPAR and α5β1 and, thus, ERK activation. Indeed, we found that incubation of uPAR-deficient cells with 5 ng/ml of suPAR-stimulated ERK activation , indicating that even when not anchored in the plasma membrane, uPAR can affect signal transduction and, possibly, in vivo proliferation of cancer cells. This may have importance in cancer progression as high levels of circulating suPAR have been shown to be associated with some cancers and may, by binding to cells in existing metastatic foci, stimulate their growth. Previous reports from our and other laboratories have shown that binding of pro-uPA to its receptor results in a de novo association of various intracellular proteins, such as nonreceptor tyrosine kinases , with uPAR (as determined by their co-IP with anti–uPAR antibodies), and that uPA binding can initiate signal transducing events . Although, the mechanism responsible for these effects has not been elucidated, one possible explanation may be that binding of pro-uPA to uPAR may change its conformation and as a result, change the nature of its interactions with specific, signal transducing transmembrane proteins such as integrins. We used several reagents, including pro-uPA, antibodies to domain 3 of uPAR , and a peptide that has been shown previously to disrupt interactions between β1 integrins and uPAR to explore their potential effect on ERK activation. Treatment of uPAR-rich cells with low pro-uPA concentrations (1 nM, equivalent to the K d value for uPAR binding in these cells) produced a level of active ERK that was 33-fold greater than the maximal level of active ERK produced by 80 times higher scuPA concentration in low uPAR cells . This supports our hypothesis that only when uPAR–integrin interactions are frequent enough, can substantial ERK activation be achieved. In uPAR-rich cells, this is achieved even when only a fraction of the receptors is uPA-bound, whereas in low uPAR cells, only when most of the uPAR is uPA-bound, some degree of stimulation is observed, explaining the need for high uPA concentrations. In support of this conclusion, we showed that the antibody to domain 3 of uPAR reduced ERK activation upon cell adhesion to FN . Also, a peptide shown previously to disrupt uPAR–β1 interaction produced a dose-dependent reduction of ERK activation in tumorigenic cells, without affecting adhesion to FN. In contrast to our findings, the authors of this study concluded that uPAR–β1 interaction deactivates the integrin and blocks adhesion to FN. While our manuscript was in preparation, Wei et al. 1999 have published results similar to ours, in which uPAR–β1 interaction led to ERK activation on FN in smooth muscle cells and peptide 25 disrupted this function without affecting adhesion. The authors ascribed this controversy in their findings to the caveolin content of different cells, suggesting that other proteins may participate in the uPAR–integrin complex. However, we found caveolin not to be present in extracts of either tumorigenic or dormant cells tested by Western blotting (results not shown), suggesting that integrin activation may depend on specific cellular contexts. In our cells, the lateral interaction of uPAR with α5β1 in the presence of uPA, is capable of keeping the MEK-ERK pathways constitutively active and the uPAR-rich cells proliferating on FN. We have only began our inquiry into the additional components of the signal transduction pathway that leads from α5β1 to ERK activation. Published reports showed that phosphorylation of the adapter molecule Shc, and recruitment of Grb2 are in this pathway, and that Shc can be coimmunoprecipitated with β1 integrins. However, we did not yet find that adhesion of uPAR-rich cells to FN leads to Shc phosphorylation or that Shc coimmunoprecipitates with β1 integrins in uPAR-rich or -poor cells. The participation of Shc may depend on the cell type or on the experimental conditions, since other authors have also failed to detect Shc phosphorylation upon adhesion to FN . It remains possible that only very potent cross-linking, such as that induced by antibodies immobilized on beads , is needed to detect Shc in anti-β1 immunoprecipitates. Similarly, we could not detect activation of Hck, the Src-like kinase, reported to occur upon DFP-uPA treatment of other tumor cells , or its association with β1 integrin. Although JNK activation may be involved in promoting cell cycle progression on LN , we did not find that JNK was activated when cells were plated on FN for 20 min, pointing so far to ERK as the main participant. We cannot exclude a role for other kinases such as Src, Fyn, Yes, Lck, FAK , or the integrin linked kinase , which have been reported to influence cell cycle progression . Finally, since we showed that a high level of uPAR, its association with uPA, and adhesion of cells to FN, converge to generate strong ERK activation, which is maintained in vivo, we tested and found that the interruption of this signal transiently inhibited in vivo proliferation. Therefore, a rapid, FN-induced ERK activation must take place for the cells to enter and progress through the cell cycle in vivo. Interruption of this signal mimics, at least transiently, the initial steps responsible for the state of dormancy. In summary, we have found that, when present at a high level, uPAR interacts and holds the integrin α5β1 in an active state. Adhesion to FN organizes a signaling complex leading to a full activation of MEK1/ERK. It appears that only when a signal above a certain threshold is generated by the combined effect of uPA/uPAR/α5β1 and FN, the cells are capable of in vivo growth. As uPAR-rich and low uPAR cells produce similar amounts of uPA , and express similar levels of α5β1 , the observed difference in signaling must be dependent on uPAR levels. When the cascade is interrupted or diminished, as with anti–uPAR or anti–β1 antibodies or by downregulation of uPAR, the signaling complex switches to the off state and the cells become unable to grow in vivo. A somewhat similar situation was described in breast cancer , where a cross-talk between the EGF receptor and β1 integrins kept the cells in a tumorigenic mode with high ERK activation. When the aberrant β1, EGF receptor, or ERK signaling was corrected, tumor cells underwent differentiation and stopped growing. However, to the best of our knowledge, ours is the first published report in which induction of tumor dormancy in vivo was shown to be dependent on blocking the function of FN-dependent uPA/uPAR/β1 signal leading to MEK/ERK activation. | Other | biomedical | en | 0.999996 |
10508859 | The incisors were dissected carefully from the lower jaws (mandibles) of 2-d-old mice (CBA × NMRI) . This stage was selected for experiments because it was easy to dissect incisors from calcified mandibular bone. The apical ends of the incisors were dissected and cultured in Trowell-type organ cultures on 0.1-μm pore-size. Nuclepore filters (Costar Corp.) supported by metal grids in a humidified atmosphere of 5% CO 2 in air at 37°C. The culture medium consisted of DME (GIBCO BRL) supplemented with 10% FCS (GIBCO BRL), penicillin/streptomycin, glutamate I (GIBCO BRL), and 100 μg/ml ascorbic acid (Sigma Chemical Co.). Calcification of the enamel and dentin matrices was visualized by alizarin red staining. For tissue recombination experiments, the apical ends of the incisors were incubated for 10 min in 2% collagenase (GIBCO BRL) in DME at 37°C, and the epithelia were mechanically separated from mesenchyme in PBS. The epithelia were placed in contact with mesenchyme and cultured for 24 h on the filters as described above. Heparin acrylic beads (Sigma Chemical Co.) releasing FGF-10 (25 ng/μl; Amgen) were placed in cultures of whole apical ends of the incisors and on isolated epithelial tissue. Control beads were incubated in BSA (Sigma Chemical Co.). The preparation of the beads has been described in detail . Freshly dissected as well as cultured tissues were fixed in 4% paraformaldehyde in PBS, pH 7.2, overnight at 4°C, dehydrated, embedded in paraffin, and serially sectioned at 7 μm. Sections were placed on triethoxysilane- and acetone-treated slides, dried overnight at 37°C, and stored at 4°C until used. For histological analysis, the sections were stained with hematoxylin/eosin. Cryostat sections were incubated with rabbit anti-CVADR, as a primary antibody for 1 h at room temperature, rinsed in PBS, and incubated with peroxidase-labeled anti–rabbit antibodies for 45 min at room temperature. For color reaction DAB (Vector Laboratories, Inc.) as chromogen was used and the sections were counterstained with hematoxylin. The fluorescent DiI label was microinjected to the epithelium of dissected apical ends of the incisors that were subsequently cultured as described above. DiI [1,1′-dioctadecyl-6,6-di(4-sulfophenyl)-3,3,3′,3′-tetramethylindocarbocyanine; Molecular Probes, Inc.] was diluted in DMSO (0.2% wt/vol). The explants were fixed after 5 d and observed using a fluorescent microscope. Cell division was analyzed by culturing the tissues in the presence of BrdU (Amersham Life Science, Inc.). BrdU (1:1,000) was present in the medium for 3, 24, or 72 h, and the tissues were fixed in 4% paraformaldehyde in PBS, pH 7.4, either directly after labeling or after a 7-d chase period. The tissues were embedded in OCT compound (Tissue Tek), and 14-μm frozen sections were cut. The incorporated BrdU was detected by indirect immunoperoxidase method with mouse mAb against BrdU (Amersham Life Science, Inc.) and peroxidase-labeled anti–mouse secondary antibody (ENVISION; DAKO). For color reaction, DAB (Vector Laboratories, Inc.) as chromogen was used and the sections were counterstained with hematoxylin. For in situ hybridization analysis, single stranded [ 35 S]UTP- and digoxigenin-labeled antisense riboprobes for mouse Notch1 , Notch2 , Notch3 , Fgf-3 , Fgfr1b , and Fgfr2b were synthesized as described. Lunatic fringe antisense probe of 570 bp was obtained by linearizing the plasmid (pBS SKII(−) with 860 bp 5′ coding region of mouse lunatic fringe ) with BamHI and transcribing with T7 RNA polymerase. For the sense probe, XhoI and T3 RNA polymerase were used, respectively. Serrate 1 probe was a gift from Domingos Henrique (University of Lisbon), Fgf-3 probe from David Wilkinson (NIMR, London), and Fgf-10 probe from Nobuyuki Itoh (Kyoto University). The procedures for in situ hybridization of paraffin sections and whole mounts of cultured explants have been described previously . The appearance of the dissected incisor tooth of a 2-d-old mouse in a stereo microscope is shown in Fig. 1 b. The cervical loop epithelium appears translucent as compared with the mesenchymal tissue. The histology of the apical end of the incisor and its schematic representation show the different cell types . The cervical loop is composed of basal epithelium and a central core of stellate reticulum. A gradient of an increasing level of cell differentiation is evident in the basal epithelium contacting the pulp mesenchyme from apical to incisal direction. The inner enamel epithelial cells differentiate into tall columnar ameloblasts secreting the enamel matrix. We devised an organ culture method for the dissected apical ends of the incisors to analyze cell kinetics and fate. The appearance of an explant as photographed through a stereomicroscope during a 7-d culture period is shown in Fig. 1e–i . The cervical loop produced new dental epithelium, which was seen as an upward movement of the apical end of the epithelium to encompass the apical mesenchyme. This differs from the in vivo situation where the growing epithelium moves towards the incisal direction, probably because of mechanical factors as in vitro the tissues are attached to the filter. After 3 d of culture, production of new enamel and dentin by differentiated ameloblasts and odontoblasts, respectively, was evident . The thickness of the enamel and dentin matrices increased with time when the explants were cultured for 5 and 7 d, respectively . Histological sections revealed normal morphology of the ameloblasts that had differentiated in vitro into tall columnar cells with nuclei polarized to their distal ends facing the normal looking stratum intermedium cells. Tomes processes were evident in the secretory ends of the ameloblasts and they appeared as a picket fence in the enamel matrix . Whole mount staining of the explants after 7 d of culture with alizarin red showed that the extracellular enamel and dentin matrices, formed in vitro, had calcified . To visualize the fate of the cervical loop cells, they were labeled with the fluorescent DiI stain at the onset of culture, and the labeled cells were examined after 5 d of culture. Care was taken to microinject DiI to the central cells in the cervical loop, and the restriction of the dye was checked at the onset of culture (not shown). After 5 d of culture, fluorescent cells were seen to extend from the cervical loop to the differentiated ameloblasts . The original site of DiI injection could still be seen , but, interestingly, the highest intensity of fluorescence was observed at some distance incisally from the original site. These presumably represent daughter cells, which proliferated rapidly. Extending incisally from this site were labeled cells that had entered the stage of ameloblast differentiation. In conclusion, the DiI injection experiments showed that the cells in the cervical loop give rise to the differentiated dental epithelial cells, in particular the ameloblasts. The DiI labeling experiments suggested that the stem cells for differentiated dental epithelium reside in the cervical loop, but they did not rule out the possibility that there would be stem cells also among the more differentiated cells. To answer this question, we experimentally removed either the cervical loop epithelium or the more differentiated epithelial cells before culture of the apical ends of the incisors. Excision of the epithelium was done at the junction of the cervical loop and the inner enamel epithelium. When the cervical loop was removed (five explants) and the explants were cultured for 9 d, it was evident that the wounded end of the epithelium did not grow like in the controls . Instead, the epithelial cells underwent differentiation into ameloblasts. The cells produced enamel matrix, and after 9 d of culture, a sharp end of matrix production was seen and its calcification was visualized by alizarin red staining . These observations indicate that the inner enamel epithelial cells did not exhibit regenerative capacity, and that they were already committed to differentiation into enamel-producing ameloblasts. In the second set of experiments, the inner enamel epithelium and ameloblast zones were removed from the apical ends of the incisors and only the cervical loop epithelium was left intact (five explants). During the 7-d culture, the cervical loop was shown to grow remarkably and to give rise to inner enamel epithelium . After 7 d of culture, matrix production by new epithelium was seen and its calcification was visualized by alizarin red staining . Therefore, these results gave evidence that cells with significant growth potential reside exclusively in the cervical loop epithelium. The kinetics of epithelial cell division was examined by labeling the apical ends of the incisors with BrdU for various times and examining the BrdU incorporating cells in tissue sections after culture. When the explants were labeled for 3 h and immediately fixed, labeled cells were seen throughout the cervical loop epithelium extending to the inner enamel epithelium zone . Presumably both stem cells and transit-amplifying cells were labeled with BrdU during this period, as recently shown for skin keratinocytes in vitro . When the explants were cultured for 24 h in the presence of BrdU, labeled cells were observed in the same locations as in the 3-h cultures and in the ameloblast zone, indicating that the inner enamel epithelium, which had undergone their last divisions during the culture, had differentiated into ameloblasts . When the labeling period was extended to 72 h, BrdU incorporation was seen to extend further to the zone of differentiated, postmitotic ameloblasts . These results are in line with the observations on DiI-labeled explants, indicating that proliferating cells in the apical region of the incisor give rise to ameloblasts. To analyze kinetics of cell division, we cultured the explants for 7 d after they had been incubated for 3 h with BrdU. The rapidly proliferating cells were, thereby, given time to dilute the label. After the 7-d chase period, very few labeled cells were seen in the inner enamel epithelium area, indicating that the vast majority of cells had diluted the label by repeated divisions or differentiated into ameloblasts after their last divisions. BrdU-labeled ameloblasts were present near the inner enamel epithelium zone, and they were grouped into clusters. Hence, they appeared as clones of cells that presumably were descendants of individual inner enamel epithelial cells, which had undergone their last cell divisions and incorporated BrdU during the 3-h labeling period . Similarly, clusters of labeled cells were seen in the epithelial cells overlying the labeled ameloblasts, suggesting that they had differentiated from precursor cells that had been labeled during the first 3 h of culture. Interestingly, intensely labeled epithelial cells were observed within the cervical loop. They were located among the peripheral stellate reticulum cells in close vicinity to the basal epithelial cells that contact the basement membrane and dental mesenchyme at their basal end . We suggest that the labeled cells represent a population of slowly dividing stem cells in the cervical loop. They did not have neighboring labeled cells, suggesting that the stem cells had underwent asymmetric cell division. It is conceivable that one daughter cell had remained as a stem cell and the other daughter cell had generated a transit-amplifying population of cells, which are the progenitors of ameloblasts. As a potential stem cell marker, we used the antibody against the adenovirus receptor CVADR (previously known as CAR), which has been associated with stem cells in the brain . Strong expression was seen in the cervical loop epithelium and, at higher magnification, the expression appeared more intense in the stellate reticulum cells than in the basal epithelium. The transit-amplifying cells of the inner enamel epithelium and the differentiated ameloblasts did not express CVADR . Hence the expression of CVADR correlated well with the suggested stem cell distribution within the cervical loop epithelium. Because the Notch pathway has an important role in cell fate determination in invertebrates and it has been specifically implicated in the determination of stem cell fate in vertebrates, we performed a careful in situ hybridization analysis of the expression patterns of several genes in this pathway. Notch receptors and ligands previously have been shown to be associated with early tooth morphogenesis and cell differentiation , and we have recently shown that lunatic fringe , a homologue of a gene that is linked with Notch signaling in Drosophila , has an expression pattern during early tooth development suggesting developmental roles (Mustonen et al., in preparation). Expression of the receptors Notch1 , 2 , and 3 mRNA, the Notch ligand Serrate1 mRNA as well as lunatic fringe mRNA was localized in longitudinal sections through the 2-d mouse incisors. In the cervical loop, Notch1 was restricted to stellate reticulum cells and was most intense in the cells facing inner enamel epithelium. On the other hand, Notch2 was expressed in the outer enamel epithelium and the underlying stellate reticulum cells, and Notch3 was not detected in the cervical loop . Expression of Serrate1 was not detected in the cervical loop . Interestingly, lunatic fringe was expressed in the inner enamel epithelium starting from the cervical loop . This correlates closely with the distribution of BrdU incorporating cells in the enamel epithelium . Furthermore, the localization of the slowly dividing putative stem cells in our BrdU incorporation study in the peripheral stellate reticulum cells correlates with the boundary between lunatic fringe and Notch1 , suggesting that the maintenance and fate of the stem cells may be influenced by Notch signaling. The expression patterns of the Notch pathway genes were also correlated with terminal cell differentiation. In the zone of ameloblast differentiation, Notch1 was expressed in the stratum intermedium cells, whereas Notch2 was intensely expressed in the stellate reticulum cells . Notch3 was expressed in stellate reticulum cells and, in addition, intense expression was seen in blood vessels and in the subodontoblastic mesenchyme . In contrast to Notch genes that were absent from ameloblasts, the ligand Serrate1 was intensely expressed in terminally differentiated ameloblasts . These findings are in line with the recent data by Mitsiadis et al. 1998 and, thus, support a role for Notch signaling in the maintenance of the differentiated state of ameloblasts. Lunatic fringe was not expressed in the zone of differentiated ameloblasts , and its functions, therefore, appear to be confined to the regulation of their determination and differentiation. Earlier tissue recombination experiments have shown that dental mesenchyme controls the morphogenesis of dental epithelium including the continuous growth of the incisor epithelium (Jernvall, J., personal communication), and our recent studies on early tooth morphogenesis have implicated FGFs-3 and -10 as mesenchymal signals regulating the early morphogenesis of dental epithelium (Kettunen, P., N. Itoh, and I. Thesleff, manuscript submitted for publication). Hence, we analyzed the patterns of the expression of these signals as well as their receptors by in situ hybridization in the 2-d-old mouse incisors. Both Fgfs were intensely expressed in a restricted area of the dental mesenchyme in the apical end of the tooth. Fgf-10 expressing cells surrounded the whole cervical loop epithelium and extended to the zone underlying inner enamel epithelium . Fgf-3 showed a more restricted pattern of expression. It overlapped with Fgf-10 in the mesenchyme under the inner enamel epithelium, but was not expressed in the mesenchyme surrounding the cervical loop . Fgfr1b was intensely expressed in the cervical loop epithelium including the basal epithelial cells and stratum intermedium, but was less intense in the stellate reticulum cells , and Fgfr2b ( Kgfr ) showed a similar pattern in the cervical loop . Hence, the patterns of the expression of Fgf-3 and Fgf-10 and their receptors were in line with the suggestion that these FGFs may be mesenchymal signals regulating the development of cervical loop epithelium. The effects of FGF-10 on the growth of cervical loop epithelium were analyzed in explant cultures of apical ends of the 2-d-old mouse incisors. FGF-10 recombinant protein was applied with beads on the explants, and the control explants received beads soaked in BSA. Two beads were placed on each explant in contact with the cervical loop epithelium, one at its apical end and one at the inner enamel epithelium. When the development of the explants with FGF-10 and BSA beads was compared during 48 h of culture, a clear acceleration of the growth of the epithelium was observed by FGF-10 . The effects of the FGF-10 and BSA beads on cell proliferation were analyzed by culturing them on isolated cervical loop epithelium. The beads were placed on different areas of the dental epithelium, and the explants were cultured in the presence of BrdU for 6 h, fixed, and processed for immunohistological analysis of BrdU incorporation. A clear zone of BrdU incorporating cells was evident around the FGF-10 beads that were placed on the inner enamel epithelium and cervical loop epithelium , but not when placed on the zone of differentiated ameloblasts (not shown). No increase in BrdU incorporation was seen around the control BSA beads . The expression of lunatic fringe in the basal epithelial cells of the cervical loop suggested that it may be regulated by the underlying mesenchyme, and the close correlation of Fgf-10 and lunatic fringe expression in mesenchyme and epithelium was suggestive of a role for FGF-10 in the regulation of lunatic fringe. We examined these possibilities in vitro in isolated epithelial and mesenchymal tissues from the apical ends of 2-d-old mouse incisors. When the isolated epithelium and mesenchyme were placed in contact on the filter and cultured for 24 h, intense expression of lunatic fringe was seen in the basal epithelial cells of the cervical loop and in the inner enamel epithelium contacting the mesenchyme . No expression was seen in the epithelium of the differentiated zone contacting mesenchyme. When the epithelium was cultured alone, lunatic fringe expression was not seen, indicating that expression had been downregulated in the absence of mesenchyme. When FGF-10 releasing beads were placed on the cervical loop epithelium and inner enamel epithelium, they caused an upregulation of lunatic fringe expression in the contacting epithelium . When FGF-10 beads were placed on the ameloblast zone, the beads had no effect . Control BSA beads had no effect on expression. Hence, these experiments demonstrated that the maintenance of lunatic fringe in the cervical loop epithelium depends on mesenchymal signals, and that this effect is mimicked by FGF-10. The incisors of rodents erupt throughout the lifetime of the animals as the wear at the incisal edge is compensated for by renewal in the apex of the tooth located deep in the jawbone. The generation of the highly specialized dental tissues including the epithelially derived enamel and mesenchymal dentin involves proliferation of progenitor cells and their differentiation, matrix deposition, and subsequent mineralization. This process is seen as an increasing gradient of cell differentiation starting from the germinative apical area and extending towards incisal direction. Although it is generally assumed that there are stem cells in the apical end of the tooth, this problem has not been actively studied and the identity of the stem cells has remained enigmatic. We present evidence that epithelial stem cells possibly reside in a specific location in the cervical loop at the apex of the tooth, and that they produce a transit-amplifying cell population that gives rise to the differentiated dental epithelial cells, in particular, the enamel-producing ameloblasts. We also show data suggesting that Notch and FGF signaling are associated with specification of these stem cells and present a putative molecular mechanism, which may be applicable to the proliferation and determination of cell fate in stem cells also in other vertebrate tissues. Our in vitro model system for the culture of the apical end of the mouse incisor supported the growth and differentiation of the germinative cells of the tooth. Like in vivo, the cervical loop epithelium proliferated, differentiated into ameloblasts, and produced enamel matrix, which underwent calcification in organ culture. A special advantage of the system was that the stereomicroscopic examination of the advancing development was easy during culture. As the cervical loop and enamel-producing cells are located at the labial side of the incisor, and the explants were oriented so that the labial surface was on one side of the explant, the growth of the cervical loop epithelium and the gradient of differentiation and matrix deposition could be visualized almost two dimensionally. Using the in vitro culture of the incisor apex, we produced two lines of evidence indicating that the epithelial stem cells reside within the cervical loop epithelium. First, when cells in the center of the cervical loop were labeled with the fluorescent dye DiI and cultured in vitro, the dye was seen in the differentiating cells after 1 d and in the ameloblasts after 2 d of culture, indicating that the ameloblast lineage starts in the cervical loop. This was supported by the analysis of BrdU incorporation during extended time periods showing that the labeled cells extended progressively more incisally and occupied the zone of postmitotic mature ameloblasts. Second, when the cervical loop was removed mechanically from the apex of the tooth, the remaining epithelium could not regenerate the cervical loop in vitro. In these explants, all epithelial cells differentiated and produced mineralized matrix forming a stunt end with no indication of new cervical loop formation. In the opposite experiment where the differentiated epithelium was removed, the remaining cervical loop generated new epithelium differentiating into secretory ameloblasts, thus, indicating that it contains a pool of immature cells competent to regenerate the dental epithelium. We identified the slowly dividing putative stem cells in organ culture experiments by labeling the cells of the incisor apex with BrdU for 3 h, followed by a 7-d chase period. Labeled cells were detected in the cervical loop among the stellate reticulum cells in close vicinity to the basal epithelium. This location is consistent with the observed strong expression of the adenovirus receptor CVADR, a putative stem cell marker in brain cells . We suggest that the BrdU-labeled cells represent the daughter cells of stem cells that had divided during the 3-h labeling period and had remained in the stem cell pool and not divided thereafter. We suggest that the other daughter cell had entered the pathway of cell differentiation, and that the cell division therefore was asymmetric. This is supported by the finding that the BrdU-labeled cells did not have labeled neighboring cells (unlike the labeled ameloblasts, which appeared in clusters as they presumably had been generated by inner enamel epithelium after the last division). Asymmetric cell division of stem cells has been demonstrated in invertebrates , and in vertebrates there is evidence that neural stem cells undergo asymmetric cell division . The Notch signaling pathway is an evolutionarily conserved cell interaction mechanism that controls fundamental aspects of cell determination during development , and it has been suggested that Notch signaling may provide a microenvironment where the stem cells are maintained. Although Notch has been shown to function in Caenorhabditis elegans and in Drosophila in the maintenance of the potential of stem cells and their undifferentiated state, respectively , definitive roles for Notch have so far not been demonstrated in vertebrate stem cells . The expression of Notch1 in skin keratinocytes and in immature neural cells including neural stem cells has been suggested to be associated with stem cell functions . Furthermore, asymmetric distribution of Notch1 has been observed in immature neural cells before their cell division, suggesting a role in regulation of different cell fates in the daughter cells, i.e., whether they are maintained as stem cells or give rise to differentiated progeny . During tooth development, Notch signaling has been associated with the differentiation of dental epithelial and mesenchymal cells. Notch1 , -2 , and - 3 are downregulated in presumptive dental epithelium already during tooth initiation and, thereafter, these genes are not expressed in the ameloblast cell lineage, but they are intensely expressed in the other dental epithelial cells, suggesting roles in the determination and maintenance of cell differentiation of the ameloblast cell lineage . Therefore, these previous studies suggested to us that Notch signaling may be involved in the regulation of the stem cells in the continuously growing incisor. Interestingly, the slowly dividing potential stem cells in the cervical loop epithelium were located within the Notch1 and Notch2 expressing cells facing basal epithelial cells not expressing Notch , which is in line with the possibility that these Notch receptors regulate fate of the stem cells at this border. The possible role of Notch signaling in the regulation of the dental stem cells is further strengthened by the expression pattern of lunatic fringe , a gene encoding a secretory molecule resembling a glycosyltransferase that modulates Notch signaling in Drosophila . Three vertebrate fringe genes are known and of these lunatic fringe has a developmentally regulated expression pattern during early tooth morphogenesis (Mustonen, T., unpublished results). We localized lunatic fringe expression to the cervical loop epithelial cells neighboring Notch expressing cells. Such expression borders are characteristic to molecules in the Notch signaling pathway in Drosophila tissues, and have been shown to be sites of specific signaling activities . An important piece of evidence supporting a role for the Notch pathway in the regulation of stem cells is the finding that the lunatic fringe expressing basal epithelial cells that contact Notch1 expressing stem cells on one side are facing the dental mesenchyme at the opposite side, and that the mesenchyme regulated lunatic fringe expression. This is significant because the dental mesenchyme controls the epithelial morphogenesis in teeth, including the type of tooth formed, i.e., whether it develops to a molar with roots or to an incisor that grows continuously . Hence, our demonstration that the mesenchyme controls epithelial lunatic fringe expression is in line with the possibility that the mesenchymal control of the continuous epithelial growth involves modulation of the Notch pathway affecting fate determination of the stem cells. It is not clear whether the maintenance and differentiation of stem cells and their cell division are regulated independently by distinct signals or whether there are signals that couple the different processes. It is also not known how similar the molecular mechanisms are between stem cells in different tissues, although there is evidence suggesting that there may be significant similarities . Of the various regenerating tissues in vertebrates, the tooth resembles developmentally other derivatives of the epithelium such as skin, gut and, in particular, hair follicles. In all these tissues, the growth and differentiation of the regenerating epithelium is controlled by interactions with the underlying mesenchymal cells . The continuously growing incisor bears significant similarities histologically to the hair follicles as in both organs the regenerating epithelium encompasses a mesenchymal papilla, which is necessary for the growth of the epithelium. Stem cells have been located in skin keratinocytes and hair follicle epithelium , but possible mesenchymal factors involved in their regulation have not been examined. Interestingly, the stem cells in skin and hair follicle epithelium as well as in the gut are located among the basal epithelial cells and are associated with the basement membrane, whereas the putative dental stem cells were located in the stellate reticulum in close vicinity to the suprabasal aspect of the basal epithelial cells and, hence, did not contact the basement membrane. Our localization of the expression of Fgf-10 and Fgf-3 , two signal molecules that we have recently identified as proliferative signals from mesenchyme to epithelium during early tooth morphogenesis (Kettunen, P., N. Itoh, and I. Thesleff, manuscript submitted for publication), suggested that they might function in the regulation of the continuous growth of the incisor epithelium. The expression of both Fgf s was restricted to the mesenchyme underlying the rapidly proliferating inner enamel epithelium. In addition, Fgf-10 expression extended more apically and surrounded the entire cervical loop. Furthermore, the FGF receptors that are known to bind these particular FGFs, namely the IIIb splice forms of FGFR1 and FGFR2 were expressed in the cervical loop epithelium, Fgfr1b mRNA being specifically intense in the basal epithelial cells facing the mesenchyme. These findings supported roles for FGF-3 and FGF-10 as signals mediating the effects of mesenchyme on epithelium, and our bead implantation experiments showed that FGF-10, in fact, stimulated proliferation in isolated cervical loop epithelium in vitro. It is apparent that they stimulated cell division in the transit-amplifying cell population in the cervical loop, and we also suggest that they stimulated proliferation of the stem cells . Interestingly, FGF-10 also stimulated lunatic fringe expression in the isolated cervical loop epithelium and, thereby, mimicked the effect of the dental mesenchyme in recombination cultures. We have recently shown that FGF-10 has a similar stimulatory effect on dental epithelial lunatic fringe expression during early budding morphogenesis of mouse teeth (Mustonen, T., unpublished results). As discussed above, the regulation of lunatic fringe expression likely affects Notch signaling and, thereby, perhaps the cell fate of the stem cells in the cervical loop . We propose that FGFs may be regulatory signals for stem cells in the tooth and perhaps also in other tissues. FGF stimulates cell division in neural stem cells, whereas removal of FGF permits their differentiation to neurons . The expression of Fgf-3 and Fgf-10 in the dental mesenchyme was downregulated exactly in the region where lunatic fringe was downregulated in the inner enamel epithelium differentiating into ameloblasts, and it is possible that this arrest in FGF signaling contributes to terminal differentiation of the cells. In conclusion, we suggest that the Notch 1 expressing stellate reticulum cells in the cervical loop of the continuously erupting mouse incisor are stem cells that have the capacity to self-renew and to generate progeny that are fated to differentiate into the various cell types of the dental epithelium, most importantly the enamel-producing ameloblasts. Based on the localization of slowly dividing cells among the peripheral stellate reticulum cells, the expression of genes in the Notch and FGF pathways, and the findings that FGF-10 stimulates both cell proliferation and expression of lunatic fringe in the cervical loop epithelium, we propose a model for the molecular mechanism of the putative dental stem cell lineage . We propose that Notch receptors influence the maintenance of the stem cell state, and that the dental mesenchyme controls their proliferation and differentiation by FGF signals directly stimulating stem cell division and regulating the Notch pathway via stimulation of lunatic fringe expression. | Study | biomedical | en | 0.999997 |
10508860 | cDNA encoding full-length PS1 was amplified from human hippocampal mRNA (Clontech) by reverse transcriptase PCR using primers F, 5′-AAAGAATTCATGACAGAGTTACCTGCACCGT-3′ and R, 5′-AAACTCGAGCCATGGGATT- CTAACCGC-3′ , and subcloned between EcoRI and XhoI sites of pEG202–LexA fusion plasmid. After confirming that cotransfection of the pEGPS1 and pJG4-5 plasmids does not show nonspecific binding, ∼5 × 10 5 clones were screened from a human embryonic brain cDNA library (provided by Dr. Roger Brent, Harvard Medical School, Boston, MA). A second screening was performed with both SG-HWUX-gal and SG-HWUL plates. Positive clones were subcloned into pBluescript KS + (Stratagene) and sequenced by using an automatic sequencer (Applied Biosystems, Inc.). 100 mg of each cerebral cortex tissue was suspended in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.8, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin), incubated at 4°C for 10 min, and disrupted by repeated aspiration through a 21-gauge needle. Cellular debris was removed by centrifugation at 10,000 g for 10 min. Aliquots of cell lysates were incubated with various antibodies for 1 h at 4°C, then precipitated with protein G–agarose (Oncogene Science). Anti–NH 2 -terminal antibody (αN) and anti-loop antibody (αL) were used finally at 1:200 dilution. Anti-QM (C-17) polyclonal antibody (Santa Cruz Biotechnology) was used at 1:1,000 dilution. αN antibody is specific for amino acids 21–80 of human PS1 . αL is a polyclonal antiserum that reacts with epitopes in the hydrophilic loop domain (amino acids 263–407) of human and mouse PS1 . The beads were washed extensively with Radio-Immuno-Protein Assay buffer, then separated by 12% SDS-PAGE, blotted to Hybond-ECL membrane (Amersham Pharmacia Biotech), incubated with anti-QM polyclonal antibody (Santa Cruz Biotechnology), then detected with ECL Western blotting analysis system (Amersham Pharmacia Biotech). Expression vectors of various forms of PS1 as well as glutathione S -transferase (GST)-QM fusion proteins were constructed as follows. Full-length PS1 cDNA was amplified with primers F182 (5′-AAACTCGAGTCTATACAGTTGCTCCAATGAC, nt 232–254) and R182 from human hippocampal mRNA, and subcloned between XhoI and XbaI sites of pCIneo mammalian expression vector (Promega). The structure and sequence were confirmed by DNA sequencing and restriction site analyses. For pCImPS1 carrying a mutation Met→Leu at codon 146 associated with early-onset familial AD, site-directed mutagenesis was performed with Transformer Site-directed Mutagenesis kit (Clontech) according to the commercial protocol. For vectors expressing GST fusion proteins in Escherichia coli , QM cDNA was subcloned between BamHI and EcoRI pGEX3X (Amersham Pharmacia Biotech) by using PCR with synthetic primers to adjust the reading frame. Fusion proteins linked to deleted QM was constructed similarly by using PCR. PS1 protein was synthesized and radiolabeled with [ 35 S]methionine (NEN) by in vitro transcription and translation with TNT T7/T3–coupled reticulocyte lysate system (Promega). Interaction between PS1 and GST-QM proteins was performed according to the reported method . 5 mm 3 of tissue was obtained from the cerebral cortex of a 37-yr-old woman 24 h after her death by loss of blood, and fixed with 4% paraformaldehyde. Sections for electronmicroscopic immunocytochemistry were postfixed in 1% osmium tetroxide, stained with 2% uranyl acetate, dehydrated using graded alcohol and propylene oxide, and embedded in Eponate 12 resin. 40-μm sections were stained by rat mAb against PS1 21–80 at 1:100–500 dilution, and anti-QM polyclonal rabbit antibody (Santa Cruz Biotechnology) at 1:1,000 dilution, simultaneously. 10 nm gold-conjugated anti–rat Ig and 5 nm gold-conjugated anti–rabbit secondary antibodies were used to localize primary antibodies. Control experiments were performed without primary antibodies. For the analyses with diseased brains, sections of formaldehyde-fixed and paraffin-embedded cortex were deparaffinized and used for the incubation with antibodies. For immunohistochemistry of the mouse brain, 40-μm sections were stained by anti-QM polyclonal antibody (Santa Cruz Biotechnology) then visualized with 0.05% 3,3′-DAB tetrahydrochloride and 0.01% H 2 O 2 in 50 mM Tris, pH 7.6, as described previously . Transfection was performed as described previously . 10 μg of reporter plasmid and 10–20 μg of effector plasmids were used. 10–20 μg pBluescript KS + (Stratagene) was added to equilibrate the total amount of plasmids for transfection. Construction of effector and reporter plasmids was reported previously . Efficiency of tranfection was verified by pCH110 (Amersham Pharmacia Biotech), a eukaryotic vector containing the simian virus early promoter and E. coli –β-galactosidase (LacZ) structural gene. Each experiment was repeated at least four times and variation of transfection efficiency was <20%. Expression vectors of fluorescent protein–QM fusion proteins were constructed by inserting various forms of QM cDNAs into pEGFPN1 (Clontech). Full-length QM cDNA was amplified by RT-PCR from human amygdala mRNA with the primers QMF (AACGAATTCCCATGGGCCGCCGCCCCGCCCGTT) and QMR (AATGGATCCGTGAGTGCAGGGCCCGCCA), and subcloned between EcoRI and BamHI sites of pEGFPN1. The effect of PS1 on c-Jun NH 2 -terminal kinase (JNK) activities was analyzed by transfecting 6 μg of T7-tagged JNK1 with 10 μg of pCIPS1 or pBS-KS as control into F9 cells. JNK1 protein was recovered by immunoprecipitation with mouse mAb against T7 epitope (Invitrogen). After the Sepharose resin was washed five times with lysis buffer containing 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM β-glycerophosphate, 0.1 mM Na 3 VO 4 , 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10% glycerol, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, the proteins were recovered with SDS sample buffer and analyzed by Western blotting with anti–T7-Tag antibodies. To make stable cells expressing antisense c- jun , pCIneo (Promega) containing the full-length c- jun cDNA at the reverse orientation was constructed and transfected into F9 cells. They were selected in α-medium (Sigma Chemical Co.) with 300 μg/ml G418 for 2 wk. Stable cells expressing normal or mutant PS1 were similarly made by transfecting pCIPS1 and pCImPS1. 1 × 10 5 F9 or stable cells were cultured in α-medium: 10% FBS with 1 μM all-trans retinoic acid (Sigma Chemical Co.) for 48 h. All the cells were collected and their genomic DNA were extracted and separated on 3% Nusieve-agarose gel (FMC BioProducts). For analysis of the effect of deletion constructs of QM on apoptosis, 1 × 10 5 F9PS1 cells were transiently transfected with 20 μg of each expression vector described below using SuperFect (Qiagen) in a 10-cm tissue culture dish. 24 h after transfection, cells were treated with 1 μM all-trans retinoic acid (Sigma) for an additional 48 h. All the cells were collected and the percentage of cell death was estimated by trypan blue dye exclusion. QM/Jif-1 cDNA fragments were amplified with RT-PCR from human hippocampal mRNA (Stratagene). The primers used were F1 , R1 (AAAGAATTCAATTCGGGCAGCCTCCA, nt 251–235), F2 (AAAGAATTCCGCATCAACAAGATGT, nt 333–348), R2 (AAATCTAGAAGCCCTCATGAGTGCA, nt 691–676), and R3 (AAATCTAGAACATC-TTGTTGATGCG, nt 348–333). Different portions of QM cDNA were amplified with F1 and R3 for Δ1 or with F1 and R1 for Δ2, digested with XhoI and XbaI or with XhoI and EcoRI, and subcloned into corresponding sites of pCIneo vector (Promega), respectively. For Δ3, two cDNA fragments amplified with F1/R1 or F2/R2 were digested with XhoI/EcoRI or with EcoRI/XbaI, respectively, then subcloned between XhoI and XbaI sites of pCIneo. We have investigated the function of PS1 by isolating the molecules that interact with PS1. We screened a human embryonic brain library using the yeast two-hybrid system. After double second screens using leucine-deficient plates and X-gal plates, we finally judged six clones showing strong interaction to be positive. In our experience with the two-hybrid system, this number of positive clones was small compared with other baits. Among them, we found a clone identical to QM/Jif-1. This molecule was isolated originally as a putative Wilms's tumor suppressor gene , then described as a transcription factor that interacts with c-Jun and inhibits its transcriptional activation . Recently, it was shown to be identical to a ribosomal protein, L10 , which was therefore suggested to be a ribosomal protein possessing multiple extraribosomal functions . This clone (PS309-4) lacked an NH 2 -terminal portion of 43 amino acids and encoded a variant Ser202Asn . QM/Jif-1 does not possess a leucine zipper but might compose a C2H2-type zinc finger . Retransformation of EGY48 yeast cells by pEGPS1 and pJGPS309-4 plasmids showed high β-galactosidase activities in independent yeast colonies . To determine the binding domain of PS1 molecule to QM/Jif-1, we performed deletion analysis of PS1 by two-hybrid assay. cDNAs corresponding to various regions of the PS1 molecule were subcloned into the pEG vector and cotransfected with pJG309-4 into yeast cells. Interestingly, any partial sequence of PS1 did not bind strongly to QM/Jif-1. Instead, full-length PS1 showed a strong interaction with QM/Jif-1 . This finding corresponds well to the results of Western blot analysis using human brains described below. To verify the interaction between QM and PS1 in vivo, we performed immunoprecipitation assay with human brains, including those of familial and nonfamilial AD patients. Approximately 50 kD full-length PS1 was detected in the precipitates by αN and by αL from normal, disease control (amyotrophic lateral sclerosis), nonfamilial AD, and PS1-linked AD brains , whereas we could not observe clear bands corresponding to the cleaved PS1 fragments. This result in human brain, together with the results from two-hybrid deletion analyses , suggest that multiple regions in the full-length structure of PS1 are necessary for tight interaction with QM/Jif-1. This idea might have some relationship to the recent finding that NH 2 - and COOH-terminal PS1 fragments reassociate and form a stable complex . Interestingly, the band was visible but very weak in lanes 6 and 7 loaded with samples from PS1-linked AD patients . Compared with the PS1 bands in Western blot analysis using the same brain samples , it is not due to difference of the PS1 protein amounts among brain samples. Instead, it could be due to the difference of residual neurons where QM and PS1 may interact, among samples, or due to the difference in affinity of QM to normal and mutant PS1. In the reverse immunoprecipitation assay, we detected QM in precipitates by αN as well as by αL , reconfirming the interaction between PS1 and QM/Jif1 in vivo. We performed immunoprecipitation with nonimmune sera and with several nonspecific antisera using the same brain samples, but did not find QM or PS1 in the precipitates (data not shown). To observe the interaction between QM and PS1 in the brain morphologically, we performed immunohistochemical analyses. As QM expression has not been reported previously, we confirmed that the QM message is widely expressed in the brain by Northern blot analysis . At the light microscopic level, anti-QM polyclonal antibody stained the cytoplasm of neurons in the mouse cerebral cortex . To further characterize subcellular localization of the QM protein, we observed the mouse brain sample with electronmicroscopy and found that regions very close to tubular membrane structures, which possessed features of smooth endoplasmic reticulum, were predominantly stained . This subcellular localization of QM was exactly like that of PS1 reported to date . Next, we asked whether the QM and PS1 proteins are colocalized in the human brain neurons. Immunohistochemical analysis was performed with secondary antibodies conjugated to different sizes of gold particles and with the brain of a 37-yr-old woman who died due to loss of blood. We observed that the 5-nm grain of PS1 and 10-nm grain of QM were located very close to each other at the edge of smooth membrane structures in the cytoplasm of cortical neurons . Most of those structures seemed to be smooth endoplasmic reticulum, whereas some of them possessed the features of Golgi apparatus (data not shown). Fewer grains were observed also in the nucleus . Although the immunoreactivity was further reduced in the formaldehyde-fixed and paraffin-embedded tissues of nonfamilial or PS1-linked AD brains, we repeatedly found that these two grains were colocalized at membrane structure in the cytoplasm . Next, we investigated the biological significance of the interaction between QM/Jif-1 and PS1. Jif-1 was isolated as a protein binding to c-Jun from a cDNA library screened with biotinylated c-Jun . Work by Monteclaro and Vogt on the function of Jif-1 has shown that GST–QM/Jif-1 fusion proteins do not bind to 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE) in gel mobility shift assays, but prevent the binding of a c-Jun homodimer to the TRE . Moreover, the c-Jun/c-Fos heterodimer can override this inhibition by Jif-1. Through this binding inhibition, Jif-1 inhibits gene transactivation by c-Jun. QM/Jif-1 is extremely conserved during evolution from yeast to mammal , suggesting that this factor is essential for basic cellular functions. From these observations, we supposed that expression of PS1 might affect the function of c-Jun through interaction with QM. To test this hypothesis, we performed cotransfection chloramphenicol acetyltransferase (CAT) assays with c- jun or c- fos expression vectors as the first effector plasmid, normal or mutant PS1 expression vectors as the second effector plasmid , and human collagenase (−517/−42) TKCAT vector containing TRE as reporter plasmid. We used F9 embryonic carcinoma cells in the assays because they possess almost no activated protein (AP)-1 activity , but express enough of the QM protein (data not shown). It was difficult to predict what kind of results would emerge with the addition of the PS1 expression vector, because the binding might keep QM in the endoplasmic reticulum where PS1 is most abundant , or it might assist the transport of QM to the nucleus where PS1 immunoreactivity is also detected . It could be that cotransfection might not affect CAT activities due to a nonfunctional binding between the two molecules. Among these possibilities, we obtained the simplest and clearest outcome. c- jun or c- jun/ c- fos transactivated the CAT gene expression as expected. The transactivation by c-Jun homodimer was suppressed by normal PS1 as well as mutant PS1, whereas the transactivation by c-Jun/c-Fos heterodimer was not affected by adding PS1 expression vectors . This suppressive effect was considered to be specific, because overexpression of a multipass transmembrane protein, glucose transporter 1 (GLUT1), which is known to move from the endoplasmic reticulum to the Golgi apparatus, did not suppress CAT activity . The weak transactivation by c-Fos, which might have been induced with a weak endogenous c-Jun activity, was not influenced by either normal or mutant PS1. These observations corresponded very well with the interacting behavior of QM to AP-1 molecules reported previously . The suppression by mutant PS1 seemed weaker than that by normal PS1 . To examine whether the effect of PS1 is mediated by TRE, we changed the reporter plasmid to those that contained only TRE . As expected, PS1 reproduced suppression of the c-Jun–mediated transactivation in collagenase 1× TRE CAT and metallothionein IIa 1× TRE CAT . These results supported our hypothesis that PS1 affects gene regulation by c- jun through TRE. Interestingly, by using 1× TRE CAT plasmids, we observed more clearly that the suppression of c-Jun–induced transactivation was weaker in mutant PS1 than in normal PS1 . Second, we tested whether PS1 affects transcriptional regulation by the other c- jun family members forming an AP-1 complex. Expression of junB and junD enhanced transcription from collagenase 1× TRE CAT reporter plasmid . Transactivation by junD was clearly suppressed by expression of normal and mutant PS1, whereas transactivation by junB was not affected . In this case, suppression of junD -mediated transactivation by normal PS1 was not remarkably different from that by mutant PS1 , in contrast to our observations with c- jun –mediated transactivation . In the CAT assays described above, we confirmed that expression of the c-Jun protein was not influenced by cotransfecting PS1, and that expression of normal and mutant PS1 proteins was equivalent . We summarized results from all the CAT assays described above with a histogram showing mean fold transactivations . From the data described above, we hypothesized that PS1 somehow promotes translocation of the QM protein from the cytoplasm to the nucleus, inhibits the binding of c-Jun homodimer to TRE, and thereby suppresses transactivation by c- jun . We used a fluorescent protein (enhanced green fluorescent protein [EGFP]) fusion reporter plasmid to observe how intracellular transport of the QM protein is modulated by PS1, and observed that translocation of the fusion protein to the nucleus is actually accelerated by cotransfecting PS1 . On the other hand, coexpression of mutant (Met146Leu) PS1 did not remarkably promote the nuclear translocation. It is interesting to note that the effects of PS1 on transcription and on protein transport were remarkable although the expression level of PS1 was not as high as that of PS1 , or as the endogenous expression level of QM/Jif-1 (data not shown). Considered with the function of PS1 assisting the nuclear transport of QM, transfected PS1 molecule might be recycled efficiently in cells, leading to the remarkable effect on c-Jun function via QM protein translocated to and accumulated in the nucleus. Next, we performed gel mobility shift assays by using nuclear extracts prepared from F9 cells expressing AP-1 transcription factors with or without PS1. We found that PS1 suppresses the binding of c-Jun homodimer to TRE and very weakly suppresses that of JunD homodimer but not that of c-Jun/c-Fos heterodimer or JunB homodimer . Furthermore, normal PS1 suppressed the binding to TRE more efficiently than mutant PS1 . These findings are consistent with the results in CAT assay and support the idea that the nuclear translocation of QM/Jif-1 is promoted by normal PS1 thereby inhibiting the binding of c-Jun homodimer to TRE. We tested another possibility that PS1 inhibits JNK and thereby suppresses transactivation by c- jun . T7-Tag-JNK1 expression vector was cotransfected with PS1, immunoprecipitated, and the JNK activity was tested with c-Jun produced in bacteria as a substrate. In this assay, we observed no change of JNK activity (data not shown). This finding indicates that PS1 represses the function of c- jun predominantly through the transport of QM/Jif-1, but not through c-Jun phosphorylation by JNK, although further analyses are necessary to determine whether or not JNK is partially involved in the suppression by PS1. c- jun had been characterized as a protooncogene promoting cellular proliferation, whereas recent data indicate that c- jun is involved in some types of apoptosis. Expression of c- jun dominant negative mutants protects sympathetic neurons against cell death induced by NGF withdrawal, and the overexpression of c- jun itself triggers apoptosis in sympathetic neurons . Transfection of a recombinant fusion protein chimera of c-Jun and hormone binding domain of estrogen receptor induces apoptosis in NIH 3T3 cells with β-estradiol added to the culture medium . Furthermore, induction of c- fos is noted as an early event of programmed cell death , and c- fos is implicated in light-induced retinal degeneration . Interestingly, the time course of the protein expression in neuronal apoptosis induced by withdrawal of trophic factor was different. c- jun is upregulated at first and c- fos follows it; expression of junD does not change . These findings indicated that AP-1 plays important roles in cell death, although each member of the AP-1 complex might play a different role. Considering these previous data, we examined whether PS1 affects c- jun– mediated apoptosis. We used F9 cells for this analysis, since retinoic acid treatment induces apoptosis and upregulation of the c- jun expression . Suppression of c- jun by the antisense transcript expression reduced the retinoic acid–induced apoptosis , indicating that c- jun mediates this type of cell death. Thus, we made stable transformants expressing >10 times the amount of PS1 protein than parental F9 cells , and observed the effect of PS1 on retinoic acid–induced apoptosis. Apoptotic cells were detected by nick end labeling assay with terminal transferase and by trypan blue dye exclusion assay then analyzed statistically . Normal PS1 significantly suppressed the percentage of apoptotic cells, whereas mutant PS1 suppressed it rather weakly. These results were consistently observed in the stable cell lines with high expression ( n = 3). In addition, we confirmed these results by DNA fragmentation in cellular nuclei . The weak antiapoptotic effect of mutant PS1 corresponds well to its weak suppressive effect on c- jun –induced transactivation in CAT assays . At the end of this study, we investigated the role of the putative zinc finger domain (zif) of QM/Jif-1 in interaction with PS1, in transcriptional regulation, and in apoptosis. First, we made various deletion constructs of QM/Jif-1 and tested their binding to PS1 by pull-down assay. Normal or mutant (Met146Leu) PS1 radiolabeled with [ 35 S]methionine by in vitro transcription/translation were interacted with GST fusion proteins of QM in vitro and pulled down by glutathione Sepharose 4B. Full-length QM interacted with normal PS1, whereas deletion constructs lacking zif did not bind to PS1 , indicating that this region is essential for interaction. Mutant PS1 binds to the GST-QM fusion proteins similarly. However, the ratio between the input and the pulled-down amounts was lower in mutant PS1 (60%) than in normal PS1 (95%). Next, we tested whether deletion of zif affects transcriptional regulation. As QM/Jif-1 is abundantly expressed in all the cell lines, we designed a dominant negative experiment. We selected F9PS1 cells in which transfected QM is translocated to the nucleus. Eukaryotic expression vectors containing the deletion constructs of QM were cotransfected with c- jun , and QM expression vectors into F9PS1 cells. Transfection of full-length QM suppressed c- jun– induced transactivation . Δ1 antagonized this suppression by QM. The CAT activities in cotransfection of Δ1 was higher than those in c- jun –induced transactivation , suggesting that Δ1 antagonized endogenous QM in addition to transfected QM. This dominant negative effect was not observed in the other constructs without zif that cannot interact with PS1 . Therefore, Δ1 probably inhibits binding of QM to PS1 in a competitive manner and represses the function of QM, since Δ1 transported to the nucleus does not have a suppressive effect on c-Jun. Consistently, translocation to the nucleus of the GFP fusion protein was observed in Δ1 but not in the other deletion constructs lacking zif . Without transfecting c- jun expression vector, transactivation by Δ1 itself was not observed in F9PS1 cells, which do not express c- jun (data not shown). Finally, we tested the role of zif in the c- jun –associated apoptosis. F9PS1 cells were transfected with the vectors expressing deleted QM/Jif-1. Transfection of Δ1 increased the percentage of cell death induced by retinoic acid treatment, whereas the other constructs did not affect the apoptosis . This result again showed the dominant negative effect of Δ1 on apoptosis. Collectively, it was concluded that zif is essential for the interaction of QM with PS1 and for the effects of QM on c-Jun derived from the interaction. This study showed that PS1 binds to a negative cofactor of c-Jun, QM/Jif-1, and that PS1 regulates the functions of c- jun in transcription and cell death. Promotion of the nuclear translocation of QM/Jif-1 by PS1 seems to be the underlying mechanism that connects the first and second conclusions. A specific point in our results is that QM/Jif-1 binds to a full-length PS1. It was reported that most PS1 molecules are cleaved into two fragments which reassociate to form a heterodimer . However, Western blot analysis in this study and in the previous reports shows that full-length PS1 also exists in the brain in vivo, although the function of this form has been unclear. Our findings suggest that the full-length form also possesses a functional role related to cell viability. So far, more than five molecules, including APP , calsenilin , β-catenin , filamin , and tau , were isolated as binding proteins to PS1. The reason why many proteins bind to PS1 is not known. However, it might be explained by supposing that PS1 functions as a kind of intracellular transporter with a relatively low specificity. The multipass transmembrane structure of PS1, especially of the full-length form, does not contradict this hypothesis, and this kind of activity was actually shown in the case of amyloid protein transport . Our observation on the nuclear translocation of QM/Jif-1 is also compatible with this idea. Activation of c- jun , especially that mediated by JNK, has been suggested to participate in various types of cell death, including TNF- or Fas-induced apoptosis . Numerous reports keep emerging on this apoptotic cascade. JNK was reported to enhance apoptosis induced by a breast cancer susceptibility gene, BRCA1, through activation of GADD45 . The JNK-dependent apoptotic pathway has been implicated in the morphogenesis of Drosophila wing . Furthermore, kainic acid–induced apoptosis in the hippocampus was prevented in the mice lacking the Jnk3 gene , indicating that JNK3 is essential for this type of apoptosis. The role of JNK in kainic acid–induced apoptosis was reconfirmed in the knock-in mice carrying mutations at JNK-phosphorylation sites of the c- jun gene . In the AD brain, the increase of c-Jun immunoreactivity was observed and strongly correlated with pathological changes . Mutations in PS1 might influence such cascades at the final step of c-Jun and thereby affect apoptosis. Since our results showed that the inhibitory effect of mutant PS1 on c- jun –mediated apoptosis was weaker than that of normal PS1, mutation in PS1 could be a promoting factor in c- jun –mediated apoptosis. However, there remain debates on the role of c- jun and JNK in the in vivo apoptosis. Although double gene disruption of Jnk1 and Jnk2 leads to severe dysregulation of apoptosis during development at specific regions in the brain, Jnk3 does not affect this apoptosis . c- jun was reported to be dispensable in developmental cell death and axogenesis of the retina . These discrepancies might suggest that the c-Jun/JNK cascade plays different roles in the developmental neuronal death and in the death of the mature neuron. Therefore, our results on the cascade from PS1 to c-Jun should be applied carefully to different types of cell death. The functions of normal and mutant PS1 in cell death reported so far are still difficult to combine . Normal PS1 had been reported to promote cell death basically via affecting the Aβ metabolism. Meanwhile, accumulating data from independent laboratories indicated that PS1 also modifies various cell death cascades. It suppresses apoptosis associated with p53 and p21 WAF1 activation and promotes the antiapoptotic function of β-catenin . In these cases, normal PS1 suppresses cell death, whereas mutations in PS1 abort this function. Suppression of c-Jun homodimer by PS1 might lead to a different outcome in signaling pathways other than the c- jun –mediated apoptosis examined in this study. For example, neurotrophins bind to tyrosine kinase–type membrane receptors and activate c-Jun through the mitogen-activated protein (MAP) kinase pathway. Many other trophic factors, including fibroblast (FGF), epidermal (EGF), platelet-derived (PDGF), and hepatocyte growth factors (HGF), induce similar signaling cascades and promote cell survival. In such a condition where the activation of c-Jun mainly contributes to survival, PS1 might promote apoptosis. Like neurotrophins which transduce survival and death signalings via the high affinity and low affinity receptors, respectively , PS1 might transduce both types of signalings via different molecules binding to PS1. In other words, our results might have proposed another type of explanation for why PS1 induces diverse effects on the cell fate. The PS1/QM/c-Jun cascade leads to the opposite outcome, survival or death, depending on the final effect of c-Jun, which is influenced by cellular conditions. Although it is not yet known which factors define the attitude of c-Jun in cells, other types of signaling cascades induced by PS1, including calcium release from endoplasmic reticulum and G protein signaling , might cross-talk and change the responding manner of cells to the c-Jun activation. In the AD brains, which usually degenerate for more than several years, it is possible that both c- jun –associated and c- jun –nonassociated neuronal deaths occur in various situations. Although apoptosis itself is a rather rapid process in a single neuron, it occurs in numerous neurons of the brain at random and so the mass degeneration of the brain proceeds gradually. Therefore, we are speculating that c- jun –mediated apoptosis influenced by PS1 might be an additive factor to modify neuronal fate in the AD brain, and could function in parallel with the amyloid deposition promoted by PS mutations as well as the pathogenic mechanisms mediated by other binding proteins. | Study | biomedical | en | 0.999996 |
10508861 | A peptide predicted to be unique among C. elegans proteins and corresponding to the 19 amino-terminal residues from DHC-1 plus a cysteine (MDSGNESSIIZPPNLKC) was synthesized, conjugated to maleimide-activated keyhole limpet hemocyanin (Pierce Chemical Co.), mixed with titer max adjuvant (Boehringer Ingelheim Ltd.), and injected into rabbits at the European Molecular Biology Laboratory animal house according to standard procedures. The third bleed was affinity-purified against a column of sulfolink coupling gel (Pierce Chemical Co.) coupled to the peptide. Anti–DHC-1 antibodies were eluted with 100 mM glycine, pH 2.5, dialyzed against PBS, and concentrated to 0.8 mg/ml in 50% glycerol. Worms from mixed developmental stages were floated off four 9-cm petri dishes with H 2 O, spun for 2 min at 2,000 rpm in a tabletop clinical centrifuge, and resuspended for a wash in 30 ml H 2 O. Worms were spun as above, resuspended in 1.5 ml H 2 O, transferred to an Eppendorf tube, and spun for 2 min in a microfuge, yielding a pellet of ∼100 μl. 200-μl modified 2× loading buffer (M2LB: 100 mM Tris, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 1 mM PMSF, 10 μg/μl of each leupeptin, pepstatin, and chemostatin) was added to the pellet. The extract was vortexed for 30 s, boiled for 2 min, supplemented with 100 μl M2LB, vortexed for 30 s, boiled for 1 min, and snap-frozen in liquid nitrogen. Cytoplasmic extracts of unfertilized Xenopus eggs arrested in metaphase of meiosis II were prepared according to standard procedures . 20-μl C. elegans extract or 1-μl Xenopus extract was loaded per lane on a 6% SDS–acrylamide gel. Proteins were transferred onto nitrocellulose in SDS gel running buffer containing 10% methanol. After blocking, the filter was incubated for 90 min at room temperature with primary antibodies (1:200 rabbit anti–DHC-1 or 1:1,000 mouse anti- Xenopus dynein heavy chain, a gift from Sigrid Reinsch, NASA Ames Research Center, Moffet Field, CA). Signal detection was performed with standard enhanced chemiluminescence kit components (Amersham Life Science, Inc.). dhc-1 (RNAi) embryos gave rise to a fully penetrant phenotype recognizable by staining with antitubulin antibodies (see Results). Therefore, levels of anti–DHC-1 reactivity were analyzed in wild-type and dhc-1 (RNAi) embryos 30 h after injection and processed on the same slide to eliminate potential slide-specific differences in staining intensities. Early embryos (as judged by the DNA stain, <30 nuclei in wild type, and the approximate equivalent in dhc-1 (RNAi) ) were examined for antitubulin reactivity. Embryos with a strong antitubulin signal were deemed to be properly fixed and stained, and were retained for subsequent analysis of anti–DHC-1 reactivity. Anti–DHC-1 reactivity was imaged with a 4912 Cohu CCD camera set on manual. Mean pixel intensity was determined for each embryo using Adobe Photoshop 4.0, and expressed as a percentage of the average staining intensity of wild-type embryos on each slide. 5–8 of each wild-type and dhc-1 (RNAi) embryos were examined per slide. Double-stranded (ds) RNA corresponding to the dynein heavy chain gene dhc-1 (T21E12.4) was generated in the following manner. A λZAPII phage containing a 1.3-kb cDNA insert (yk161f11) was obtained from Yuji Kohara (National Institute of Genetics, Mishima, Japan). The insert was PCR-amplified from ∼2.4 × 10 4 phage particles using primers corresponding to vector sequences flanking the insert and that contain consensus sequences for T3 (forward primer) or T7 (reverse primer) RNA polymerases. The PCR product was purified using the QIAquick PCR purification kit (Qiagen). About 0.5 μg was used as a template in 20 μl T3 and T7 RNA polymerase reactions to generate sense and antisense single-stranded (ss) RNAs (RiboMAX™; Promega Corp.). After treatment for 15 min at 37°C with 0.5 U RQ1 DNAse, the RNAs were extracted with phenol/chloroform and resuspended in 20 μl H 2 O. An aliquot was run next to RNA standards on a 1% TBE agarose gel to estimate the quality and quantity of RNA generated. Typically, 20–50 μg (1–2.5 μg/μl) of RNA was produced per reaction. To generate dsRNA, equal volumes of sense and antisense ssRNAs were mixed with 1 vol of 3× injection buffer , incubated 10 min at 68°C and 30 min at 37°C. The resulting dsRNA was aliquoted, snap-frozen in liquid nitrogen, and stored at −70°C. The same batch of yk161f11 dsRNA was used to quantify all the phenotypic manifestations reported in the text. However, other batches of yk161f11 dsRNA gave identical phenotypes, as did dhc-1 dsRNAs generated from four other sources: (1) a 1.6-kb cDNA insert in λZAPII from Yuji Kohara, yk166g8; (2) T21E12.4-5, a PCR fragment corresponding to exon 2 and part of exon 3 of dhc-1 , positions 358–1803 in cosmid T21E12; (3) T21E12.4-M, a PCR fragment corresponding to the end of exon 7 and most of exon 8, positions 6636–7844 in T21E12; and (4) T21E12.4-3, a PCR fragment corresponding to exons 13–15, positions 13420–14854 in T21E12 dsRNAs 2–4 were generated by Alan Coulson at the Sanger Center. dsRNA (2) was used for injections that yielded dhc-1 (RNAi) embryos shown in Fig. 6 and Fig. 8 . To generate dsRNA corresponding to p150 Glued ( dnc-1 ) and p50/dynamitin ( dnc-2 ), wild-type (N2) genomic DNA was PCR-amplified with primers corresponding to fragments of either gene plus 30 nucleotides for binding of T3 (forward primer) or T7 (reverse primer) RNA polymerases. The following primer pairs were used: (1) dnc-1 (A), covering exons 4, 5, and 6 of dnc-1 , positions 18537–20065 in cosmid ZK593; (2) dnc-1 (B), covering exons 8, 9, 10, and 11, positions 20843–22155 in cosmid ZK593; and (3) dnc-2, corresponding to all five exons of the dnc-2 gene, positions 41836–43012 in cosmid Z28H8. Generation of dsRNA was as described above. All primer sequences can be obtained upon request. Wild-type (N2) adult hermaphrodites were injected bilaterally in the gonads according to standard procedures, and placed at 20°C. Animals were dissected 24–30 h after injection and their embryos analyzed by time-lapse DIC microscopy (1 frame every 5 s) or indirect immunofluorescence as previously described . The following primary antibodies were used: 1:100 or 1:200 rabbit anti–DHC-1, 1:100 rabbit anti–ZYG-9 , 1:5,000 rabbit anti–PGL-1 , and 1:400 mouse antitubulin (clone DM1A; Sigma Chemical Co.). For peptide blocking experiments, slides were preincubated for 15 min with 0.1 mg/ml DHC-1 peptide, and then incubated with anti–DHC-1 antibody in the continued presence of peptide. Secondary antibodies were 1:800 goat anti–mouse Alexa488 (Molecular Probes Inc.) and 1:1,000 donkey anti–rabbit Cy3 (Dianova). Slides were counterstained with Hoechst 33258 (Sigma Chemical Co.) to reveal DNA. Indirect immunofluorescence data were gathered on a confocal microscope (LSM510; Carl Zeiss). All high magnification images are 1.2-μm confocal slices; the stage was refocused slightly between channels in some cases. Images were processed with Adobe Photoshop 4.0. Time-lapse DIC microscopy was performed at 1 frame every 0.5 s to determine the velocity of the fast minus end–directed movements of yolk granules towards the center of asters. In wild type, the focal plane was that of the center of the anterior aster. In dhc-1 (RNAi) embryos, the focal plane included the center of both asters, which are together at the very posterior of these embryos (see Results). The analysis was carried out during the ∼2 min separating pronuclear envelope breakdown from anaphase in wild-type, when these motility events are most frequent, and the corresponding time interval in dhc-1 (RNAi) embryos. The analysis was restricted to motility events that lasted 2 s or more. Average peak velocities were determined using the public domain NIH Image program 1.62b7 (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/). Time-lapse DIC microscopy was performed at 1 frame every 5 s to determine the velocity of the posteriorly directed flows of yolk granules that occur just before pronuclear migration in the cytoplasm posterior of the pseudocleavage furrow . As noted previously , these movements are sometimes turbulent, so that calculated velocities should be considered only as best estimates. Average peak velocities were determined as above. We sought to determine the function of conventional cytoplasmic dynein in MTOC positioning in the one cell stage C. elegans embryo. There are two cytoplasmic dynein heavy chain genes in the C. elegans genome, dhc-1 and che-3 (gene F18C12.1). We focused our analysis on dhc-1 because the corresponding protein is more similar to the vertebrate conventional cytoplasmic dynein heavy chain MAP1C (57% amino acid identity along the entire protein as opposed to 30% for CHE-3). Moreover, a putative null allele of che-3 is viable and has defects restricted to sensory neuron structure and function (Wicks, S., C. de Vries, and R.H.A. Plasterk, personal communication). Accordingly, we did not observe a phenotype in early embryos when che-3 expression was silenced with RNAi (data not shown). We began our study by raising polyclonal antibodies against an amino-terminal peptide of DHC-1 (see Materials and Methods). Affinity-purified anti–DHC-1 antibodies recognized two bands on a Western blot of total C. elegans proteins: a very high molecular mass species and a species of ∼180 kD . The very high molecular mass species most likely corresponds to the dynein heavy chain, which is predicted to be 512 kD in size . Compatible with this view, this species comigrated with a Xenopus protein recognized by anti- Xenopus dynein heavy chain antibodies . The lower molecular mass species may correspond to a degradation product of the dynein heavy chain or a distinct cross-reactive species. We tested whether anti–DHC-1 antibodies specifically recognize dynein heavy chain protein in C. elegans embryos in two ways. First, we compared the immunofluorescence staining intensity observed with anti–DHC-1 antibodies in wild type to that seen in embryos in which dhc-1 gene expression was silenced in a sequence-specific manner using RNAi (hereafter referred to as dhc-1 (RNAi) embryos; see Materials and Methods). As shown in Fig. 1B and Fig. c , 88% of the anti–DHC-1 signal was lost on average in dhc-1 (RNAi) embryos. Residual staining might be due to incomplete silencing of the dhc-1 gene by RNAi. Second, we determined that anti–DHC-1 immunostaining was entirely absent from embryos incubated with anti–DHC-1 antibodies in the presence of 0.1 mg/ml DHC-1 peptide (data not shown). Taken together, these results demonstrate that most, if not all, of the signal detected with anti–DHC-1 antibodies in wild-type embryos is specific for the cytoplasmic dynein heavy chain. We used anti–DHC-1 antibodies to determine the subcellular distribution of cytoplasmic dynein in early wild-type embryos by immunofluorescence microscopy . We found that cytoplasmic dynein was present in a punctate manner throughout the cytoplasm at all stages of the cell cycle. In addition, a stronger signal was detected at the periphery of pronuclei in one cell stage embryos and of nuclei in later stage embryos . Moreover, cytoplasmic dynein was present at the cell cortex; this was especially apparent at boundaries between cells, for instance, between the AB and P 1 blastomeres of the two cell stage embryo . The distribution of cytoplasmic dynein changed as cells progressed through mitosis. During prometaphase, cytoplasmic dynein accumulated along both sides of prometaphase chromosomes . Since chromosomes in C. elegans are holocentric , this possibly corresponds to kinetochore staining. During metaphase, cytoplasmic dynein became enriched on the spindle . During early anaphase , strong spindle signal was still detected, both between segregating chromosomes and spindle poles, as well as centrally , between the two sets of chromosomes . A similar staining pattern persisted throughout anaphase . At telophase, cytoplasmic dynein was enriched in two areas of the cytoplasm adjacent to the spindle poles . In addition, a strong signal was detected at the periphery of reforming nuclei . A subcellular distribution analogous to the one reported here was observed in C. elegans embryos using polyclonal antibodies raised against purified dynein heavy chain protein (Lye, J., personal communication). This confirms that the distribution described here truly reflects that of dynein heavy chain and not of an unrelated protein. We wanted to determine if cytoplasmic dynein function is essential in C. elegans . To this end, we specifically silenced the expression of the conventional dynein heavy chain gene dhc-1 using RNAi. Hermaphrodites were injected with dsRNA corresponding to a segment of the dhc-1 gene (see Materials and Methods). Such animals gave rise to 100% dead embryos 20 h or more after injection ( n = 268 embryos over three experiments). Thus, dynein heavy chain is essential for C. elegans embryogenesis. In addition, dynein heavy chain is required for fertility, as mature oocytes ceased being produced 35–40 h after injection. We addressed whether minus end–directed motor activity was indeed abolished in dhc-1 (RNAi) embryos. A manifestation of minus end–directed motility in wild-type one cell stage embryos is the fast movement of yolk granules 0.3–1 μm in diameter towards the center of the asters along linear paths, suggestive of movements along astral microtubules . We determined the average peak velocity of these motility events to be 1.44 μm/s , which is in the range of velocities that have been reported for dynein-dependent motility events in other systems . During the ∼2 min separating the breakdown of the pronuclear envelopes from anaphase, 10 or more such motility events that lasted at least 2 s could be typically observed in a given focal plane in wild-type embryos. We investigated whether these fast minus end–directed motility events were altered in dhc-1 (RNAi) embryos. Of the five dhc-1 (RNAi) embryos examined in detail, three displayed no such movement, whereas the remaining two each had a single instance of fast minus end–directed motility event. In contrast to wild-type, however, these two motility events lasted <2 s. The lack of motility events in dhc-1 (RNAi) embryos was not merely due to an absence of astral microtubules, as asters in dhc-1 (RNAi) embryos were observed both by DIC and immunofluorescence microscopy (see below). Lack of motility events was not due either to a general inability of yolk granule movement because the slower posterior-directed flow of yolk granules that occurs in the cytoplasm of wild-type embryos just before pronuclear migration was not affected in dhc-1 (RNAi) embryos . Consistent with this observation, segregation of P granules towards the posterior of the embryo, which may be driven by this flow , was also not affected (32/32 one cell stage dhc-1 (RNAi) embryos examined; Fig. 4 B). Taken together, these findings demonstrate that fast minus end–directed motility of yolk granules is specifically abolished in dhc-1 (RNAi) embryos and suggest that cytoplasmic dynein drives this form of cellular transport. To determine the consequences of the loss of cytoplasmic dynein motor activity on MTOC positioning, we examined dhc-1 (RNAi) one cell stage embryos by time-lapse DIC microscopy. This approach is well-suited to examine MTOC positioning because yolk granules are excluded from areas of high microtubule density, such as the center of asters and the spindle, as well as from pronuclei and nuclei. Fig. 5A–D , shows the relevant sequence of events in wild type. After fertilization, the two meiotic divisions are completed in the one cell stage embryo. The resulting female pronucleus lies slightly off the anterior cortex , whereas the male pronucleus is tightly apposed to the posterior cortex . The sperm contributes the single centrosome of the one cell stage embryo . After duplication, the two daughter centrosomes separate, while remaining closely associated with the male pronucleus. The separated centrosomes migrate slightly anteriorly, along with the male pronucleus, whereas the female pronucleus migrates posteriorly towards the centrosomes. As a result, the male and female pronuclei meet at ∼70% egg length . We found that dhc-1 (RNAi) one cell stage embryos displayed several striking phenotypes when examined by time-lapse DIC microscopy . First, dhc-1 (RNAi) embryos often had multiple female pronuclei and displayed aberrant polar body formation, both indicative of defects during the female meiotic divisions. The role of cytoplasmic dynein during the meiotic divisions is beyond the scope of this work and will not be discussed further here. Second, migration of the male and female pronuclei never took place in dhc-1 (RNAi) embryos . The nuclear envelope of the male pronucleus broke down 1–2 min before that of the female pronuclei . Such asynchrony is characteristic of mutants defective in pronuclear migration . Third, after breakdown of the pronuclear envelopes, a bipolar spindle was not apparent by DIC microscopy in dhc-1 (RNAi) embryos. While an area devoid of yolk granules did extend towards the anterior of the embryo over time , consistent with an underlying high density of microtubules, no aster was apparent at the anterior end of this area . Instead, both asters appeared to be located at the very posterior of dhc-1 (RNAi) embryos . Fourth, proper cell division did not occur in dhc-1 (RNAi) embryos . The absence of cleavage furrow specification was expected given the apparent absence of bipolar spindle. While some furrowing activity did take place towards the anterior of the embryo, this rarely resulted in productive cleavage, like in embryos lacking a spindle after nocodazole treatment . Numerous small nuclei reformed in dhc-1 (RNAi) embryos as the cell returned into interphase , indicative of failure in chromosome segregation. Quicktime movies of a wild-type and a dhc-1 (RNAi) embryo can be viewed on the Hyman lab web site (http://www.embl-heidelberg.de/ExternalInfo/hyman/Data.htm) to help compare the sequence of events observed with time-lapse DIC microscopy. In summary, these observations demonstrate that cytoplasmic dynein is essential for pronuclear migration in the one cell stage C. elegans embryo. In addition, they suggest a possible role in centrosome separation, since both asters are located together at the very posterior of dhc-1 (RNAi) embryos. To test whether centrosome separation was indeed defective in dhc-1 (RNAi) embryos, we determined the position of centrosomes by antitubulin staining; in addition, some of the embryos were simultaneously labeled with antibodies against ZYG-9, a centrosomal marker in C. elegans . In prophase, daughter centrosomes have separated to opposite sides of the male pronucleus in wild type . In contrast, in dhc-1 (RNAi) embryos, daughter centrosomes failed to separate and remained positioned posterior of the male pronucleus . After breakdown of the pronuclear envelopes, the two centrosomes were still in close proximity of one another and located at the very posterior of dhc-1 (RNAi) embryos . In contrast to wild type, a bipolar spindle was never observed in dhc-1 (RNAi) embryos, and chromosomes were never located in the very small space between centrosomes (46/46 dhc-1 (RNAi) embryos examined after breakdown of the male pronucleus). Bundles of microtubules up to 20 μm in length emanated from the posterior where the centrosomes were located and extended anteriorly towards a set of chromosomes . These microtubules most likely correspond to the area devoid of yolk granules that had been observed extending towards the anterior by time-lapse DIC microscopy . These findings suggest that chromosomes from the male pronucleus are pushed towards the anterior by growing microtubules after breakdown of the pronuclear envelope. Importantly, these results demonstrate that cytoplasmic dynein is required for centrosome separation in the one cell stage C. elegans embryo. To confirm that the absence of pronuclear migration and centrosome separation were a result of interfering with cytoplasmic dynein function, we examined the phenotype of embryos depleted of dynactin components by RNAi. Dynactin has been shown to be required for proper cytoplasmic dynein function in several systems . Therefore, silencing of dynactin components by RNAi in C. elegans might be expected to result in a similar phenotype to that observed in dhc-1 (RNAi) embryos. Contrary to this prediction, however, it has been reported that injection of ssRNA corresponding to the dynactin components p150 Glued or p50/dynamitin yield embryos that undergo pronuclear migration and form a bipolar spindle . However, these embryos may have had residual p150 Glued and p50/dynamitin function since ssRNA is much less potent than dsRNA in silencing gene expression . Therefore, we tested whether pronuclear migration and centrosome separation were affected after silencing of p150 Glued or p50/dynamitin gene expression with dsRNA. As reported in Table and shown in Fig. 7A and Fig. D , 12/20 p150 Glued (dsRNAi) and 8/20 p50/dynamitin (dsRNAi) embryos had a pronuclear migration phenotype indistinguishable from that of dhc-1 (RNAi) embryos by time-lapse DIC microscopy. Like for dhc-1 (RNAi) embryos, no bipolar spindle was apparent after breakdown of the pronuclear envelopes in these p150 Glued (dsRNAi) and p50/dynamitin (dsRNAi) embryos, and both asters remained in close proximity to one another at the very posterior of the embryos . Staining with antitubulin antibodies confirmed that centrosomes were close to one another at the very posterior of 13/34 p150 Glued (dsRNAi) and 14/24 p50/dynamitin (dsRNAi) embryos examined after breakdown of the male pronucleus . The remainder of p150 Glued (dsRNAi) and p50/dynamitin (dsRNAi) embryos had milder phenotypes, resembling in part those obtained after injections of single-stranded material . The fact that some p150 Glued (dsRNAi) and p50/dynamitin (dsRNAi) embryos underwent pronuclear migration and centrosome separation may be because of incomplete gene silencing, even by dsRNA. Importantly, these results demonstrate that the dynactin components p150 Glued and p50/dynamitin are required, at least in part, for pronuclear migration and centrosome separation in the one cell stage C. elegans embryo. Our observations of dhc-1 (RNAi) embryos with time-lapse DIC microscopy and indirect immunofluorescence revealed that cytoplasmic dynein is also involved in the mechanisms that maintain centrosome association with nuclei. In wild-type one cell stage embryos, the separated daughter centrosomes are initially tightly associated with the male pronucleus, and with both pronuclei after pronuclear meeting. This tight association is apparent by DIC microscopy because yolk granules are excluded both from pronuclei and the center of asters , as well as by staining with antibodies against tubulin or the centrosomal marker ZYG-9 and counterstaining with Hoechst 33258 to visualize DNA . While the majority of dhc-1 (RNAi) embryos maintained association between the unseparated centrosomes and the male pronucleus , this was not always the case. In ∼15% of dhc-1 (RNAi) embryos , the centrosomes were not in the immediate vicinity of the male pronucleus , but instead located 3–11 μm away . In addition, we noted that centrosomes remained at the posterior cortex in these embryos , even though the male pronucleus was not present anterior to them. This suggests that cytoplasmic dynein is required for movement of centrosomes away from the posterior cortex. These results indicate that cytoplasmic dynein is required, at least in part, for proper association between centrosomes and the male pronucleus in the one cell stage C. elegans embryo. Cytoplasmic dynein appears to play a role in maintaining this association, rather than in establishing it, because asters initially in close proximity to the male pronucleus can be observed drifting away in time-lapse DIC recordings of dhc-1 (RNAi) embryos (data not shown). We wanted to test whether cytoplasmic dynein is required for the positioning of centrosomes onto the longitudinal axis that leads to proper spindle orientation in the one cell stage C. elegans embryo. However, the lack of centrosome separation in dhc-1 (RNAi) embryos precludes addressing this question because of the resulting absence of spindle assembly. Therefore, we sought to generate weaker phenotypes with RNAi to bypass the early requirement for centrosome separation. Weaker phenotypes were produced by injecting undiluted ssRNA and examining embryos 12–16 h after injection or by injecting 16-fold diluted ssRNA and examining embryos 24–30 h after injection. The resulting dhc-1 (ssRNAi) embryos reproducibly fell into one of three broad phenotypic classes, corresponding to the equivalent of an allelic series. First, embryos that were wild type. In these cases, the RNAi effect was probably too weak to significantly deplete dynein heavy chain. Second, embryos that had phenotypes akin to those obtained after injection of double-stranded material. In these cases, the RNAi effect was probably strong enough to deplete a substantial fraction of dynein heavy chain. Third, embryos that exhibited milder phenotypes that probably resulted from intermediate diminution of cytoplasmic dynein function; MTOC positioning in embryos of the third class is described below. In wild type, the centrosome pair is positioned at 70% egg length and transverse to the longitudinal axis after pronuclear meeting . The centrosome pair and associated pronuclei subsequently move to the embryo center while undergoing a 90° rotation . As a result, after breakdown of the pronuclear envelopes, the spindle is positioned in the cell center and oriented along the longitudinal axis . We found that the third class of dhc-1 (ssRNAi) embryos underwent pronuclear migration as in wild type , but failed to undergo subsequent centration and rotation of centrosomes . As a result, the spindle was set up at ∼70% egg length, perpendicular to the longitudinal axis . However, the spindle was typically rescued onto the longitudinal axis by the end of anaphase, presumably because of the physical constraints of the eggshell . An identical phenotype has been reported previously for p150 Glued (ssRNAi) and p50/dynamitin (ssRNAi) embryos , and was observed in this study for some p150 Glued (dsRNAi) and p50/dynamitin (dsRNAi) embryos ( Table ). These results demonstrate that cytoplasmic dynein, like dynactin components, is required for centration/rotation of centrosomes and, thus, proper spindle orientation, in the one cell stage C. elegans embryo. By using RNAi, we have demonstrated that cytoplasmic dynein is required for all three major aspects of centrosome positioning that occur in the one cell stage C. elegans embryo: centrosome separation, movement of centrosomes away from the posterior cortex accompanying male pronuclear migration, and subsequent positioning of centrosomes onto the longitudinal axis. In addition, we found that cytoplasmic dynein is required for female pronuclear migration and plays a role in maintaining association between centrosomes and male pronucleus. The function of cytoplasmic dynein in MTOC positioning in complex eukaryotes has not been unambiguously determined in the past, owing largely to experimental difficulties associated with loss-of-function studies. Both in Drosophila and mice, cells bearing strong mutations in the heavy chain gene fail to proliferate or survive , severely hampering investigation of cytoplasmic dynein function. Such difficulties can be circumvented by using RNAi in C. elegans . Germ cells targeted by RNAi undergo no divisions between the time of injection and fertilization. Therefore, even if cytoplasmic dynein is essential for an aspect of cell division, this alone cannot interfere with analyzing its function in the one cell stage embryo. Cytoplasmic dynein does play a role during the meiotic divisions that take place shortly after fertilization, since dhc-1 (RNAi) one cell stage embryos often possess multiple female pronuclei. However, this does not prevent scoring cell division processes in the remainder of the first cell cycle, and cannot explain the subsequent defects of centrosome positioning. Indeed, the same defects are observed in those dhc-1 (RNAi) embryos that have a single female pronucleus. Conversely, centrosome positioning defects are not apparent in a number of mutant strains with multiple female pronuclei . One potential limitation of using RNAi resides in the possibility that the component under study is also required to generate mature oocytes, in which case function in the one cell stage embryo may not be assessed. In fact, cytoplasmic dynein does play some role in gametogenesis, since oocyte production ceases 35–40 h after injection of dhc-1 dsRNA. Nonetheless, this has not hampered our analysis, because reproducible phenotypes were observed in one cell stage embryos 24–32 h after injection. Therefore, RNAi in C. elegans offers an excellent opportunity to analyze the in vivo requirements of cytoplasmic dynein in MTOC positioning in a complex eukaryote. Our results unequivocally establish that cytoplasmic dynein and dynactin are required for centrosome separation in the one cell stage C. elegans embryo. A similar conclusion had been reached for cytoplasmic dynein from experiments in vertebrate cells that made use of function-blocking antibodies . However, subsequent studies suggested that dynein and dynactin are not needed for centrosome separation . These apparently conflicting data may be reconciled if the majority of cells in the later studies retained sufficient motor activity to permit centrosome separation and proceed through to subsequent stages of mitosis, where there may be a higher requirement for dynein and dynactin function. Interestingly, cytoplasmic dynein is not required for MTOC separation in S. cerevisiae . Therefore, whereas many cell division processes have been conserved throughout evolution of eukaryotic cells, the use of cytoplasmic dynein to separate MTOCs may be specific to metazoans. Perhaps different mechanisms of MTOC separation have been imparted by the fact that spindle pole bodies are embedded in the nuclear envelope, in contrast to centrosomes that are simply associated with nuclei. Two conditions must be met for proper centrosome separation to take place in complex eukaryotes. First, centrosomes must move until they are diametrically opposed on the nucleus. Second, separating centrosomes must remain tightly associated with the nucleus. Two types of mechanisms have been invoked to explain centrosome separation. In one, separation results from pushing forces acting on overlapping antiparallel microtubules emanating from the two centrosomes. Plus end–directed motors are expected to generate the force driving separation in this case. The requirement for plus end–directed kinesins like Xklp2 in centrosome separation lends support to this view . A minus end–directed motor such as cytoplasmic dynein may still be essential in this scenario by transporting effector molecules like Xklp2 . However, this type of mechanism requires the existence of an extranuclear spindle during centrosome separation, which has not been observed in several vertebrate cells or in the C. elegans embryo . Moreover, this type of mechanism predicts that centrosomes move apart in a coordinated fashion, whereas there is evidence to the contrary in newt cells . Finally, such a mechanism alone does not explain how separating centrosomes remain tightly associated with the nucleus. In the second type of mechanism, separation results from pulling forces acting on astral microtubules in front of the moving centrosomes. Minus end–directed motors are expected to generate the force driving separation in this case. The requirement for cytoplasmic dynein uncovered in this study is fully compatible with this view. Dynein could generate such pulling forces by being anchored throughout the cytoplasm or at the cell cortex, as has been discussed previously . Here, we propose an alternative model in which pulling forces result from interactions between cytoplasmic dynein anchored on the nucleus and astral microtubules . Supporting evidence for such a model comes from the presence of cytoplasmic dynein at the periphery of nuclei, both in MDCK cells and in C. elegans . This model is attractive because it provides a single mechanism to explain both how centrosome separate and how they remain tightly associated with the nucleus. While we have no direct data to support this model at present, evidence that interactions between cytoplasmic dynein anchored on nuclei and astral microtubules can generate force comes from our discovery that female pronuclei fail to migrate in dhc-1 (RNAi) embryos. In wild type, cytoplasmic dynein is enriched at the periphery of the female pronucleus, and may, thus, drive migration of this organelle toward centrosomes by minus end–directed motility. Additional evidence compatible with this mechanism comes from Xenopus in which a reconstituted system that mimics female pronuclear migration has been shown to require cytoplasmic dynein function . Why would centrosomes in the model presented in Fig. 10 move apart until they are diametrically opposed to one another? The role of cytoplasmic dynein suggests a possible mechanism involving length-dependent forces. In this scenario, the minus ends of astral microtubules, along with the centrosome, are pulled when they encounter anchored cytoplasmic dynein on the nucleus. Longer astral microtubules encounter more anchored motors and, thus, experience a stronger pulling force than shorter ones. After centrosome duplication, microtubules extending away from the centrosomes along the nucleus are long, whereas those projecting towards the other centrosome are short. Thus, length-dependent forces could ensure that centrosomes move away from each other until such pulling forces are balanced, which occurs when they are diametrically opposed. In this model, the initial position of daughter centrosomes after duplication determines the final position of separated centrosomes. Such a mechanism for centrosome separation would simultaneously ensure association between separating centrosomes and the nucleus. The nature of the association between centrosomes and nuclei is poorly understood. It has been postulated that organelle-like motility of nuclei along microtubules may serve to maintain this association . The presence of cytoplasmic dynein on nuclei in MDCK cells and in C. elegans is compatible with this postulate . Importantly, our finding that the association between centrosomes and male pronucleus is lost in some dhc-1 (RNAi) embryos provides the first evidence that cytoplasmic dynein may indeed play a role in maintaining the nucleus tightly associated with centrosomes by minus end–directed motility. We have shown that cytoplasmic dynein and dynactin are required for the movement of centrosomes away from the posterior cortex that accompanies male pronuclear migration. Perhaps centrosomes remain at the posterior cortex in the absence of cytoplasmic dynein function as a secondary consequence of defective separation. In wild type, microtubule polymerization forces that act against the posterior cortex are thought to push away centrosomes and associated male pronucleus . In the absence of centrosome separation, perhaps the close proximity of two MTOCs somehow prevents microtubule polymerization forces from efficiently acting against the posterior cortex. Another possibility is that movement of centrosomes away from the posterior cortex occurs via pulling forces exerted by dynein anchored throughout the cytoplasm. Separated centrosomes alter their position before spindle assembly in many cells, thus ensuring proper spindle orientation during mitosis . In the wild-type one cell stage C. elegans embryo, centration/rotation of centrosomes ensures that the spindle sets up along the longitudinal axis. By using ssRNA to bypass the requirement for centrosome separation, we found that centration/rotation and spindle orientation in the one cell stage embryo require cytoplasmic dynein function. A similar result is obtained when p150 Glued or p50/dynamitin are subjected to weak RNAi effects . Cytoplasmic dynein and dynactin are also required for proper orientation of the spindle at the bud neck in S. cerevisiae . It has been proposed that dynactin at the bud cortex tethers cytoplasmic dynein, which captures astral microtubules and translocates the associated spindle pole body by minus end–directed motility towards the bud neck . An analogous cortical capture mechanism could account for centration/rotation in the one cell stage C. elegans embryo, if cytoplasmic dynein were tethered somewhere in the anterior of the embryo. We have shown that cytoplasmic dynein is present at the cortex of one cell stage embryos, albeit at low levels. Since p150 Glued is enriched at the site of polar body extrusion, at the very anterior cortex , it is formally possible that cytoplasmic dynein at this site mediates centration/rotation. However, we think this is unlikely because this process still occurs in rare embryos in which polar bodies are positioned away from the anterior cortex (Gönczy, P., unpublished observations). Evidence from S. cerevisiae suggests an alternative to the cortical capture model. In this organism, microtubule dynamics are affected in dynein heavy chain mutants, and the average length of microtubules is altered in mutants of other motor proteins that play a role in spindle orientation . While the exact role of microtubule dynamics in S. cerevisiae spindle orientation remains to be determined, these observations raise the possibility that cytoplasmic dynein could similarly influence microtubule dynamics in C. elegans . This, in turn, may be responsible for some of the phenotypic manifestations reported in this work, including improper spindle orientation in the one cell stage embryo. Separated centrosomes in the P 1 blastomere of the two cell stage C. elegans embryo also undergo a 90° rotation that aligns them along the longitudinal axis. It has been suggested that P 1 rotation also results from a cortical capture mechanism . In this case, laser microsurgery experiments identified the requirement for a discrete cortical site, which overlaps with the cell division remnant generated after cleavage of the one cell stage embryo . The dynactin components actin capping protein and p150 Glued are enriched at this site, lending support to the hypothesis that cytoplasmic dynein anchored at this site drives P 1 rotation . Compatible with this view, we found that cytoplasmic dynein is present all along the cortex in the P 1 blastomere, including the cortical site. However, despite this correlative finding, we could not assess the role of cytoplasmic dynein in P 1 rotation with certainty. While P 1 rotation was defective in some dhc-1 (ssRNAi) embryos (Gönczy, P., S. Pichler, and M. Kirkham, unpublished observations), this may not reflect a direct requirement for cytoplasmic dynein function. Indeed, the first cleavage furrow typically ingressed sooner on one side of dhc-1 (ssRNAi) embryos after rescue of spindle orientation onto the longitudinal axis . As a result, the cortical site is predicted to be eccentrically located. A similar asynchronous ingression of the first cleavage furrow has been observed in weak p150 Glued (RNAi) and p50/dynamitin (RNAi) embryos . In this case, eccentric location of the cortical site has been directly demonstrated by anti-p150 Glued staining in p50/dynamitin (ssRNAi) embryos . Therefore, we suggest that the failure of P 1 rotation with diminished cytoplasmic dynein or dynactin function may result from the eccentric location of the cortical site caused by asynchronous ingression of the first cleavage furrow. We believe that demonstration of a direct requirement of cytoplasmic dynein and dynactin function in P 1 rotation awaits further experiments, including the use of temperature-sensitive alleles or local inactivation of protein function. The development of such experimental approaches will be important to further dissect the requirement of cytoplasmic dynein in C. elegans and other metazoans where RNAi is not available. | Study | biomedical | en | 0.999997 |
10508862 | Immunoscreening of a chicken brain cDNA expression library in λgt11 with a rabbit serum against the aqueous Triton X-114 fraction of chicken brain synaptic plasma membranes identified a strongly immunopositive clone designated 5.8. Starting from clone 5.8 , full-length mouse cDNA and nearly full-length chicken (lacking ∼80 NH 2 -terminal codons) cDNA contigs were built by several rounds of hybridization and PCR screening of cDNA libraries from chicken and mouse brain (newborn whole brain, Stratagene; adult whole brain, Clontech). Database searches identified a human expressed sequence tag (EST) that was obtained through Genome Systems, Inc., and completely sequenced. Total and poly(A) + RNA preparation from chicken tissues and Northern blot analysis with 32 P-labeled hybridization probes were performed according to conventional procedures. Human Northern blots were purchased from Clontech and hybridized according to the manufacturer's instructions. Chicken blots were hybridized with clone 5.8 and human blots with IMAGE clone 192540. Two sequence regions that have little or no similarity with Bassoon and Rim were amplified from mouse brain RNA and cloned into the His-tag vectors pQE31 and pQE30 (Qiagen). Subclone inserts were sequenced to confirm the absence of mutations. His-tag fusion proteins were expressed in bacteria, purified on nickel agarose, and used to immunize rabbits. Sera were affinity-purified with the same fusion proteins coupled to tresyl chloride–activated Sepharose (Sigma Chemical Co.). Commercial mAbs for mannosidase II (clone 53FC3; BAbCO), Na/K-ATPase β subunit (Upstate), Rab3 (clones 42.1 and 42.2; Transduction Laboratories, Inc., and Synaptic Systems), Rab5 (Transduction Laboratories, Inc.), transferrin receptor (clone H68.4; Zymed), and α-tubulin (Amersham Pharmacia Biotech), and sera for synaptophysin (Biometra) and rabphilin-3A (Synaptic Systems) were purchased from the sources indicated. An affinity-purified Mena antibody (LKE) was donated by Frank Gertler (Massachusetts Institute of Technology, Cambridge, MA), isoform-specific antisera against profilins I and II were gifts of Walter Witke (European Molecular Biology Laboratory, Heidelberg, Germany), and a KDEL receptor mAb was the gift of Wanjin Hong (University of Singapore, Singapore). To determine the tissue distribution of aczonin, tissues were homogenized in 0.32 M sucrose, 1 mM EDTA, 10 mM Tris, pH 7.4, 0.5 mM PMSF, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, with a glass-Teflon homogenizer, or for muscle and heart, a turning-knife homogenizer. After spinning for 3 min at 900 g , 80 μg protein of each supernatant was resolved by SDS-PAGE (5% polyacrylamide), transferred to nitrocellulose, and the blot developed with affinity-purified rabbit antiaczonin and the ECL kit (Amersham Pharmacia Biotech). For 120,000 g fractionation and reextraction experiments, 900 g supernatants of brain homogenates (in 150 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 7.4, 0.5 mM PMSF, 2 μg/ml pepstatin A, 2 μg/ml leupeptin) were subjected to a 120,000 g centrifugation for 30 min at 4°C. Pellets were washed by resuspending in homogenization buffer followed by a second 120,000 g spin, and then resuspended either in homogenization buffer or in various solubilization buffers , either for 20 min at 4°C or for 30 min at room temperature. The 120,000 g centrifugation was then repeated. Equal aliquots of pellets and supernatants were analyzed by immunoblotting as described above. Preparative subcellular fractionation procedures for the purification of synaptic vesicles or synaptic plasma membranes were performed according to Hell et al. 1988 and Babitch et al. 1976 , respectively . Immunohistochemical procedures for light and electron microscopical analysis of rat brain were as described previously . Identical results were obtained with affinity-purified antibodies against two different aczonin sequence regions (see above). Images shown in Fig. 4 were obtained with serum 2. Preimmune antibodies and preincubation of the immune antibodies with an excess of the recombinant antigen were employed as controls. Cell culture, immunofluorescence analysis, and brefeldin A treatment were performed according to conventional procedures. PC12 and NS20Y cells were fixed in 4% paraformaldehyde in PBS, and permeabilized with either 0.04% saponin or 0.2% Triton X-100. For double-labeling experiments, cells were incubated simultaneously with both primary antibodies. Antiaczonin was visualized with a biotinylated goat anti–rabbit secondary antibody (Vector Labs) followed by streptavidin-FITC. Antimannosidase II, anti-KDEL receptor, or antitransferrin receptor marker antibodies were visualized with a Cy3-conjugated goat anti–mouse antibody (Dianova). The mouse Rab3A sequence was taken from Baumert et al. 1993 and the mouse profilin I sequence from Sri Widada et al. 1989 . The mouse profilin II sequence was identified by expressed sequence tag (EST) database screening . Full-length coding sequences of these three proteins were amplified from mouse brain RNA and subcloned, with NH 2 -terminal His-tags, into pQE-31 (Qiagen). Sequences encompassing codons 374–654 and codons 863–1115 of mouse aczonin, and codons 11–399 of rat Rim were amplified from mouse brain and subcloned into pGEX-4T (Amersham Pharmacia Biotech). All subclones employed for expression were verified by sequencing to be free of mutations. Mouse brain was homogenized in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 4 mM MgCl 2 , 2 mM EDTA, 10 mM NaF, 1 mM Na 3 VO 4 , 2 mM PMSF, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 mM benzamidine, and 0.2% Triton X-100). The 120,000 g supernatant was adjusted to 4 mg/ml total protein. The pellet was resuspended in 0.1 M Na 2 CO 3 (pH 11.5) for 30 min at room temperature, dialyzed against several changes of lysis buffer with stepwise decreasing pH until pH 7.4, and adjusted to 6 mg/ml. Recombinant profilins or BSA (MBI Fermentas) were coupled to N -hydroxysuccinimide–activated Hi-Trap columns (Amersham Pharmacia Biotech) following the manufacturer's instructions. 20-μl aliquots of protein-coupled resins were preblocked with 3% BSA in PBS, washed with lysis buffer, and incubated with brain lysate for 4 h at 4°C under constant agitation. After spin, pellets were washed six times in lysis buffer and resuspended in SDS sample buffer. 1/20 vol of supernatants and 1/2 vol of pellets were analyzed by SDS-PAGE and immunoblotting. In poly-amino acid blocking experiments, resins were preincubated with 100 μl of 5 mg/ml polyproline (1–10 kD; Sigma Chemical Co.) or polyalanine (1–5 kD; Sigma Chemical Co.) in lysis buffer overnight at 4°C and washed once with 1 ml lysis buffer before incubation with lysates. Alternatively, profilins and other His-tagged protein constructs (see Results) were immobilized on nickel agarose (20 μl of resin per sample), incubated with lysates additionally containing 5 mM imidazole, and washed with lysis buffer additionally containing 50 mM imidazole. For Rab3A overlay assay, equal quantities (2 μg) of glutathione S -transferase (GST) 1 fusion proteins with similar-sized inserts from aczonin , Rim (11–399) as positive control, and paralemmin and HSB as negative controls, were resolved by SDS-PAGE and transferred to nitrocellulose. Blots were renatured overnight at 4°C in PBS with 50 μM ZnCl 2 and 0.5 mM MgCl 2 , blocked for 2 h in overlay buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM MgCl 2 , 0.1% Tween 20, and 1% BSA) with 5% nonfat dry milk, rinsed in water, and incubated overnight at 4°C with 12 μg/ml of His-tagged Rab3A in the overlay buffer containing 2 μg/ml each of pepstatin A, leupeptin, and aprotinin, in the presence of 0.5 mM GTPγS, 1 mM GDP, or without nucleotides. Blots were washed with overlay buffer and processed for immunodetection using His-tag antibody (Qiagen) or Rab3A antibody (Synaptic Systems) and the ECL kit (Amersham Pharmacia Biotech). Recombinant Rab3A was also immobilized on nickel agarose and employed in precipitation experiments with brain lysates as described above for profilin, in the presence of either 0.5 mM GTPγS or 1 mM GDP. Immunoprecipitations with affinity-purified antiaczonin were performed with Pansorbin (Calbiochem) according to conventional procedures using mouse brain lysate prepared as described above, with the addition of 0.5% Triton X-100 and 0.5% BSA, in the presence of either 0.5 mM GTPγS or 1 mM GDP. Aczonin was identified as a new molecular constituent of neuronal synapses by immunoscreening a brain cDNA expression library with antisera raised against synaptic plasma membranes . An alignment of the cDNA-deduced mouse and chicken aczonin sequences with Bassoon is presented in Fig. 1 , and an overview of the regional organization of the aczonin sequence and its similarities with Bassoon and Rim is given in Fig. 2 . The longest splicing variant of mouse aczonin is predicted to be a 550-kD hydrophilic polypeptide of balanced charge (pI 6.4) featuring two mutually homologous pairs of Cys 4 zinc fingers in the NH 2 -terminal region, a polyproline stretch in the middle (22 uninterrupted proline residues in the mouse and 11 in chicken), and a PDZ domain and two C2 motifs in the COOH-terminal region. Sequence comparison between mouse and chicken reveals an organization into regions of high or low interspecies conservation . In particular, the sequences surrounding the two zinc finger pairs and the polyproline stretch are poorly conserved, suggesting that they mainly serve as spacers. This interpretation is further supported by the finding that the two zinc finger pairs are flanked by blocks of sequence repeats. Upstream of the first zinc finger pair is a series of degenerate proline- and glutamine-rich 10-mer repeats (consensus, KPxPQQPGPx, other residues often small or aliphatic amino acids). These repeats are found in different numbers (15–25) in chicken, mouse, and humans. We also isolated mouse cDNA sequence variants differing in the presence or absence of three 10-mer repeat units , which may reflect differential splicing or a genomic polymorphism. Downstream from the second zinc finger pair, the chicken and mouse sequences diverge again for several hundred amino acids, and the mouse sequence is shorter, whereas the chicken sequence has expanded by eight lysine-rich ∼22-mer repeats (core motif, VQKED … SADKI). We suppose that both repeat series served to expand the spacer sequences during evolution, although it remains possible that the 10-mer repeats have an additional function. Aczonin shares regions of homology with two other proteins concentrated at active zones, Rim and Bassoon . Rim possesses a similar COOH-terminal array of one PDZ and two C2 domains, and both Cys 4 zinc finger pairs of aczonin have sequence similarity with the single pair of Cys 4 zinc fingers of Rim. Outside these circumscript molecular modules, we do not detect significant sequence similarity between aczonin and Rim, except a motif of 19 amino acids with 68% identity close to the NH 2 termini of both proteins . Upstream of the PDZ domain, many but not all sequences highly conserved between mouse and chicken aczonin are homologous to Bassoon. This includes the two zinc finger pairs and the extreme NH 2 terminus, but not the polyproline stretch and another proline-rich region downstream from it. A highly charged region between amino acids 1550–1700 of mouse aczonin is particularly conserved between mouse and chicken aczonin and Bassoon. In summary, the aczonin sequence can be structured into Rim-related, Bassoon-related, and aczonin-specific regions or motifs. The zinc finger motifs are similar in all three proteins. Two sequence regions near the COOH terminus are subject to differential splicing. Splicing at codon 4829 of the mouse sequence can abort the downstream 210 amino acids, including the C2B domain, and replace them by a short SKRRK COOH terminus. Out of nine adult mouse brain cDNAs from this region that were sequenced, eight encode the shorter (aczonin-S) and one the longer COOH terminus (aczonin-L). From chicken, only one cDNA was isolated. It encodes the longer COOH terminus plus 61 additional codons (AHKS … PEGA) inserted at a position corresponding to mouse codon 4829 (aczonin-XL). Reverse transcription PCR with primers between positions corresponding to codons 4751 and 4946 of the mouse sequence indicated that mRNAs with and without this insert are expressed in similar quantities in chicken brain. In the human aczonin gene (see Discussion), the sequence encoding the short SKRRK COOH terminus is contiguous with the sequences immediately upstream in the cDNA, whereas the alternative COOH-terminal sequences, including the XL insert, are encoded by exons further downstream. Northern blot analysis of RNAs from various chicken and human tissues showed that aczonin mRNA, which is very large and therefore partially degraded, is most highly expressed in the brain and detectable at low abundance in several endocrine glands but in none of the other tissues analyzed . In both species, testis mRNA gave a unique band pattern, with smaller molecular sizes than in the other tissues. Antisera were raised against recombinant aczonin partial sequences and employed for Western blot analysis of mouse tissue homogenates. A very large protein far above the 206-kD marker, apparently partially degraded, was detected in different brain regions and, after longer exposure, very weakly in stomach but in none of the other tissues analyzed, including adrenal gland, testis, and pancreas . In endocrine cells, aczonin protein may be poorly translated from the mRNA or rapidly degraded. Aczonin mRNA and protein are found in similar abundances in forebrain, cerebellum, and brainstem , indicating expression throughout the brain. Antisera raised against aczonin-specific sequences labeled neuropil-rich areas throughout the rat brain. Cell bodies and myelin-rich areas were spared . The examination of the cerebellum and the dentate gyrus by electron microscopy revealed that immunoreactivity concentrates at the presynaptic side of synaptic specializations. Immunoperoxidase reaction product is restricted to a space reaching from the plasma membrane of the active zone into the interior of the synapse by only a few neurotransmitter vesicle diameters . Aczonin immunoreactivity was found in many but not all synapses. In the glomeruli of the cerebellum, all mossy fiber terminals were decorated, whereas only a fraction of synapses between Golgi and granule cells showed reaction product . Terminals of parallel fibers, which form contacts with Purkinje cell dendrites in the molecular layer of the cerebellum, were also labeled in many but not all cases. In the dentate gyrus only a subpopulation of granule cell mossy fiber terminals was immunopositive. 120,000 g fractionation of brain homogenate showed that ∼90% of total aczonin was recovered in the pellet and ∼10% in the supernatant . From the pellet, aczonin could not be extracted with 1 M NaCl or with 1% Triton X-100, but could be with 0.1 M sodium carbonate (pH 11.5). In this behavior, aczonin differed from the intrinsic membrane protein, synaptophysin, which was almost completely solubilized by the detergent but not by sodium carbonate, and was similar to the cytoskeletal control protein, tubulin . This result suggests that aczonin is associated through polar interactions with a detergent-resistant cytoskeletal-like subcellular fraction. The distribution of aczonin in a subcellular fractionation course leading to the purification of synaptic vesicles closely follows that of the plasma membrane marker, the Na/K-ATPase β subunit, and differs from the synaptic vesicle marker, synaptophysin, which partitions markedly also into the light fractions. In the final step of this procedure, controlled-pore glass gel filtration, synaptophysin is enriched in the small-vesicular fraction permeating the gel (fraction P II P), whereas aczonin is enriched in the P I P exclusion peak, even more so than the plasma membrane marker. Codistribution with a plasma membrane marker was also observed in a fractionation procedure leading to synaptic plasma membranes (data not shown). These observations indicate that aczonin is not firmly associated with free synaptic vesicles, but rather with larger structures that sediment faster than vesicles in centrifugation and are excluded by the controlled-pore glass chromatography matrix, such as large cytoskeletal aggregates, the plasma membrane, or both. Neuronal cell lines also express aczonin. In these cells, it is associated with endomembrane structures within the cell body. In PC12 neuroendocrine cells, aczonin immunofluorescence is congruent with that of mannosidase II, a marker of the Golgi complex . This aczonin and mannosidase II–positive membrane structure is fragmented within ∼5 min by brefeldin A, but is unaffected by wortmannin (not shown), further supporting its identification as the Golgi complex or a closely apposed structure such as the TGN. In NS20Y neuroblastoma cells, aczonin instead decorates more finely punctate structures that cluster around the nucleus or at the bases of processes but spare the Golgi region. This apparently vesicular compartment is not labeled by KDEL receptor (marker for ER–Golgi intermediate compartment and cis -Golgi) or transferrin receptor (recycling endosomes) immunofluorescence (data not shown). These observations suggest that aczonin, although not an intrinsic membrane protein, associates with membranes, membrane proteins, or the membrane-associated cytoskeleton during earlier stages of the secretory pathway, and in this way probably reaches its presynaptic destination in neurons. A polyproline tract is conserved between chicken, mouse, and human aczonin, whereas extensive flanking regions are not or poorly conserved . Synthetic polyproline and proline tracts in a number of proteins are known to bind profilin, a small protein that is implicated in actin cytoskeletal dynamics, and which in neurons is concentrated in synaptic terminals . Efficient profilin binding is mediated by homoproline tracts of >10 residues or PPPPPG tandem repeats. Because it is well-established that isolated homopolymeric proline tracts like those in mouse and chicken aczonin bind profilin, we concentrated on probing for profilin binding to native, full-length aczonin. Profilin exists in two isoforms, profilin I being expressed in many tissues including brain, and profilin II predominating in brain and skeletal muscle . We expressed both isoforms as His-tag fusion proteins, coupled them to Sepharose resin, and could specifically precipitate aczonin from brain lysates with both . Aczonin precipitation is blocked by preincubation of the profilin resins with synthetic polyproline but not by polyalanine, indicating that polyproline and aczonin compete for the same binding site on profilin, aczonin probably through its polyproline tract . In several independent experiments with equal quantities of covalently immobilized proteins as shown in Fig. 7 , profilins I and II precipitated aczonin with similar efficiencies. In another set of experiments where equal quantities of recombinant profilins were immobilized on nickel agarose, profilin II consistently precipitated more aczonin than profilin I did. The established profilin-binding protein, Mena , for which we probed as a positive control target protein in these experiments, was also more efficiently precipitated from brain lysate by profilin II than by profilin I (data not shown). The specificity of profilin binding to aczonin was underpinned by additional controls (data not shown). Two additional His-tagged negative control constructs immobilized on nickel agarose, recombinant Rab3A (see below), and a 91 amino acid sequence from neurobeachin (construct C3; Wang, X., and M.W. Kilimann, unpublished data) did not precipitate either aczonin or Mena. A negative control target protein, neurobeachin, probed for by Western blotting, was not precipitated by the profilin constructs or by the negative control constructs. Profilins I and II bound both aczonin from the soluble fraction of a brain lysate , and aczonin from the sedimentable fraction solubilized by 0.1 M Na 2 CO 3 and then back-dialyzed against lysis buffer. The two zinc finger pairs of aczonin, but not their flanking sequences, have significant sequence similarity to the zinc finger structures of rabphilin-3A and Rim. Sequence regions from these two proteins encompassing the zinc fingers have been shown to bind Rab3A, a small G protein involved in synaptic vesicle trafficking. However, in three different types of experiments we were unable to detect binding of Rab3A and related Rab proteins to aczonin (data not shown). We expressed the two zinc finger pairs from mouse aczonin together with extensive flanking sequences as GST fusion proteins, and probed for binding to recombinant Rab3A by blot overlay assay. No binding of recombinant Rab3A to these constructs was detected, in contrast to a corresponding Rim sequence (amino acids 11–399 fused to GST) employed as a positive control, which displayed pronounced GTP-dependent binding of Rab3A on the same blot. Recombinant His-tagged Rab3A immobilized on nickel agarose also did not precipitate detectable amounts of holo-aczonin from brain lysate; however, as positive controls, Rab3A precipitated rabphilin-3A, and resin-bound recombinant profilin (see above) precipitated aczonin in the same experiment. Finally, immunoprecipitation of aczonin from brain lysate did not bring down Rab3A, Rab3B, Rab3C, or Rab5 in quantities detectable by Western blot analysis. Aczonin is a very large multidomain protein of ∼5,000 amino acids which is specifically concentrated at presynaptic active zones and firmly associated with a detergent-resistant, cytoskeletal-like subcellular fraction. It may be a scaffolding protein that structures the presynaptic cortical cytoplasm, interacts with multiple partner molecules, and orchestrates the interplay of neurotransmitter vesicles with the cytoskeleton, the plasma membrane, and probably cytosolic proteins at the active zone. From the aczonin sequence, several motifs stand out that also suggest links to plasmalemmal, vesicular, and cytoskeletal proteins. The overall pattern of one PDZ and two C2 domains in the COOH-terminal region is similar to Rim. Whereas many PDZ domain proteins have been identified that are involved in the organization of postsynaptic protein arrays , few presynaptic PDZ proteins are yet known. Rim and aczonin are the only two proteins known to contain both PDZ and C2 domains. C2 domains may bind proteins or membrane lipids. Alignment of the aczonin C2 domains with those of various other proteins shows that those of Rim are also the closest relatives in terms of sequence similarity (47% identity in the C2A and 42% identity in the C2B domains). Synaptotagmins are also close relatives with 35–42% identity, whereas Munc13 C2 sequences are less related, with 17–29% identity. All five aspartate residues critical for calcium chelation by C2 domains are conserved in the aczonin C2A domain. In the aczonin C2B domain, four of them are replaced by uncharged residues, as is the case in both C2 domains of Rim. Differential splicing of sequences between the two C2 modules is another feature that aczonin shares with Rim. In addition, the C2B domain of aczonin is subject to differential splicing, and the relative frequencies of short and long variants isolated from a mouse brain cDNA library suggest that aczonin-S, with a single COOH-terminal C2A domain, is more common. PDZ domains of other proteins are known to bind to the cytoplasmic COOH termini of transmembrane proteins or to signaling proteins . According to sequence alignment with a number of PDZ domains, that of Rim again seems to be the closest relative (36–41% identity with mouse and chicken aczonin, respectively). The PDZ and/or C2 domains are likely to anchor aczonin to the plasma membrane, and it will be of interest to identify their binding partners. The two Cys 4 zinc finger pairs of aczonin are homologous to each other, to two similar motifs in Bassoon, and to the single Cys 4 zinc finger pairs of Rim, rabphilin-3A, and Noc2. According to cysteine residue spacing (CX 2 CX 17 CX 2 CX 4 CX 2 CX 15 CX 2 C in aczonin) and additional sequence similarity, the zinc finger pairs of these proteins constitute a distinct subfamily of zinc finger motifs, and the three-dimensional structure of its prototype, the rabphilin-3A zinc finger pair, has been solved recently . A larger, related subfamily with the same cysteine residue spacing (four-residue interval between the two central cysteines) but a different sequence signature binds to the membrane lipid, phosphatidylinositol 3-phosphate, and is found, for example, in the synaptosomal-associated protein of molecular mass 25 kD (SNAP-25)–binding protein Hrs-2 and the Rab5-binding protein EEA1 . Rim, rabphilin-3A, and Noc2 have been shown to be functionally implicated in calcium-stimulated exocytosis, and sequence regions encompassing the zinc finger motifs of Rim and rabphilin-3A bind Rab3A, but Noc2 does not . We were unable to detect binding of Rab3A to aczonin, suggesting that the Bassoon/aczonin/rabphilin/Rim/Noc2-type zinc fingers (proposed designation: “BARRN fingers”) are not sufficient, and perhaps not necessary for Rab3A binding. They may be, at least in rabphilin-3A and Rim, effector domains that mediate an interaction between Rab3A binding and other molecular targets such as synaptic vesicle proteins or lipids. In agreement with this notion, the three-dimensional structure of the Rab3A-binding region of rabphilin-3A in complex with Rab3A shows that Rab3A is contacted by sequences upstream and downstream from the zinc finger, but not by the zinc finger motif itself . The SGAWFF sequence motif that is part of the downstream Rab3A-binding interface of rabphilin-3A and is conserved in Rim but also in Noc2, is not present in either aczonin or Bassoon. Aczonin contains several proline-rich regions that may include targets for the binding of SH3- or WW-domain–containing proteins, such as the NH 2 -terminal 1,100 amino acids around and upstream of the zinc fingers or a conserved sequence region at amino acids 2380–2500. Most strikingly, a polyproline stretch and short flanking sequences in the middle of the aczonin molecule are conserved between chicken, mouse, and humans, whereas several hundred amino acids around them are not or poorly conserved. This suggested an interaction with profilin, and we could indeed demonstrate that both profilin isoforms bind to aczonin and that this binding is blocked by homopolymeric proline but not by polyalanine. Profilin is an actin and phosphoinositide-binding protein expressed in many cell types, including neurons, where it is concentrated in synaptic terminals . It is believed to play an important role in the local remodeling of the actin cytoskeleton, although its exact mechanistic role(s) in interplay with actin, membrane lipids, and multiple profilin-binding proteins is insufficiently understood. Depending on specific conditions, profilin may either promote actin polymerization or depolymerization . Actin filaments are abundant in synaptic terminals but may be rarefied at the active zone . Control of the presynaptic microfilament architecture may be important for neurotransmitter vesicle dynamics, particularly in the vesicle reserve pool . Binding of profilin to several presynaptic proteins, including the synapsins, clathrin, and dynamin has been described recently . In interplay with other presynaptic profilin-binding proteins, and possibly also with other proteins binding to its proline-rich sequences , aczonin may recruit profilin to or control its availability at the active zone. At the gene level, a polyproline tract is encoded by a triplet repeat that has an intrinsic tendency to expand or contract. Therefore, it may be argued that the polyproline tract of aczonin has arisen by serendipity and may be physiologically meaningless at the protein level, although like any polyproline sequence, it binds profilin. However, it is clearly accessible for profilin binding in the context of the complete aczonin molecule, and it is highly conserved in evolution, even between birds and mammals (chicken, 11; mouse, 22; humans, 22 uninterrupted proline residues and additional prolines immediately upstream or downstream), whereas its flanking sequences are not. Leucine residues at or near the ends of proline runs, as in the aczonin sequences, are also found in other profilin-binding proteins . In contrast, for example, a polyglutamine tract in Bassoon is more heterogeneous in length, with 11 residues in the mouse, 24 in the rat, but only 5 in the human sequence , whereas its flanking sequences are highly conserved. Therefore, the Bassoon polyglutamine is more likely to represent an autonomously fluctuating microsatellite-repeat DNA element with little physiological significance at the protein level. The zinc finger region of aczonin is flanked by oligopeptide repeats. Dekapeptide repeats upstream of the first zinc finger motif differ in number between chicken, humans, and mouse and even among different mouse cDNAs, whereas 22-mer repeats downstream from the second zinc finger pair are found only in chicken but not in mouse. Therefore, it seems likely that these repeats primarily served as an evolutionary mechanism to rapidly expand spacer sequences around the zinc finger modules. The 10-mer repeat region is similar (45% predicted amino acid sequence identity) to an untranslated repetitive sequence region in the bovine herpesvirus type 1 BICP22 gene . The Bassoon sequence contains a tract of heptapeptide repeats downstream from its second zinc finger pair (three copies in rat and five in mouse, unrelated in sequence to the aczonin repeats) that may also serve as a spacer. Long spacers could position binding partners of the zinc finger modules and of the polyproline tract at defined distances from the main body or along the backbone of aczonin, or they could facilitate the accommodation of bulky binding partners such as vesicles or large protein complexes. We have also determined a partial cDNA sequence from the NH 2 -terminal region of human aczonin (codons 34–794). A partial human cDNA sequence representing the 1,213 COOH-terminal amino acids of the short splicing variant of human aczonin has been reported . Human aczonin genomic sequences connecting both partial cDNAs are also available , extending over several hundred kilobases and revealing a remarkable gene structure with some very large exons. The Bassoon gene also has unusually large exons . Several sequence-tagged site markers from both human cDNA sequence regions have been concordantly mapped to a region on human chromosome 7 corresponding to cytogenetic bands 7q11.23–q22.1 (NCBI GeneMap'98). However, no human neurological disease loci have yet been mapped to this region according to Online Mendelian Inheritance in Man (OMIM) (all databases accessible via http://www.ncbi.nlm.nih.gov). Computer-assisted secondary structure prediction from the aczonin sequence indicates a high potential of flexibility, but also some sequence stretches with high coiled-coil potential that are shared with Bassoon. For example, anchored at the plasma membrane to ion channels or other transmembrane proteins through its PDZ domain, aczonin could potentially reach into the synaptic terminal across several neurotransmitter vesicle diameters. To give an upper-limit estimate, 5,000 amino acids could form an extended α-helix 750-nm long, i.e., 15 vesicle diameters. Thus, aczonin may play a role in organizing the supramolecular structure of the cortical cytomatrix at the active zone. It could constitute or be part of the longer strands seen in quick-freeze deep-etch electron microscopy to tether synaptic vesicles to the active zone . In lamprey reticulospinal axons, Pieribone et al. 1995 observed a 300-nm-thick layer of synaptic vesicles adhering to the active zone independently of synapsin I. Aczonin, alone or together with proteins like Bassoon and Rim, may be responsible for the plasma membrane anchoring of this pool. Proteins tethering vesicles to target membranes at distances longer than the reach of soluble N -ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), as observed recently at the Golgi complex , may be important for rapid and efficient vesicle trafficking and also for targeting specificity . The synaptic target membrane SNAREs, syntaxin-1 and SNAP-25, are distributed all over the axonal plasmalemma , so aczonin and related proteins may be essential to specifically target synaptic vesicles to the active zone. According to immunoblot analysis and immunolight microscopy, aczonin is found throughout the brain. By immunoelectron microscopy of selected brain areas, we detect it very consistently in the mossy fiber terminals of cerebellar glomeruli, but only in a fraction of the terminals of other synapse populations. It remains to be clarified whether aczonin is present in all synapses, albeit at different concentrations, or only in specific populations, and with what functional features of synapses its level of expression and its different splicing variants correlate. It will be of particular interest to understand the relationship between aczonin and its partial homologues, Bassoon and Rim. It will also be interesting to see whether Piccolo, which resembles both aczonin and Bassoon in molecular size, subcellular distribution, and immunomorphology, is identical to aczonin or whether it constitutes an additional member of this protein family. It is conceivable that these proteins can partially substitute for each other in different types of synapses, that their homologous domains interact with different isoforms or homologues of partner proteins, or that they work hand in hand within the same synapse. For example, aczonin, with a longer reach into the presynaptic cytoplasm than Rim, may usher vesicles towards the plasma membrane without interfering with Rab3 bound to them, and hand them over to Rim immediately before docking. | Study | biomedical | en | 0.999997 |
10508863 | The S . cerevisiae strains and plasmids used in this study are listed in Table and Table , respectively. All strains are isogenic derivatives of JK9-3d. Rich media (YPD) or synthetic minimal media (SD, SRAFF, and SGAL) complemented with the appropriate nutrients for plasmid maintenance were as described . Latrunculin-A (200 μM) was added to cultures from a 200-mM stock in DMSO. A culture of cells was treated with Zymolyase 20T (7.5 U/ml; Seikagaku Corp.) added from a 750-U/ml stock in YPD at 30°C for 20 min. A culture of cells was treated with SDS (0.02%) added from a 10% stock in water at 24 or 30°C for 10 min. For all heat shock experiments, 2-ml aliquots were removed from a logarithmic phase culture grown at 24°C and incubated at 37°C for the indicated time. Plasmid DNA was isolated as described . Yeast transformation was performed by the lithium acetate procedure . Escherichia coli strain MH1 was used for propagation and isolation of plasmids . The entire open reading frames of WSC1 , WSC2 , WSC3 , BCK1 , and MKK1 were replaced by PCR-generated kanMX4 or HIS3MX6 cassettes, as described . Disruptions were verified by PCR. A 0.45-kb fragment encoding a triple hemagglutinin (HA) tag followed by the CYC1 transcription terminator was generated by PCR using the plasmid pYADE4 , containing the CYC1 terminator sequence, as a template and the following two oligonucleotides: 5′-ACAT GCATGC TACCCATACGATGTTCCTGACTATGCGGGC - TATCCCTATGACGTCCCGGACTATGCA GGA TATCCATATGA - CGTTCCAGATTACGCT TAACCAAGATGGCCTTTGGTGGGTTG- AAGAAG-3′ (SphI site in bold and HA epitope coding sequence underlined) and 5′-ATAGCAAAGATTGAATAAGGC-3′. The 0.45-kb fragment was cut with SphI and HindIII, and the resulting fragment was cloned into YEplac181 (2μ LEU2 ), YCplac111 ( CEN LEU2 ), YCplac33 ( CEN URA3 ), and YEplac195 (2μ URA3 ) , creating plasmids pPAD80, pPAD81, pPAD82, and pPAD83, respectively. These plasmids, containing a 3×HA coding sequence preceded by a multiple cloning site and followed by a termination codon and the CYC1 terminator sequence, are useful for constructing COOH terminally 3×HA–tagged proteins. pPAD86 (pPAD81:: WSC1 ) ( CEN LEU2 ) contains WSC1 as a 1.6-kb SacI-SmaI PCR fragment. The SacI site was introduced 460 nucleotides upstream of the WSC1 initiation codon, and the SmaI site replaced the termination codon. Logarithmically growing cells were fixed for 2 h in the growth medium supplemented with formaldehyde (3.7%) and potassium phosphate buffer (100 mM, pH 6.5). Cells were washed and resuspended in sorbitol buffer (1.2 M sorbitol, 100 mM potassium phosphate, pH 6.5). Cell walls were digested for 60–120 min at 37°C in sorbitol buffer supplemented with β-mercaptoethanol (20 mM) and Zymolyase 20T (0.1 mg/ml; Seigagaku Corp.) or lyticase (gift from P. Ernst, Biozentrum Basel). Spheroblasts were attached on poly- l -lysine–coated glass slides and permeabilized in PBT (53 mM Na 2 HPO 4 , 13 mM NaH 2 PO 4 , 75 mM NaCl, 0.1% Triton X-100). Immunofluorescent detection of FKS1 was performed with the T2B8 anti–FKS1 mAb . HA-tagged WSC1 and RHO1 were detected with a high affinity monoclonal anti–HA antibody (clone 16B12; BAbCO) at a final dilution of 1:1,000 in PBT. The samples were treated with primary antibody for 2 h and subsequently with Cy3-conjugated rabbit anti–mouse IgG (Molecular Probes, Inc.), diluted 1:1,000 in PBT for 90 min. Washed cells were examined with a Zeiss Axiophot microscope (100× objective) and a video imaging system (MWG Biotech). Logarithmically growing cells were fixed in formaldehyde (3.7%) and potassium phosphate buffer (100 mM, pH 6.5), and stained with TRITC-phalloidin (Sigma Chemical Co.) to visualize actin, as described previously . The actin cytoskeleton is transiently depolarized upon heat shock . To investigate this effect further, we examined the actin cytoskeleton in wild-type cells shifted from 24°C to 37°C for different time intervals (see Materials and Methods). The percentage of small- and medium-budded cells (cells that normally exhibit a polarized actin cytoskeleton) containing a depolarized actin cytoskeleton was determined. Cells containing <50% of their actin patches in the bud were considered to have a depolarized actin cytoskeleton. The maximum percentage of depolarized cells (∼95%) was reached after ∼30 min of heat shock, with complete repolarization occurring after ∼120 min . The maximally depolarized state persisted for ∼15 min. Thus, as seen previously, heat shock induces a rapid and severe, but transient, depolarization of the actin cytoskeleton. Interestingly, heat shock did not depolarize actin patches at the mother–bud neck in large-budded cells. Heat shock is thought to cause a cell wall weakening . To determine if heat-induced actin depolarization is a consequence of a cell wall defect, we examined if osmotic stabilization of the cell wall by 1 M sorbitol blocked the actin depolarization response. The response was partly blocked in cells shifted to a high temperature in the presence of 1 M sorbitol (data not shown). To determine further if actin depolarization is a consequence of a cell wall defect, the actin cytoskeleton was examined in cells in which a cell wall defect was induced either by mild treatment with Zymolyase (β-1,3-glucanase), which digests the cell wall, or by treatment with the anionic detergent SDS (see Materials and Methods) . Essentially all cells treated with 7.5 U/ml Zymolyase or with 0.02% SDS exhibited a depolarized actin cytoskeleton (data not shown). However, unlike heat-induced depolarization, the Zymolyase- and SDS-induced depolarization persisted for the duration of the experiment (120 min), possibly because cells cannot adapt to these treatments as they can adapt to heat shock. These results suggest that depolarization of the actin cytoskeleton is a response to cell wall stress. The above findings suggested that cells might delocalize the actin cytoskeleton as a homeostasis mechanism to repair cell wall damage. To investigate this possibility, we first examined the cellular localization of FKS1 in heat shocked cells by immunofluorescence (see Materials and Methods). FKS1, the presumed catalytic subunit of the cell wall biosynthetic enzyme β-1,3-glucan synthase, normally localizes with its regulatory subunit, RHO1, and actin patches at sites of cell growth . As expected, FKS1 displayed a polarized distribution in cells growing at 24°C, found associated with the plasma membrane exclusively in the bud. Upon heat shock, the distribution of FKS1 was transiently depolarized, with a time course similar to that of the actin cytoskeleton . After 35 min at 37°C, the FKS1 signal decreased in the bud and appeared uniformly at the periphery of the entire cell. After 120 min at 37°C, FKS1 was relocalized exclusively to the bud. A depolarization of FKS1 was also observed in SDS-treated cells (data not shown). We also examined the cellular localization of the glucan synthase regulatory subunit RHO1 in heat shocked cells. Cells expressing HA-tagged RHO1 (PA120-3b) were shifted from 24 to 37°C and further incubated for 35 or 120 min. Visualization of RHO1 by indirect immunofluorescence revealed a transient depolarization similar to that observed for FKS1 . Thus, glucan synthase is redistributed evenly throughout the cell periphery in response to cell wall stress. This depolarization of cell growth may be to repair general cell wall damage. To investigate further the possibility that actin delocalization is part of a homeostasis mechanism to depolarize cell growth and repair general cell wall damage, we examined if FKS1 de- and repolarization are dependent on the actin cytoskeleton. To determine if the actin cytoskeleton is required for FKS1 depolarization, FKS1 localization was examined in cells treated simultaneously with heat and latrunculin-A (LAT-A), a toxin that disrupts the actin cytoskeleton by sequestering actin monomers . LAT-A prevented the heat-induced depolarization of FKS1, indicating that the actin cytoskeleton is required for FKS1 depolarization . To determine if the actin cytoskeleton is also required for FKS1 repolarization, we examined the effect of LAT-A on depolarized FKS1. Cells previously incubated at a high temperature (37°C) for 35 min were treated with LAT-A and examined for localization of FKS1. After 120 min of heat shock and 85 min of LAT-A treatment, FKS1 remained depolarized, whereas FKS1 repolarized in mock-treated cells . Thus, the actin cytoskeleton is required for both FKS1 de- and repolarization, suggesting that cells depolarize their actin cytoskeleton in response to cell wall damage as a mechanism to depolarize cell growth and repair general cell wall damage. RHO1 and the PKC1-activated MAP kinase cascade are activated by cell wall damage . To determine if RHO1 or the PKC1-activated MAP kinase cascade mediates the stress-induced depolarization of the actin cytoskeleton, we examined this response in null mutants individually lacking a WSC family member (WSC1, WSC2, WSC3, WSC4, or MID2), a RHO1 exchange factor (ROM1 or ROM2), the nonessential GTPase RHO2, a RHO1 effector (BNI1, SKN7, or FKS1), or a member of the PKC1-activated MAP kinase pathway (BCK1 or MPK1). rho1 and pkc1 null mutants were not examined because RHO1 and PKC1 are essential for growth. The wsc1 , wsc2 , wsc3 , wsc4 , mid2 , rom1 , rom2 , rho2 , bni1 , skn7 , fks1 , bck1 , and mpk1 null mutants were grown at 24°C, shifted to 37°C for different time intervals, and processed for visualization of the actin cytoskeleton. The wsc1 and rom2 mutants were severely, but not completely, defective in heat-induced depolarization of the actin cytoskeleton . A maximum of ∼55% and ∼40% of the wsc1 and rom2 cells (versus ∼95% for wild-type cells), respectively, exhibited a depolarized actin cytoskeleton after 30 min at 37°C. This defect was not simply a delay in depolarization of the actin cytoskeleton, as a higher percentage of depolarized cells was not observed at later time points. Furthermore, wsc1 and rom2 mutants were sensitive to SDS for growth, and exhibited a defect in FKS1 depolarization that correlated with these mutants' defect in actin depolarization (data not shown). Because a wsc1/wsc1 homozygous diploid strain has a more severe growth defect than a wsc1 haploid , we also examined the actin cytoskeleton in a heat shocked wsc1/wsc1 diploid. A maximum of ∼30% of the wsc1/wsc1 cells (versus ∼95% for wild-type diploid cells) exhibited a depolarized actin cytoskeleton, after 30 min at 37°C, indicating that the block in depolarization of the actin cytoskeleton was indeed more pronounced in the homozygous diploid mutant. Surprisingly, all of the other mutants listed above exhibited a wild-type–like depolarization response. These observations suggest that stress-induced actin and growth depolarization requires WSC1 and ROM2, but not the PKC1-activated MAPK kinase pathway or any RHO1 effector other than PKC1. The previous observation that cell wall defects increase ROM2 exchange activity toward RHO1 and the above finding that a wsc1 or rom2 mutation reduces stress-induced depolarization suggest that the depolarization process requires RHO1 hyperactivation. To examine this further, we investigated the effect of overexpressing constitutively activated RHO1 (RHO1*) on the actin cytoskeleton and FKS1 localization. As RHO1* is toxic, we constructed a plasmid-borne RHO1* allele under control of the strong and galactose-inducible GAL1 promoter (pGAL-RHO1*). This construct was not toxic in cells grown on a carbon source other than galactose (data not shown). Cells containing pGAL-RHO1* were grown to logarithmic phase in medium containing raffinose as a carbon source, and galactose was added to a final concentration of 2%. Aliquots of cells were removed at different time points after addition of galactose and processed for visualization of the actin cytoskeleton. In raffinose-containing medium, cells displayed a normal vegetative distribution of actin patches. After 1.5 h in galactose, the number of patches in the mother cell increased (data not shown). After 2.5 h in galactose, the actin cytoskeleton was depolarized in essentially all cells . FKS1 was also depolarized, with similar kinetics and severity as observed for the actin cytoskeleton (data not shown). Expression of wild-type RHO1 from the GAL1 promoter (pGAL-RHO1) had no depolarizing effect (data not shown). Thus, RHO1 hyperactivation is sufficient to induce actin and FKS1 depolarization. This finding, combined with the previous findings that cell wall stress hyperactivates ROM2 exchange activity toward RHO1 and that a rom2 mutation blocks depolarization, suggests that depolarization results from RHO1 hyperactivation. We examined the effect of overexpressed and constitutively activated PKC1 (PKC1*), BCK1 (BCK1*), and MKK1 (MKK1*) on the actin cytoskeleton and FKS1 distribution. Like pGAL-RHO1*, pGAL-PKC1*, pGAL-BCK1*, and pGAL-MKK1* were also toxic uniquely in cells grown on galactose-containing medium (data not shown). The effect of pGAL-PKC1* on the actin cytoskeleton and FKS1 distribution was indistinguishable from that of pGAL-RHO1*, indicating that hyperactivation of PKC1 is also sufficient to elicit the depolarization response . Hyperactivation of BCK1 or MKK1 did not induce a depolarization of the actin cytoskeleton or FKS1 in cells shifted to galactose for 2.5 (as performed above for RHO1* and PKC1*) or 5 h . These results further indicate that the PKC1-activated MAP kinase cascade is not involved in stress-induced depolarization of growth, and suggest that a yet-to-be defined PKC1 effector pathway mediates the depolarization response. Expression of wild-type WSC1, WSC2, WSC3, RHO2, or ROM2 from the GAL1 promoter had no effect on the actin cytoskeleton (data not shown). The previous findings that mutants defective in MPK1/SLT2 exhibit a depolarized actin cytoskeleton and overexpression of MPK1 suppresses the actin polarization defect of a rho1 mutant suggested that the PKC1-activated MAP kinase pathway might be required for repolarization. To investigate this further, we examined repolarization of the actin cytoskeleton and FKS1 in bck1 and mpk1 null mutants. As described above, the actin cytoskeleton and FKS1 were normally depolarized in bck1 and mpk1 cells shifted to 37°C for 35 min . However, after 120 min and longer, when the actin cytoskeleton is repolarized in wild-type cells, depolarization of the actin cytoskeleton persisted in most bck1 and mpk1 cells . A similar effect was obtained when examining FKS1 distribution (data not shown). It is important to note that bck1 and mpk1 null mutations in our strain background do not confer a severe temperature sensitive growth defect, as reported for other strain backgrounds, and therefore the persistent depolarization of the actin cytoskeleton and FKS1 cannot be attributed to cell death. Thus, the PKC1-activated MAP kinase cascade is required for repolarization rather than depolarization of cell growth. To determine if cell wall stress affects localization of the plasma membrane protein WSC1, we constructed a COOH terminally, 3×HA-tagged, functional WSC1 (pWSC1-HA) (see Materials and Methods). Visualization of WSC1 by indirect immunofluorescence revealed a polarized distribution similar to that of FKS1 and other proteins found at a growth (bud) site . Surprisingly, WSC1 had previously been reported to be evenly distributed throughout the cell periphery . This discrepancy may be because of the earlier study using overexpressed WSC1, whereas we examined WSC1 expressed from its own promoter on a centromeric plasmid. To examine WSC1 localization in response to stress, growing wild-type cells containing pWSC1-HA were shifted from 24 to 37°C, incubated at the higher temperature for 35 and 120 min, and processed for visualization of WSC1 (WSC1-HA). After 35 min, WSC1 was dispersed throughout the cell periphery . After 120 min at 37°C, WSC1 was repolarized (data not shown). Thus, like the actin cytoskeleton and FKS1, WSC1 displayed a transient depolarization upon heat shock. Interestingly, WSC1 was also found in internal compartments at both low and high temperatures. We asked if, like for FKS1, stress-induced depolarization of WSC1 is LAT-A sensitive and, thus, dependent on the actin cytoskeleton. Interestingly, WSC1 was depolarized in LAT-A–treated cells grown at both low and high temperatures, indicating that LAT-A treatment induces WSC1 depolarization independently of stress . Thus, LAT-A could not be used to determine if stress-induced WSC1 depolarization requires the actin cytoskeleton. However, this experiment demonstrates that WSC1 responds to a change in the actin cytoskeleton in addition to controlling the actin cytoskeleton, as shown above. To determine if the stress-induced depolarization of WSC1 is dependent on a depolarized actin cytoskeleton, we investigated WSC1 distribution in heat shocked rom2 cells. As reported above, a rom2 mutation blocks stress-induced depolarization of the actin cytoskeleton and FKS1. WSC1 distribution was still depolarized in a rom2 mutant shifted to a high temperature for 35 min . Thus, WSC1 depolarization is independent of depolarization of the actin cytoskeleton, suggesting that WSC1 may be a stress-specific actin landmark in addition to a signal transducer . The above results taken together suggest that WSC1, like mammalian integrins, both controls and responds to the actin cytoskeleton (see Discussion). We have shown that the actin cytoskeleton and the distribution of the cell wall synthesis enzyme glucan synthase (FKS1 and its regulatory subunit RHO1) are transiently depolarized in response to cell wall stress. In heat shocked or SDS-treated cells, actin patches and glucan synthase are redistributed from a polarized growth site (bud) to the periphery of the entire cell. This stress-induced depolarization of cell growth, possibly as a mechanism to repair general cell wall damage, is mediated by a putative signaling pathway consisting of the plasma membrane protein WSC1, the RHO1 exchange factor ROM2, RHO1, PKC1, and a yet-to-be identified PKC1 effector branch. The PKC1-activated MAP kinase pathway mediates repolarization of the actin cytoskeleton and FKS1. The de- and repolarization of FKS1 is dependent on the actin cytoskeleton. A model summarizing our results is shown in Fig. 6 . The type I transmembrane protein WSC1 is possibly a signal transducer that senses and signals the integrity of the cell wall. A wsc1 mutant is defective in stress-induced depolarization and in activation of MPK1, sensitive to cell wall stress (SDS and heat), and is suppressed by overexpression of ROM2, RHO1, or PKC1 . The mechanisms by which WSC1 senses and signals cell wall integrity remain to be determined. WSC1 might sense and respond to a turgor pressure–induced outward stretching of the plasma membrane that results from a cell wall defect. Consistent with this possibility, several treatments that stretch the plasma membrane induce a depolarization response. Chlorpromazine, a cationic amphipath that inserts into and stretches the plasma membrane, induces depolarization of the actin cytoskeleton (Delley, P.-A., and M.N. Hall, unpublished results) and MPK1 activation . Hyperosmotic shock also induces depolarization of the actin cytoskeleton . Depolarization of the actin cytoskeleton in response to chlorpromazine or hyperosmotic shock is partly blocked in a wsc1 or rom2 mutant (Delley, P.-A., and M.N. Hall, unpublished results). Alternatively, WSC1 might interact with and directly sense the cell wall . It also remains to be determined if WSC1 indeed controls ROM2 activity and, if so, how this might be achieved. In addition to its role as a putative sensor and signal transducer, WSC1 might also serve as a stress-specific actin landmark. WSC1 also depolarizes upon heat shock and this depolarization is independent of a depolarization of the actin cytoskeleton. Interestingly, although WSC1 may control the actin cytoskeleton both as a signal transducer and a landmark, it also responds to the actin cytoskeleton as LAT-A–induced depolymerization of the actin cytoskeleton also causes WSC1 depolarization. Thus, there appears to be a bidirectional signaling between WSC1 and the actin cytoskeleton. A similar bidirectional signaling between integrins and the actin cytoskeleton has been observed in mammalian cells . The RHO1 GTPase switch and the RHO1 effector PKC1 mediate the transient depolarization response. ROM2 is necessary and hyperactivated RHO1 or PKC1 is sufficient to induce depolarization of the actin cytoskeleton and FKS1. Interestingly, RHO1 and PKC1 are also required for polarization of the actin cytoskeleton. First, loss-of-function rho1 mutants are defective in polarization of the actin cytoskeleton . Second, overexpression of PKC1 restores actin organization in a rho1 mutant . Third, overexpression of RHO1 or PKC1 rescues a tor2 mutant defective in polarized organization of the actin cytoskeleton . The finding that activated RHO1 and its effector PKC1 control both polarization and depolarization of the actin cytoskeleton suggest that RHO1 may be a more finely-tuned switch than previously thought. RHO1 in a hyperactive state, as suggested by our findings, may cause depolarization, whereas RHO1 in a normally active state mediates polarization. Thus, RHO1 may function as a molecular rheostat, with more than one on position, rather than as a simple on-off switch. A second on position could require another RHO1 regulator in addition to ROM2. Alternatively, RHO1 may still function as a simple on-off switch, and depolarization may result from activation of a larger or a specific pool of RHO1. PKC1, like RHO1, also appears to mediate both depolarization and repolarization of the actin cytoskeleton and FKS1. However, unlike RHO1, PKC1 may mediate these two different responses via different effectors. First, mutants defective in MPK1/SLT2 exhibit a depolarized actin cytoskeleton . Second, overexpression of MPK1 suppresses the actin polarization defect of an rho1 mutant . Third, bck1 and mpk1 mutants are not defective in stress-induced actin and FKS1 depolarization . Fourth, BCK1 and MPK1 are necessary to restore polarization of the actin cytoskeleton and FKS1 after cell wall stress . Furthermore, hyperactivated BCK1 or MKK1 does not induce actin and FKS1 depolarization . These findings suggest that the PKC1-activated MAP kinase cascade mediates repolarization, and that a PKC1 effector branch other than this well characterized MAP kinase cascade mediates depolarization. Although the components of a second PKC1 effector branch have not been identified, the existence of a signaling bifurcation below PKC1 has been proposed previously . RHO1 and PKC1 may control both depolarization and repolarization by activating the two PKC1 effector pathways at different times. Activation of the yet-to-be defined PKC1 effector branch, which causes depolarization (primary response), may occur rapidly and only upon stress-induced hyperactivation of RHO1. Activation of the PKC1-activated MAP kinase cascade may be a delayed response that results in repolarization (secondary response). Interestingly, Kamada et al. 1995 reported that the MPK1 MAP kinase is maximally activated after 30 min of heat shock, which is significantly delayed compared with the rapid induction (1–2 min) of MPK1 or the HOG1 MAP kinase by hypotonic or hypertonic stress, respectively . When we use the same regimen to heat shock cells as that used by Kamada et al. 1995 , we find that the maximum percentage of depolarized cells is reached within ∼15 min (Delley, P.-A., and M.N. Hall, unpublished results), suggesting that the actin depolarization response precedes activation of MPK1. The actin-dependent depolarization of glucan synthase (FKS1 and RHO1) in response to cell wall stress may be a homeostasis mechanism to repair general cell wall damage. Lillie and Brown 1994 reported that MYO2, a yeast class V myosin, and SMY1, a MYO2-interacting protein, disappear from and reappear at the bud upon heat shock, within a time course similar to that of actin de- and repolarization. MYO2 plays a role in the transport of secretory vesicles along actin cables to the growth site, and is, thus, involved in polarized growth and secretion. It will be of interest to determine if depolarization of the integral membrane protein FKS1 involves depolarized secretion of newly made FKS1 or dispersal of previously polarized FKS1. Attempts to address this issue by examining FKS1 localization in heat-stressed and cycloheximide-treated cells were inconclusive because cycloheximide alone affects actin organization. Furthermore, the mechanism by which the peripheral membrane protein RHO1 is redistributed may be different from that of FKS1. Unlike depolarization of FKS1, depolarization of RHO1 was not prevented by LAT-A treatment or by a rom2 or wsc1 mutation (Delley, P.-A., and M.N. Hall, unpublished results). The regulation of FKS1 de- and repolarization by RHO1 is the third level on which RHO1 controls FKS1. In addition to controlling FKS1 localization, RHO1 also controls transcription of glucan synthase encoding genes , and is a regulatory subunit of the glucan synthase complex . | Study | biomedical | en | 0.999996 |
10508864 | Monolayers of baby hamster kidney (BHK) cells were grown and maintained as described in Glasgow minimal essential medium (GMEM; Sigma Chemical Co.) supplemented with 5% FCS, 2 mM l -glutamine under standard tissue culture conditions. Triton X-100 Ultra Pure was purchased from Pierce Chemical Co., BSA from Biomol, and saponin, β-methyl cyclodextrin (β-MCD), compactin, trypsin, and trypsin/chymotrypsin inhibitor from Sigma Chemical Co. Antibodies against the human transferrin receptor and proaerolysin were gifts from I. Throwbridge (The Salk Institute for Biological Studies, San Diego, CA) and J.T. Buckley (University of Victoria, British Columbia, Canada), respectively. mAbs against caveolin-1 were purchased from Transduction Laboratories. BHK cells were grown, as described, for 16 h. Before the addition of the toxin, the cells were washed three times with PBS containing 1 mM CaCl 2 , 1 mM MgCl 2 , and 1 μg/ml of trypsin/chymotrypsin inhibitor (PBS 2+ ), and treated with either 0.4% saponin in PBS 2+ for 1 h at 4°C or with 10 mM β-MCD for 1 h at 37°C in incubation medium (IM) containing GMEM buffered with Hepes, pH 7.4, and 1 μg/ml of trypsin/chymotrypsin inhibitor. Proaerolysin was purified as described previously . Concentrations were determined by measuring the OD at 280 nm, considering that a 1 mg/ml sample has an OD of 2.5 . Proaerolysin was labeled with 125 I using iodogen reagent (Pierce Chemical Co.) according to the manufacturer's recommendations. The radio-labeled toxin was separated from the free iodine by gel filtration on a PD10-G25 column (Pharmacia Biotech, Inc.) equilibrated with 150 mM NaCl, 20 mM Hepes, pH 7.4. We consistently obtained a specific activity of ∼2.10 6 cpm/μg of proaerolysin. Radiolabeled proaerolysin ran as a single band on an SDS gel. To activate proaerolysin into aerolysin in vitro, the protoxin was treated with trypsin (1:20; mol:mol ratio) for 10 min at room temperature. The reaction was stopped by addition of a 10-fold excess of trypsin/chymotrypsin inhibitor. Confluent monolayers of BHK cells were washed three times for 5 min with ice-cold PBS 2+ . Cells were then incubated at 4°C with 125 I-proaerolysin in IM. Cells were then washed three times for 10 min with PBS 2+ at 4°C and further incubated for various times at 37°C. Cells were then rinsed three times with PBS 2+ at 4°C, scraped from the dish, and collected by centrifugation at 1,500 rpm for 5 min. A Triton X-100 insoluble fraction was prepared as described below or a postnuclear supernatant (PNS) was prepared as follows. Cells were gently homogenized in 250 mM sucrose, 3 mM imidazole, pH 7.4, by passage through a 22G injection needle. The PNS was obtained by centrifugation (2,500 rpm) and analyzed for the presence of proaerolysin and aerolysin by SDS-PAGE, followed by autoradiography. The detergent extraction was performed as described previously . BHK cells were treated with 125 I-proaerolysin and harvested as described above, and resuspended in 0.5 ml of cold buffer containing 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, as well as Complete, a cocktail of protease inhibitors (Boehringer Mannheim Corp.). Membranes were solubilized by rotary shaking at 4°C for 30 min. The detergent insoluble fractions were obtained either by high-speed centrifugation (30 min, 4°C, at 55,000 rpm in a TLS55 Beckman rotor) or purified on a sucrose gradient as follows. The solubilized cell lysate sample was adjusted to 41% sucrose (in 10 mM Tris-HCl, pH 7.4) in a SW40 Beckman tube, overlaid with 8.5 ml of 35% sucrose, topped up with 16% sucrose, and centrifuged for 18 h at 35,000 rpm at 4°C. The initial load (1 ml), as well as 12 fractions of 1 ml, were collected. Each of the fractions were counted in a Packard auto-γ scintillation spectrometer and/or precipitated with 6% trichloroacetic acid in the presence of 375 μg of sodium deoxycholate as a carrier. Each fraction was analyzed by SDS-PAGE, followed by autoradiography or Western blot analysis. BHK cells were grown for 16 h on 12 × 12-mm glass coverslips in GMEM medium complemented with 2.5% FCS. Cells were left untreated or treated with saponin or β-MCD, as described above. After three washes with PBS 2+ at 4°C, cells were incubated for 1 h at 4°C with 2 nM of proaerolysin, then washed three times for 10 min with PBS 2+ complemented with 0.5% BSA at 4°C, further incubated for 1 h at 4°C with mAbs against proaerolysin , and then washed briefly at 4°C in PBS 2+ –BSA. Further cross-linking with FITC secondary antibodies (1:50) was performed at 4°C for 1 h. After washing, the cells were fixed first in 3% paraformaldehyde in PBS for 4 min at 8°C and then in methanol for 5 min at −20°C. Cells were visualized using a Zeiss Axiophot fluorescence microscope equipped with a cooled CCD camera (Princeton Instruments), controlled by a Power Macintosh. The IPLab Spectrum 3.1 software (Signal Analytics Corp.) was used for data acquisition. Potassium efflux measurements were performed by flame photometry using a Philips PYE UNICAM SP9 atomic absorption spectrophotometer as described previously . Protein concentrations of cellular fractions were determined with bicinchoninic acid (BCA; Pierce Chemical Co.). Proaerolysin overlays, to detect proaerolysin receptors, were performed as described previously . In brief, membrane fractions were run on a 12.5% SDS gel . The gel was incubated in 50 mM Tris-HCl, pH 7.4, and 20% glycerol for 15 min. Proteins were then blotted onto a nitrocellulose membrane for 15 h at 100 mA in the cold using a BioRad wet transfer chamber with a transfer buffer containing 10 mM NaHCO 3 and 3 mM Na 2 CO 3 , pH 9.8. The nitrocellulose membrane was incubated in a binding buffer containing 50 mM NaH 2 PO 4 , pH 7.5, and 0.3% Tween-20 for 20 min followed by a 2 h incubation in the presence of 1.4 nM 125 I-proaerolysin diluted in the same buffer. The membrane was then washed six times for 5 min with toxin-free binding buffer. Binding of proaerolysin was revealed by autoradiography using BioMax films (Kodak). When performing Western blot analysis, HRP-labeled secondary antibodies were revealed using enhanced chemiluminescence (Pierce Chemical Co.). Total cholesterol contents of cells were determined using a colorimetric method following the instructions of the manufacturer (Boehringer Mannheim Corp.). Aerolysin is able to form heptamers in solution in the absence of membranes . However, this process is not very efficient. As can be seen in Fig. 1 a, at a toxin concentration of 4 μM, <50% of the toxin had oligomerized, even after 1 h incubation at 37°C. In marked contrast, oligomerization occurred efficiently on the surface of target cells, such as BHK cells. When BHK cells were incubated with a 10 4 -fold lower concentration of toxin (0.4 nM), 25 min at 37°C was sufficient to observe almost complete oligomerization . Oligomerization and channel formation occurred at even lower doses, such as 1 pM of aerolysin, as witnessed by measuring the intracellular potassium concentration 1 h after toxin addition . Thus, oligomerization at the cell surface can occur at bulk toxin concentrations 10 5 –10 6 -fold lower than those required to detect oligomerization in solution. Previous observations indicated that the toxin was associated with DIGs of the plasma membrane . Here we investigated whether all forms of the toxin, i.e., not only proaerolysin, but also mature aerolysin and the heptamer, were enriched in DIGs. 125 I-Proaerolysin was bound to cells at 4°C. Cells were then incubated either with proaerolysin at 4°C or with aerolysin (obtained by in vitro activation with trypsin) at 4°C followed by 10 min at 37°C to allow heptamerization . Cells were subsequently solubilized in Triton X-100 and DIGs were purified on a sucrose flotation gradient as described in Materials and Methods. As shown in Fig. 2 , not only proaerolysin was highly enriched in the low-density DIGs in agreement with our previous observations , but also mature aerolysin and the heptamer. We next quantified the fraction of the total toxin that was associated with detergent insoluble domains as a function of the incubation time at 37°C. As previously shown , 45% of the toxin initially bound to the cells at 4°C was released into the medium upon incubation at 37°C, the remaining 65%, however, was stably bound to the cells . We then investigated whether the distribution of the cell-bound toxin would change with time. In these experiments, toxin-treated cells were incubated for different times at 37°C, then solubilized in cold Triton X-100 and submitted to a high-speed centrifugation. The amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As can be seen in Fig. 3 b, the amount of toxin associated with detergent-resistant membranes increased with time, consistent with the notion that the toxin was being recruited into these microdomains, presumably because of oligomerization. Since all forms of aerolysin were found in DIGs, we investigated whether disruption of these microdomains had any influence on the protoxin cleavage and oligomerization processes. The integrity of microdomains has been shown to depend on cholesterol . Moreover, cholesterol has been shown to be implicated in the detergent insolubility of GPI-anchored proteins . We therefore analyzed the effect of cholesterol-affecting drugs on the association of proaerolysin with detergent insoluble membrane domains. It is important to note, that, in contrast to what is observed with pore-forming toxins, such as streptolysin O and other members of the thiol-activated toxin family, no requirement for cholesterol has ever been observed for channel formation by aerolysin in artificial phospholipid membranes, suggesting that cholesterol does not play any crucial structural role in aerolysin channel formation and can, therefore, be manipulated in the present experiments. Two drugs were tested: β-MCD and saponin. β-MCD is an effective extracellular cholesterol acceptor that can extract cholesterol from membranes . Saponin, in contrast, binds to cholesterol and sequesters it away from other interactions, but does not extract it from the membrane . Under the experimental conditions used, 50 ± 2% ( n = 4) of total cellular cholesterol was removed by β-MCD, whereas the cholesterol content of saponin-treated cells was the same as that of control cells, as expected. After drug treatment, cells were incubated with 125 I-proaerolysin for 1 h at 4°C. Neither drug prevented binding of the toxin to the cells . Toxin-treated cells were then solubilized in cold Triton X-100, submitted to a high-speed centrifugation, and the amounts of toxin in the detergent insoluble pellet and in the solubilized fraction were determined. As shown in Fig. 4 a, β-MCD led to a mild redistribution of proaerolysin to the detergent soluble fraction. However, after saponin treatment, >80% of cell bound proaerolysin was associated with the detergent soluble fraction. Similar observations were made when purifying DIGs by sucrose density centrifugation from proaerolysin-treated cells. The distribution of proaerolysin along the gradient after β-MCD treatment (results not shown) was similar to that observed for control cells . Therefore, the toxin was still highly enriched in DIGs after β-MCD treatment, but this was no longer the case after saponin treatment . However, saponin did not disrupt all DIGs since the caveolar marker caveolin-1 was still highly enriched in the light density detergent insoluble fractions of the gradient, as also observed for control cells and cells treated with β-MCD (results not shown). Previously, we have shown that receptor bound proaerolysin can be clustered using antitoxin antibodies, and that clustering leads to a characteristic punctate distribution by immunofluorescence . Within these clusters, the toxin was found to fully colocalize with antibody cross-linked alkaline phosphatase and partially with caveolin-1 . Similar antibody-induced clustering has been observed for a variety of GPI-anchored proteins . We have investigated whether treatments with β-MCD or saponin would affect this antibody-induced clustering capacity. The staining pattern found on β-MCD–treated cells was similar to that observed on control cells. In contrast, a diffuse staining was observed when cells were treated with saponin indicating that, under these conditions, even a sandwich of primary and secondary antibodies, before cell fixation, could not induce clustering of proaerolysin bound to its receptor. The above experiments indicate that cholesterol removal with β-MCD did not alter the ability of proaerolysin to associate with microdomains, but that cholesterol clustering with saponin led to a redistribution of the receptor-bound toxin over the entire plasma membrane. These experiments also suggest that β-MCD and saponin affect different pools of cholesterol within the plasma membrane. Despite the lack of effect of β-MCD on steady state proaerolysin distribution, we analyzed the effect of the drug on the cleavage and oligomerization processes. Interestingly, we found that β-MCD treatment led to a dramatic acceleration of the proteolytic processing of proaerolysin into aerolysin . In contrast, there was no effect on the oligomerization kinetics, as can be seen when treating cells with aerolysin that had been activated in vitro to bypass the cell surface activation step . The same observation was made when β-MCD was present throughout the experiment (including incubation with the toxin at 37°C) in addition to the cholesterol synthesis inhibitor compactin. Our interpretation of the effects of β-MCD is that the drug favored the interaction between the enzyme and protoxin because the drug affected regions of the plasma membrane that contain the proaerolysin-processing enzyme, but does not significantly affect the microdomains enriched in GPI-anchored proteins . Previously, we have shown that processing of proaerolysin at the cell surface is primarily performed by the endoprotease furin in both CHO and BHK cells (our unpublished results). We could not detect any in vitro processing of proaerolysin bound to DIGs, suggesting that furin is not present to any significant extent in microdomains (results not shown). As shown in Fig. 4 a, saponin led to a dramatic redistribution of proaerolysin to the detergent soluble fraction of Triton X-100–treated cells. This treatment, however, did not lead to solubilization of the plasma membrane since the total phospholipid content of saponin-treated cells was the same as that of control cells, as measured using the Fiske-Subbarow phosphore determination method (results not shown). As mentioned above, the total cholesterol content was similar to that of control cells. Also, the plasma membrane distribution of the transferrin receptor was not affected by the treatment (results not shown). Finally, toxin receptors were not solubilized by this treatment since binding of aerolysin was not affected, and the same amount of receptors could be detected in control and saponin-treated cells using our previously established toxin overlay assay . This overlay assay led to the identification of various GPI-anchored proaerolysin binding proteins, including a 130- and a 80-kD protein, the 80-kD protein being apparently the most abundant receptor . The redistribution of proaerolysin to the detergent soluble fractions upon saponin treatment was paralleled by a redistribution of the proaerolysin receptors , indicating that solubilization of the toxin is not due to an effect of the drug on the toxin-receptor interaction. Previously, it has been shown that saponin treatment led to the Triton X-100 solubilization of other GPI-anchored proteins . We next tested the effects of saponin both on cleavage of proaerolysin by furin and on heptamerization kinetics. Cells were treated with saponin at 4°C, then incubated in the presence of proaerolysin at 4°C, and further incubated for various times at 37°C. As shown in Fig. 8 a, the kinetics of cleavage were not affected by saponin, in contrast to what we observed with β-MCD . This observation indicates that overall cell surface dynamics were not significantly affected by saponin-treatment. In contrast, oligomerization was dramatically inhibited, as can clearly be seen when treating cells with aerolysin activated in vitro with trypsin . This was not due to some effect of saponin on the toxin itself, since saponin had no effect on oligomerization kinetics in vitro (results not shown). Similar observations were made on other cell types, such as CHO cells or the polarized human intestinal cell line, CaCoII (results not shown). Saponin prevented clustering of cell surface-bound toxin and dramatically inhibited the oligomerization process. Since the toxin was no longer found in microdomains and showed uniform cell surface labeling, yet remained bound to its receptor, it is likely that saponin caused the receptors and their bound toxin molecules to become evenly distributed in the plane of the plasma membrane by preventing any clustering. The absence of clustering due to saponin would reduce the probability of encounter of toxin monomers and thereby the efficiency of oligomerization. If so, one might expect to, at least to some extent, counteract the action of saponin by increasing the amount of cell-bound toxin, hence the probability of toxin monomer–monomer interactions. As can be seen in Fig. 8 c, this is indeed the case. When higher toxin concentrations were added to the saponin-treated cells, oligomerization did occur. The efficiency of the process, however, remained much lower than on control cells, further strengthening the importance of toxin concentration within microdomains on the oligomerization process. The present work illustrates the surface dynamics of proaerolysin upon binding to its GPI-anchored receptor on target cells. Proaerolysin must first be proteolytically processed to become active. The toxin then oligomerizes into a heptameric ring, a step that precedes membrane insertion and channel formation . Using two cholesterol-perturbing drugs, we could selectively affect the kinetics of processing or of oligomerization, suggesting that the two events occur in topologically distinct areas of the plasma membrane. Processing appeared to occur in the phosphoglyceride region, whereas oligomerization was most efficient in cholesterol-rich microdomains, which act as concentration devices at the cell surface. Previously, we have shown that processing of proaerolysin at the cell surface is primarily performed by the endoprotease furin . Since furin contains a canonical Tyr-based and a dileucine-like internalization signal in its cytoplasmic tail, it is thought to be internalized via clathrin-coated pits . Furin is therefore expected to be in the phosphoglyceride region of the membrane and not in cholesterol-rich microdomains. In agreement, we could not detect any proaerolysin converting activity in purified microdomains. We found that β-MCD treatment of cells, which led to the removal of ∼50% of total cholesterol, accelerated the processing of proaerolysin, presumably by increasing the lateral mobility of both furin and receptor-bound proaerolysin. In contrast, the drug did not affect the ability of the toxin to associate with detergent resistant microdomains. Our interpretation is that β-MCD extracted cholesterol from a pool that is not involved in the association of GPI-anchored proteins with microdomains. Cholesterol is indeed not only found in raft-like microdomains, but also in the fluid phosphoglyceride regions , and has also been predicted to accumulate at the edge of microdomains . In agreement with our observations, Ilangumaran and Hoessli 1998 suggested that β-MCD only affects cholesterol surrounding and outside of GPI-containing microdomains. However, effects of β-MCD on the distribution of GPI-anchored proteins have been observed also . These differences in the observed effects of β-MCD might be due to differences in cell lines and/or in culture conditions. The next crucial step in the channel formation process of aerolysin is oligomerization. Previously, we have shown that in vitro, in the absence of membranes, at physiological salt concentrations and temperature, heptamers can only form when the toxin concentration is higher than 1 μM . On target cells, however, oligomerization was found to occur even when adding a 10 5 -fold lower aerolysin concentration. Several factors might contribute to this increased efficiency of oligomerization. Binding of the toxin to its receptors directs it to its target cell. Moreover, since the estimated binding dissociation constant ( K d ) is low , it can be expected that binding also concentrates the toxin. Indeed, as discussed first by Adam and Delbrück 1968 and later by McLaughlin and Aderem 1995 , membrane binding reduces the dimensionality from three to two. Using the Guggenheim model of a surface , we calculated that toxin binding to its receptor leads to an increase in concentration by a factor of ∼1,500 (see ). Although this estimate may be approximate, it strongly suggests that binding cannot solely account for increased efficiency of oligomerization observed at the cell surface, as compared with in solution. Additional factors are likely to contribute. Here, we show that cholesterol-rich microdomains play an important role. Due to the long and saturated acyl chains of the GPI-moiety, GPI-anchored proteins have the capacity to associate transiently with lipid rafts . We found that on cells that had been treated with the cholesterol-binding drug saponin, proaerolysin lost its capacity to associate with microdomains and to cluster upon cross-linking with antibodies . Concomitantly, saponin led to a dramatic inhibition of aerolysin oligomerization, without altering toxin activation kinetics, suggesting that the concentration threshold required for heptamer formation could no longer be reached, even locally. Oligomerization could, however, be forced when adding higher amounts of toxin to saponin-treated cells, but remained far less efficient, as expected. These results show that microdomains act as concentration platforms at the cell surface, due to their ability to recruit GPI-anchored proteins, and that aerolysin has hijacked this device to suit its own purpose. When the same number of toxin receptors were dispersed at the plasma membrane and prevented from clustering by saponin treatment, oligomerization either did not occur at all or kinetics were dramatically inhibited, depending on the toxin concentration. The corollary of these observations is that having surface receptors that can cluster renders cells more sensitive to low doses of toxin. Recently, it has been proposed by Bray et al. 1998 that receptor clustering could be a cellular mechanism to control the sensitivity of a cell to a given ligand. Backed-up by a mathematical model, they proposed that the sensitivity and response range of chemotatic bacteria to attractants depend on the clustering of the chemotatic receptors on the cell surface . The present findings demonstrate experimentally that this proposal is correct for pore-forming toxins, such as aerolysin. We believe the importance of microdomains for efficient oligomerization will extend to other pore-forming proteins, both toxins and proteins, from the immune response . Vibrio Cholera cytolysin has been shown to require both cholesterol and sphingolipids to form channels in vitro . Lysenin, a pore-forming toxin for earthworms, has been shown to bind to sphingomyelin . Both these toxins, which require oligomerization for channel formation, are likely to bind to cholesterol-sphingolipid–rich microdomains on living cells. A major class of pore-forming toxins is composed of the so-called thiol-activated toxins, which include streptolysin O, lysteriolysin O, and perfringolysin O . Members of this family require cholesterol to form pores , even though cholesterol might not be the receptor, as initially thought . Cholesterol binding to these toxins probably triggers a conformational change necessary for membrane insertion. We would like to speculate that cholesterol, in addition, targets thiol-activated toxins to microdomains to increase in local toxin concentration to promote oligomerization. Clustering of thiol-activated toxins is probably even more important than for aerolysin, since the pores contain an average of 50 monomers . Similarly, the pores formed by the ninth component of complement contain ∼20 monomers . In the present work, we describe the journey of proaerolysin on the plasma membrane of the target cell and how the toxin makes use of the peculiar properties of its GPI-anchored receptors to optimize its toxic action. Importantly, GPI-anchored proteins can be found both scattered in the phosphoglyceride region of the plasma membrane and clustered in the microdomains. Indeed, only ∼35% of the GPI-anchored protein Thy-1 was found in microdomains at steady state, as judged by a single particle tracking analysis . Once bound to its receptor, proaerolysin must move about to encounter its processing enzyme furin in the phosphoglyceride region of the plasma membrane. This lateral movement is favored by the lipid anchor of the receptor. It has indeed been shown that GPI-anchored proteins have a higher lateral mobility within a phospholipid bilayer than their transmembrane counterpart . Subsequent encounters between toxin subunits, required for the aerolysin oligomerization step, is favored by the capacity of the GPI-anchored receptors to associate with cholesterol-rich microdomains and to remain confined to them for several seconds . This clustering device enables the toxin to concentrate locally and therefore to oligomerize efficiently. Cholesterol-rich microdomains, thus, appear to act as concentration platforms and offer sites of assembly for the aerolysin heptamer. In addition, microdomains might provide an ideal location for membrane insertion of the toxin. At the junctures between cholesterol-sphingolipid–rich domains and fluid phase phosphoglyceride domains, there are probably unfavorable energetic effects that locally weaken the lipid bilayer and might favor membrane penetration. The route followed by aerolysin at the cell surface nicely illustrates the highly dynamic behavior of raft components and the transitory nature of the interaction of GPI-anchored proteins with cholesterol-rich microdomains. The present work also provides functional evidence for the existence of rafts at the surface of living cells. | Study | biomedical | en | 0.999997 |
10508865 | Rat antioccludin mAb (MOC37) was described previously . Anti–VE-cadherin mAb was a kind gift from Dr. N. Matsuyoshi (Kyoto University, Kyoto, Japan). Anti–platelet endothelial cell adhesion molecule (PECAM) polyclonal antibody (pAb) was purchased from PharMingen. Mouse L cells and their transfectants were cultured in DME supplemented with 10% FCS. Two polypeptides, KYSAPRRPTANGDYDKKNYV and YSTSVPHSRGPSEYPTKNYV, which correspond to the COOH-terminal cytoplasmic domains of mouse claudin-5/TMVCF (aa 199–218) and claudin-6 (aa 200–219), respectively (a cysteine residue was added at their NH 2 termini), were synthesized and coupled via the cysteine residue to keyhole limpet hemocyanin. These peptides were injected into rabbits as antigens. Rabbit antisera against the former and latter peptides were affinity-purified on nitrocellulose membranes with glutathione S -transferase (GST) fusion proteins with claudin-5/TMVCF and claudin-6Δ to obtain anti–claudin-5/6 pAb and anti–claudin-6 pAb, respectively . To construct claudin-5/TMVCF or claudin-6 expression vectors with or without a FLAG-tag at their COOH termini, EcoRI sites were introduced at 3′ ends of claudin cDNAs by PCR, and amplified fragments were subcloned into pBluescript SK(−)–FLAG or SK(−). The inserts were excised by SaII-XbaI digestion followed by blunting with T4 polymerase, and then introduced into pCAGGSneodelEcoRI , provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan). Mouse L cells were used for transfection. Aliquots of 1 μg of each expression vector were introduced into L cells in 1 ml of DMEM using Lipofectamine Plus (GIBCO BRL). After 24- or 48-h incubation, cells were replated and cultured in DMEM containing 10% FCS and 500 μg/ml of Geneticin (GIBCO BRL) to select stable transfectants. For whole-mount staining, mouse 12.5-d embryos were killed. Samples were pretreated by microwaving in PBS for 20 s and fixed in 4% paraformaldehyde/PBS for 30 min. They were dehydrated in methanol and bleached with 30% H 2 O 2 . Samples were then rehydrated, blocked with PBS-MT (0.2% Triton X-100 and 1% skimmed milk/PBS) and incubated overnight with primary antibodies followed by secondary antibodies. HRP-conjugated goat anti–rabbit Ig (Chemicon International, Inc.) was used as a secondary antibody. They were then washed with PBS-MT and PBS-T (0.2% Triton X-100/PBS), each for 5 h. Bound antibodies were visualized by incubating with 0.025% diaminobenzidene, 0.08% NiCl 2 , and 30% H 2 O 2 in PBS-T. Mouse brain, lung, kidney, and intestine were frozen using liquid nitrogen. Frozen sections ∼6 μm thick were cut on a cryostat, mounted on glass slides, air-dried, and fixed in 95% ethanol at 4°C for 30 min followed by 100% acetone at room temperature for 1 min. They were then rinsed in PBS containing 0.2% Triton X-100 for 15 min, blocked with 1% BSA/PBS for 15 min, and incubated with primary antibodies. After washing with PBS three times, samples were incubated for 30 min with secondary antibodies. Cy3-conjugated goat anti–rat Ig (Amersham Pharmacia Biotech) and Cy2-conjugated goat anti–rabbit Ig (Jackson ImmunoResearch Laboratories, Inc.) were used as secondary antibodies. Samples were washed three times with PBS, then mounted in 90% glycerol/PBS containing 0.1% paraphenylenediamine and 1% n -propylgalate. Specimens were observed using a Zeiss Axiophot photomicroscope (Carl Zeiss, Inc.). Lysates of Escherichia coli expressing GST–claudin fusion proteins were subjected to one-dimensional SDS-PAGE (12.5%) according to the method of Laemmli 1970 , and gels were stained with Coomassie brilliant blue R-250. For immunoblotting, proteins were electrophoretically transferred from gels onto nitrocellulose membranes, which were then incubated with the first antibody. Bound antibodies were detected with biotinylated secondary antibodies and streptavidin-conjugated alkaline phosphatase (Amersham Pharmacia Biotech). Nitroblue tetrazolium and bromochloroindolyl phosphate were used as substrates for detection of alkaline phosphatase. Immunoelectron microscopy to examine freeze–fracture replicas was performed as described . The mouse lung was cut into small pieces and quickly frozen in high pressure liquid nitrogen with an HPM 010 High Pressure Freezer (BAL-TEC). The frozen samples were fractured at −110°C and platinum-shadowed unidirectionally at an angle of 45° kin Balzers Freeze–Etch System (BAF 060; BAL-TEC). The samples were immersed in a sample lysis buffer containing 2.5% SDS, 10 mM Tris-HCl, and 0.6 M sucrose (pH 8.2) for 12 h at room temperature, and then replicas floating off the samples were washed with PBS. Under these conditions, integral membrane proteins were captured by replicas, and their cytoplasmic domains were accessible to antibodies. The replicas were incubated with anti–claudin-5/6 pAb for 60 min, then washed with PBS several times. They were then incubated with goat anti–rabbit Ig coupled to 10 nm gold (Amersham Pharmacia Biotech). The samples were washed with PBS, picked up on formvar-filmed grids, and examined in a JEOL 1200EX electron microscope at an accelerating voltage of 100 kV. The GST fusion protein with the cytoplasmic domain of claudin-5/TMVCF was produced in E. coli and used as an antigen to generate specific pAbs in rabbits. Several pAbs that recognized claudin-5/TMVCF were obtained, but all of them cross-reacted with claudin-6 on immunoblotting as well as immunofluorescence microscopy . As shown in Fig. 1 A, the COOH-terminal KNYV sequence was shared between claudin-5/TMVCF and claudin-6. The GST fusion protein with the cytoplasmic domain of claudin-6 lacking these four aa (GST–claudin-6Δ) was not detected by these pAbs , indicating that they specifically recognized the COOH-terminal KNYV. These pAbs were then referred to as anti–claudin-5/6 pAbs. On the other hand, when the GST fusion protein with the cytoplasmic domain of claudin-6 was used as an antigen, several pAbs, which recognized claudin-6 but not claudin-5/TMVCF on immunoblotting as well as immunofluorescence microscopy , were obtained (anti–claudin-6 pAb). As expected, these pAbs recognized GST–claudin-6Δ . Therefore, to examine the expression and localization of claudin-5/TMVCF in various tissues, these anti–claudin-5/6 pAbs and anti–claudin-6 pAbs were used in combination; if some cells were anti–claudin-5/6 pAb-positive and anti–claudin-6 pAb-negative, we concluded that they expressed claudin-5/TMVCF. Fortunately, Northern blotting revealed that in most organs of adult mice the expression of claudin-6 was fairly restricted . We first examined the distribution of claudin-5/TMVCF in the brain, which does not contain epithelial cells but expressed claudin-5/TMVCF as detected by Northern blotting . When 12.5-d mouse embryos were labeled by whole-mount immunostaining with anti–claudin-5/6 pAb, a characteristic tree-like staining pattern was detected in the head portion . Anti–claudin-6 pAb gave no significant staining in the head portion , and a pAb specific for PECAM, a specific marker for endothelial cells of blood vessels , showed a very similar staining pattern to anti–claudin-5/6 pAb . These findings indicated that claudin-5/TMVCF was expressed exclusively in endothelial cells of all segments of blood vessels in the fetal brain. Frozen sections of adult brain were then immunofluorescently double stained with anti–claudin-5/6 pAb and anti–VE-cadherin mAb. VE-cadherin was reported to be specifically expressed in endothelial cells . As shown in Fig. 2e and Fig. f , all blood vessels were exclusively costained with these antibodies. These anti–VE-cadherin mAb-positive endothelial cells were negative for staining with anti–claudin-6 pAb . Furthermore, at higher magnification, both claudin-5/TMVCF and VE-cadherin were shown to be precisely coconcentrated at cell–cell borders of endothelial cells of blood vessels . Taken all together, we concluded that claudin-5/TMVCF is expressed and localized at cell–cell contact sites of endothelial cells of all segments of blood vessels in the adult brain. Next, we examined the distribution of claudin-5/TMVCF in the lungs, which contained both epithelial and endothelial cells and expressed fairly large amounts of claudin-5/TMVCF as detected by Northern blotting . When frozen sections of the lung were double stained with anti–claudin-5/6 pAb and anti–VE-cadherin mAb, both signals completely overlapped . In contrast, on double staining with anti–claudin-5/6 pAb and antioccludin mAb, the two signals did not overlap at all . Furthermore, anti–claudin-6 pAb gave no detectable signals (data not shown). Considering that occludin was expressed in epithelial cells but not in most of the endothelial cells in nonneuronal tissues, these findings indicated that claudin-5/TMVCF was expressed in endothelial cells of all segments of blood vessels but not in epithelial cells delineating the alveolar space in the lung. Furthermore, immunoreplica analysis with anti–claudin-5/6 pAb revealed that claudin-5/TMVCF was localized on the TJ strands of the endothelial cells of the lung . In the kidney, claudin-5/TMVCF did not appear to be expressed in endothelial cells of all segments of the blood vessels . When frozen sections of the kidney cortex were doubly-stained with anti–claudin-5/6 pAb and antioccludin mAb, some blood vessels running in the connective tissues surrounding renal tubules were stained with anti–claudin-5/6 pAb but not with antioccludin mAb . Instead, antioccludin mAb labeled TJs of distal tubules intensely . Anti–claudin-6 pAb showed no significant signals , indicating that only claudin-5/TMVCF was detected in the kidney by the anti–claudin-5/6 pAb. Judging from the density/number of the claudin-5/TMVCF–positive blood vessels, only some selected vessels appeared to be stained with anti–claudin-5/6 pAb. Frozen sections of the kidney were double stained with anti–claudin-5/6 pAb and anti–VE-cadherin mAb . Anti–VE-cadherin mAb stained numerous capillaries surrounding renal tubules and in glomeruli. These intertubular and glomerular capillaries were not stained by anti–claudin-5/6 pAb. Interestingly, afferent as well as efferent arterioles of glomeruli were reproducibly stained with anti–claudin-5/6 pAb . Furthermore, thicker arteries (probably interlobular arteries) but not veins were also intensely stained with anti–claudin-5/6 pAb . Taken together, we concluded that in the kidney, claudin-5/TMVCF constituted TJs only in endothelial cells of the arteries. The expression and distribution of claudin-5/TMVCF were also examined in other organs, among them the intestine, skeletal muscle, skin, liver, testis. In these tissues claudin-5/TMVCF was specifically detected in endothelial cells in some segments of blood vessels, but not in epithelial cells. The endothelial cell–specific localization of claudin-5/TMVCF in the intestine was presented in Fig. 5i and Fig. j . We reported previously that claudin-1 and -2 reconstituted TJ strands between adjacent transfectants when introduced into L fibroblasts . Then, we examined the ability of claudin-5/TMVCF to reconstitute TJ strands in L fibroblasts. When stable L transfectants expressing claudin-5/TMVCF were stained with anti–claudin-5/6 pAb, claudin-5/TMVCF was shown to be concentrated at cell–cell borders as planes, similar to claudin-1 and -2 . Conventional freeze–fracture electron microscopy of glutaraldehyde-fixed L transfectants revealed that huge well-developed networks of TJ strands were formed in the cell–cell contact planes . As shown previously in L transfectants expressing claudin-1 and -2 , claudin-1–induced strands were largely associated with the P-face as mostly continuous structures with vacant grooves at the E-face (P-face–associated TJs), whereas claudin-2–induced strands were discontinuous at the P - face with complementary grooves at the E-face that were occupied by chains of particles (E-face–associated TJs). As shown in Fig. 6 c, the claudin-5/TMVCF–induced TJs were an extreme case of the E-face–associated TJs; almost all particles were associated with the E-face, leaving particle-free ridges on the P-face . TJs are thought to be involved in the barrier and fence functions in both epithelial and endothelial cells, but to date no differences have been reported in the molecular architecture of TJs between epithelial and endothelial cells . In this study, we found that claudin-5/TMVCF was expressed specifically in endothelial cells, not in epithelial cells, and that this molecule constituted TJ strands in endothelial cells. The human TMVCF gene was originally found to be localized to chromosome 22q11, which is frequently deleted in velo-cardio-facial/DiGeorge syndrome patients , and we proposed to designate it as claudin-5/TMVCF since it showed significant similarity to claudin-1 and -2 . This gene was also identified as a membrane protein named MBEC1 that was expressed in the brain endothelial cells . In both studies, the concentration of the product of TMVCF/MBEC1 at TJs was not examined, probably due to the difficulty generating antibodies specific for this protein. We avoided this technical difficulty by generating and using anti–claudin-5/6 pAb and anti–claudin-6 pAb in combination. Immunofluorescence microscopy with these pAbs revealed that in the brain and the lung, the endothelial cells of all segments of blood vessels, which were positive in the anti–VE-cadherin mAb staining, expressed significant amounts of claudin-5/TMVCF. In contrast, in the kidney, veins and capillaries lacked the expression of claudin-5/TMVCF, and its expression was restricted to arteries. In addition, in all other tissues we examined, the expression of claudin-5/TMVCF was restricted to endothelial cells of some segments of blood vessels. Therefore, we concluded that claudin-5/TMVCF is an endothelial cell–specific component of TJ strands, but that in contrast to VE-cadherin , claudin-5/TMVCF is not expressed in all types of endothelial cells. Since it is widely accepted that TJs play central roles in the regulation of vascular permeability, the structure of endothelial TJs has been extensively examined, mainly by freeze–fracture electron microscopy . In general, in glutaraldehyde-fixed endothelial cells in nonneuronal tissues, TJ strands in endothelial cells were characterized by particle-free ridges on the P-face and continuous grooves at the E-face, which were densely occupied by chains of particles (E-face–associated TJs) . Interestingly, the claudin-5/TMVCF–based TJ strands reconstituted in L transfectants showed the same morphological characteristics , favoring the notion that claudin-5/TMVCF is a major constituent of endothelial TJ strands of nonneuronal tissues in situ. Furthermore, in previous freeze–fracture studies, TJs were shown to be developed also in veins and capillaries in various tissues . Therefore, some species of claudins other than claudin-5/TMVCF must be involved in the formation of TJ strands in veins and capillaries. To date, in addition to pAbs used in this study, pAbs specific for claudin-2, -3, -4, -8, -11, and -14 were available. Preliminary immunofluorescence microscopy with these pAbs showed that these claudins were not detected in endothelial cells in all of the organs we examined. Among the other claudin species (claudin-1, -7, -9, -10, -12, -13, and -15), claudin-1 is expected to be expressed in endothelial cells, since this claudin species is expressed rather ubiquitously, even in organs lacking epithelial tissues as shown by Northern blotting . However, in contrast to claudin-5/TMVCF, claudin-1 appeared to be expressed also in epithelial cells, because this claudin species was first identified from hepatocytes and was expressed in large amounts in the liver and the kidney . Further identification and characterization of claudin species expressed in endothelial cells will be important in future studies. Endothelial cells in the adult brain, which form the blood–brain barrier (BBB), are coupled by TJs of extremely low permeability that are more like those of epithelial barriers . Despite the large numbers of in vivo studies that have been performed, the developmental timing of the formation and maturation of BBB in vivo is still controversial, but the TJs of brain endothelial cells are thought to be leaky in embryos . As shown in this study, claudin-5/TMVCF was clearly detected in endothelial cells of both the fetal and adult brain , suggesting that the formation and maturation of BBB is not attributable to the developmental changes in the expression level of claudin-5/TMVCF. TJ strands of endothelial cells in the fetal brain are E-face–associated similarly to those in nonneuronal tissues as discussed above, whereas those in the adult brain are P-face–associated . Therefore, it is tempting to speculate that during development the claudin-5/TMVCF–based, E-face–associated TJ strands are modified by addition of other claudin species or by some inside-out signaling, to be switched to the P-face–associated forms. Although occludin and claudins were identified as components of epithelial TJ strands , our knowledge of the molecular architecture of endothelial TJs is limited. Occludin was reported to be expressed in large amounts in brain endothelial cells, but was undetectable in most endothelial cells in nonneuronal tissues . Now that claudin-5/TMVCF has been identified as a specific component of endothelial TJ strands, it will be possible to examine and modulate the mechanism of regulation of vascular permeability in molecular terms. For example, vascular endothelial cell growth factor has been shown to elevate vascular permeability through binding to its tyrosine kinase–type receptors flt-1 and flk-1 , and to cause disorganization of interendothelial junctions . Thus, it would be intriguing to examine whether and how activation of these receptors modulates claudin-5/TMVCF. Furthermore, it will be important to analyze the possible involvement of claudin-5/TMVCF in vasogenic edema and inflammation in various pathological states. Further detailed analyses of claudin-5/TMVCF along these lines will lead to a better understanding of the molecular mechanisms behind vascular permeability. | Study | biomedical | en | 0.999995 |
10508866 | Guinea pig anti–mouse claudin-1 pAb was raised against the GST fusion protein with the COOH-terminal cytoplasmic domain of claudin-1. Rabbit anti–mouse claudin-2 pAb, rabbit anti–mouse claudin-3 pAb, and rabbit anti–mouse claudin-4 pAb were raised and characterized, previously . These pAbs were affinity purified on nitrocellulose membranes with MBP (maltose-binding protein) fusion protein with the cytoplasmic domain of claudin-1, or GST fusion proteins with the cytoplasmic domains of claudin-2 to -4. L transfectants expressing claudin-1 (C1L), claudin-2 (C2L), and claudin-3 (C3L) were established previously , and those expressing claudin-4 (C4L) were obtained in this study. L transfectants were cultured on coverslips in DME supplemented with 10% FCS. MDCK I cells were cultured in Transwell™ chambers (polycarbonate membrane, filter pore size, 0.4 μm; Costar Corp.) in DME supplemented with 10% FCS. CPE was purified by the method of Sakaguchi et al. 1973 . The COOH-terminal fragment (184-319 amino acids) of CPE with a 10-histidine tag was produced in E . coli and purified as described previously . The cytotoxic effect of CPE on L transfectants and MDCK I cells was determined by examining their morphological alterations 24 h after addition of CPE (500 ng /ml) to the culture medium. The binding of 125 I-CPE to L transfectants expressing respective claudins was measured, and Scatchard analysis was performed as described previously . For treatment of C3L cells on coverslips, purified C-CPE was added into culture medium at a final concentration of 2.5 μg/ml. When MDCK I cells were cultured on Transwell™ chamber, purified C-CPE was added into the basolateral compartment at a final concentration of 2.5 μg/ml. These C-CPE–treated cells were processed for immunofluorescence microscopy, immunoblotting, and freeze-fracture replica electron microscopy. SDS-PAGE was performed according to the method of Laemmli 1970 , and proteins were electrophoretically transferred from gels onto nitrocellulose membranes. The membranes were soaked in 5% skimmed milk and incubated with the primary antibodies. After washing, the membranes were incubated with the second antibodies to rabbit (Amersham-Pharmacia Biotechnology), or guinea pig (Chemicon) IgG as appropriate followed by incubation with streptavidin-conjugated alkaline phosphatase (Amersham-Pharmacia Biotechnology). Nitroblue tetrazolium and bromochloroindolyl phosphate were used as substrates to visualize the enzyme reaction. MDCK I cells cultured on filters and L transfectants on coverslips were fixed with 10% trichloroacetic acid for 30 min on ice and 1% formaldehyde for 10 min at room temperature, respectively. Cells were then washed with PBS, treated with 0.2% Triton X-100 in PBS for 10 min, washed with PBS several times, and soaked in 1% BSA in PBS. Samples were incubated with primary antibodies for 30 min in a moist chamber. Cy3-conjugated goat anti–rabbit IgG (Amersham-Pharmacia Biotechnology), rhodamine-conjugated goat anti–guinea pig IgG (Chemicon) and Cy2-conjugated goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories Inc.) were used as secondary antibodies. Cells were washed three times with PBS and then mounted in 90% glycerol-PBS containing para-phenylenediamine and 1% n- propylgalate. Specimens were observed using a fluorescence Zeiss Axiophot photomicroscope (Carl Zeiss, Inc.), and the images were recorded with a Semsys™ cooled CCD camera system (Photometrics). Cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) overnight, washed with 0.1 M sodium cacodylate buffer three times, immersed in 30% glycerol in 0.1 M sodium cacodylate buffer for 2 h, and then frozen in liquid nitrogen. Frozen samples were fractured at −100°C and platinum-shadowed unidirectionally at an angle of 45° in Balzers Freeze Etching System (BAF060; BAL-TEC). Samples were immersed in household bleach, and the replicas floating off the samples were picked up on formvar-filmed grids, and examined with a JEOL 1200 EX electron microscope at an acceleration voltage of 100 kV. Morphometrical analysis was performed on freeze-fracture replica images of TJs, which were printed at a final magnification of 20,000. The mean TJ strand number was determined by taking numerous counts along a line drawn perpendicular to the junctional axis at 200-nm intervals . The complexity of TJ strand networks was defined as the number of branch points per unit length (1 μm) of TJ strands . The free end number of TJ strands was defined here as the number of free ends per unit length (1 μm) of TJ strands. Confluent monolayers of MDCK I cells grown in Transwell™ chamber were used. Transepithelial electric resistance (TER) was measured using a Millicell-ERS epithelial volt-ohmmeter (Millipore Corp.) and normalized by the area of the monolayer. The background TER of blank Transwell™ filters was subtracted from the TER of cell monolayers. For paracellular tracer flux assay, FITC-dextran with a molecular mass of 4, 10, or 40 kD (Sigma Chemical Co.) was dissolved in P buffer (10 mM Hepes, pH 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl 2 , and 145 mM NaCl) at the concentration of 1 mg/ml. MDCK I cells on filters were pretreated with 2.5 μg/ml C-CPE from the basolateral compartment for 24 h. The media in apical and basal compartments were then replaced with 200 μl of P buffer containing one type of FITC-dextran and 600 μl of P buffer, respectively. To evaluate the permeability of monolayers, basal compartment media were collected after 3-h incubation with FITC-dextran, and the amount of FITC-dextran in the media was measured with a fluorometer (excitation, 485 nm; emission, 535 nm). All values are given as mean ± SD. Statistical analysis was performed using the nonparametric Mann-Whitney test. In this study, to examine whether claudins are involved in the barrier function of TJs, as described below, we analyzed the effects of the COOH-terminal fragment of CPE on cultured MDCK I cells, which showed very high transepithelial electric resistance (TER) . First, we examined what types of claudins are expressed in this cell line. Preliminary immunofluorescence microscopic observations using available antibodies revealed that at least claudin-1 and -4 were expressed in large amounts. Since CPE was reported to bind to claudin-3/RVP1 and claudin-4/CPE-R, we focused on claudin-1 to -4 in MDCK I cells in this study. As shown in Fig. 1 A, in the total cell lysate of MDCK I cells, claudin-1 and -4 were reproducibly detected by immunoblotting as bands with expected molecular masses. When confluent cultures of MDCK I cells were immunofluorescently stained with antibodies for claudin-1 to -4, intense signals for claudin-1 and -4 and very weak signals for claudin-3 were detected at cell–cell borders, but claudin-2 was undetectable . Confocal microscopy confirmed that claudin-1 and -4 were concentrated at the most apical part of lateral membranes of MDCK I cells (data not shown). We next examined the sensitivity of claudin-1 to -4 to full-length CPE using L transfectants expressing respective claudins (C1L, C2L, C3L, and C4L cells). When purified CPE at 500 ng/ml was added into the culture medium followed by 1-h incubation, C3L and C4L cells formed characteristic bleb balloons and underwent cell death as expected from the results of previous studies , whereas either C1L or C2L cells did not exhibit any morphological changes up to 24-h incubation , suggesting differences in the affinity to CPE between claudin-1/2 and claudin-3/4. We then compared the binding kinetics of CPE to claudin-1/2 with those of claudin-3/4 by incubating L transfectants with various concentrations of 125 I-labeled CPE . Scatchard plot analyses indicated the binding of 125 I-CPE to claudin-1 and -2 to be negligible, whereas claudin-3 and -4 gave Ka values of 8.4 × 10 7 M −1 and 1.1 × 10 8 M −1 for 125 I-CPE binding, respectively . Therefore, these findings suggested that MDCK I cells mainly expressed CPE-insensitive claudin-1 and CPE-sensitive claudin-4. In good agreement, MDCK I cells exhibited typical bleb formation in the presence of 500 ng/ml CPE (data not shown). In good agreement with previous observations , the COOH-terminal fragment (184-319 amino acids) of CPE (C-CPE) did not show any cytotoxicity to C3L or C4L cells . The non-cytotoxicity of C-CPE allowed us to follow the subsequent behavior of claudin-3 and -4 after their specific binding to C-CPE within cells. Thus, in C1L, C2L, and C3L cells, we examined the effects of C-CPE on the subcellular distribution of claudin-1 to -3, respectively, by immunofluorescence microscopy, since for as yet unknown reasons in C4L cells exogenous claudin-4 was not concentrated efficiently into cell–cell borders. Similarly to claudin-1 and -2 , claudin-3 was also concentrated at cell–cell contact planes to reconstitute TJ strand networks when introduced into L fibroblasts. When these L transfectants were incubated with 2.5 μg/ml C-CPE for 4 h, claudin-3, but not claudin-1 and -2, which were concentrated at cell–cell contact planes, showed punctate distribution and gradually disappeared . We then observed the cell–cell contact planes of C3L cells before and after 4-h incubation with C-CPE by conventional freeze-fracture replica electron microscopy. As shown in Fig. 3 d, in the presence of C-CPE, the well-developed network of claudin-3–based TJ strands disintegrated into thick belt-like aggregates of intramembranous particles of various lengths, and then disappeared. Next, in MDCK I cells plated at confluent density on filters, we examined the effects of C-CPE on the expression and distribution of claudin-1 and -4 . Double-staining immunofluorescence microscopy with anti–claudin-1 and anti–claudin-4 pAbs revealed that C-CPE did not affect the subcellular distribution of claudin-1 even after 24-h incubation, but that within 4-h incubation claudin-4 began to be distributed in the cytoplasm with concomitant gradual disappearance from the junctional complex areas . At 24 h after incubation, the claudin-4 signal became undetectable, but when C-CPE was washed out at this time point, the concentration of claudin-4 at the junctional region was completely recovered within 24 h. In good agreement, immunoblotting revealed that in the presence of C-CPE, claudin-4, but not claudin-1, decreased in amount with a similar time course to the disappearance of the immunofluorescence signal of claudin-4 . These findings favored the notion that also in MDCK I cells claudin-1 and -4 were insensitive and sensitive for CPE, respectively. Furthermore, these findings were observed only when C-CPE was added to the basolateral compartment of MDCK I cells. C-CPE added to the apical compartment did not affect the subcellular distribution or the expression level of claudin-4 (data not shown). The question naturally arose as to what types of morphological changes of TJ strands are associated with the C-CPE–induced disappearance of claudin-4 in MDCK I cells. MDCK I cells were incubated with C-CPE in their basolateral compartment for 4, 8, and 24 h, fixed with glutaraldehyde, and then examined by conventional freeze-fracture replica electron microscopy. TJs in nontreated MDCK I cells were characterized by well-developed anastomosing networks of TJ strands . The mean strand number, which was determined by taking numerous counts along a line drawn perpendicular to the junctional axis , was 4.0 ± 1.3, and the complexity of TJs, defined as the number of branch points per unit length (1 μm) of TJ strands , was 11.4 ( Table ). At 4 h after incubation with C-CPE, TJ strands facing toward the basolateral membrane domains began to disintegrate to thick belt-like aggregates of intramembranous particles . Around 8 h after incubation, these belt-like particle aggregates mostly disappeared, leaving a fairly simple TJ strand network . The mean strand number and the complexity of TJs at this time point were 2.6 ± 0.9 and 5.9, respectively ( Table ). The number of free ends per unit length (1 μm) of TJ strands also increased with incubation period ( Table ). This type of simple network of TJs was maintained until 24 h after addition of C-CPE ( Table ). Next, we examined the effects of C-CPE on TER of MDCK I cells plated at confluent density on filters. When 2.5 μg/ml C-CPE was added in the apical compartment, the TER was not affected but remained at a level of 8,000–10,000 Ωcm 2 . In contrast, addition of C-CPE to the basolateral compartment resulted in an ∼4.5-fold reduction in TER from ∼9,000 Ωcm 2 to ∼2,000 Ωcm 2 within 4 h, and removal of C-CPE from the compartment induced gradual recovery of TER to the level of nontreated controls within one day . Furthermore, this C-CPE–induced reduction of TER was dose dependent . Finally, to exclude the possibility that this reduction of TER was caused by an increase in transcellular plasma membrane permeability to ions, we assessed the flux of membrane-impermeable paracellular tracers (FITC-dextran 4K, 10K, and 40K) across MDCK I cell monolayers. As shown in Fig. 6 D, C-CPE in the basolateral compartment caused an approximately twofold increase in the flux of FITC-dextran 4K and 10K, but not 40K. These findings indicated that C-CPE in the basolateral compartment downregulated the TJ barrier itself of MDCK I cells significantly with a similar time course to the disappearance of claudin-4 . In previous studies, we demonstrated that claudin-1, -2, and -11 reconstituted strands/grooves within plasma membranes, which were morphologically indistinguishable from in situ TJ strands/grooves when introduced into L fibroblasts . Furthermore, we recently confirmed that other claudin species also have the ability to reconstitute TJ stands in L fibroblasts (data not shown). In contrast, occludin, another TJ-specific integral membrane protein, did not reconstitute a well-developed network of TJ strands in L fibroblasts, although a small number of fragmented TJ strand-like structures were induced. Interestingly, when occludin was cotransfected into L fibroblasts together with claudin-1, occludin was incorporated into well-developed claudin-1–based strands . Therefore, at least from the structural point of view, we concluded that claudins, but not occludin, are major integral membrane proteins constituting the backbone of TJ strands . To date, however, there have been no reports concerning the functional aspects of claudins. In this study, to examine the functions of claudins we used Clostridium perfringens enterotoxin (CPE), which has been reported to bind specifically to claudin-3/RVP1 and claudin-4/CPE-R (see introduction). The COOH-terminal half fragment of CPE (C-CPE), which did not show any cytotoxicity, did not bind to claudin-1 or -2, and caused disintegration reconstituted claudin-3–based TJ strands in L fibroblasts, but not those based on claudin-1 or claudin-2. Interestingly, TJ strands of MDCK I cells, which were composed of at least claudin-1 and -4, were also partly disintegrated in the presence of C-CPE with concomitant disappearance of claudin-4 from TJs. This C-CPE–induced disintegration of TJ strands and disappearance of claudin-4 were associated with a rapid increase in epithelial permeability, and removal of C-CPE from the medium resulted in reconcentration of claudin-4 at TJs as well as recovery of the epithelial barrier. Taking the specificity and high affinity of the binding between C-CPE and claudin-3/4 into consideration, we concluded that claudins are not only structural but also functional components of TJ strands, which are involved in the TJ barrier. Occludin has been also shown to be involved in the barrier functions of TJs . When occludin lacking the COOH-terminal cytoplasmic domain was introduced into MDCK cells, endogenous occludin as well as introduced occludin mutant were aggregated in a punctate manner at cell–cell borders, but the morphology and continuity of TJ strands were not affected . Since claudins are now thought to be the major structural components of TJ strands, in these MDCK transfectants it is likely that the introduced occludin mutant removed endogenous occludin from the claudin-based TJ strands to aggregate in a punctate manner. Interestingly, in these transfectants, TER was not significantly affected, although the permeability for dextran was increased, suggesting that occludin itself is not directly involved in development and maintenance of TER. In contrast, as shown in this study, when claudin-4 was selectively removed from TJ strands in MDCK cells, their TER dropped rapidly. These findings indicated that claudins, but not occludin, play a central role in TJ barrier function. In good agreement, occludin-deficient visceral endoderm appeared to bear functional TJs , and the very tight TJs in Sertoli cells in human testis lacked occludin . The molecular mechanism behind the C-CPE–induced disintegration of TJ strands in C3L and MDCK cells remains unclear. There are two alternative possible mechanisms. First, the direct binding of C-CPE to claudin-3 or -4 within TJ strands may induce depolymerization of the strands themselves, which may be homopolymers of claudin-3 in C3L cells and heteropolymers of at least claudin-1 and -4 in MDCK I cells . In L transfectants expressing exogenous claudins, strands were not observed on the free surface of the plasma membranes as a single strand, but instead were restricted to cell–cell adhesion sites to form strands paired laterally with those in apposing membranes . Therefore, if C-CPE causes dissociation of these paired strands into single strands by binding to the extracellular loops of claudin-3 or -4, it would result in the further depolymerization of single strands. The characteristic morphological changes of TJ strands of C3L and MDCK cells during the course of C-CPE–induced disintegration favored this depolymerization mechanism. Alternatively, C-CPE may bind to claudin-3 and -4 molecules on nonjunctional areas, which constitute the nonpolymerized pool, and this binding may suppress polymerization of these claudins into strands. If the network of TJ strands is dynamically maintained by the equilibrium between polymerization and depolymerization, this sequestering mechanism could explain the C-CPE–induced destruction of TJ strands. Interestingly, C-CPE downregulated the TJ barrier only when it was applied in the basolateral compartments of MDCK cells . The sequestering mechanism, but not the depolymerization mechanism, would readily explain this finding if claudins target the basolateral membranes as monomers or small oligomers and are then integrated into TJ strands similarly to occludin . These two depolymerization and sequestering mechanisms must be evaluated in future studies, but in either case C-CPE binding appeared to facilitate the degradation of claudin-4 (and probably also claudin-3) within cells as shown by immunoblotting in Fig. 4 B. In good agreement, the diffuse cytoplasmic distribution of claudin-4 in MDCK cells was observed within 4 h after addition of C-CPE to the culture medium . It should be clarified how these C-CPE–bound claudins were internalized and degraded in future studies. The downregulation of TJs induced by binding of C-CPE to specific claudins does not appear to be the principal molecular mechanism responsible for the cytotoxicity of CPE itself. It is widely accepted that the NH 2 -terminal half of CPE increases membrane permeability by forming small pores in the plasma membrane . Therefore, binding of the COOH-terminal half of CPE to specific claudins may facilitate pore formation by its NH 2 -terminal half. Furthermore, the reason remained unclear why CPE, a luminal enterotoxin, binds only to the basolateral membranes of epithelial cells, but not to their apical membranes. It also remained elusive what types of claudins expressed in intestinal epithelial cells are responsible for the CPE-binding in situ. As shown in this study, however, C-CPE can be used as a powerful tool to modulate TJ barrier function. C-CPE bound with high affinity to claudin-3 and -4 but not to claudin-1 or -2, suggesting that claudins are subclassified into CPE-sensitive and nonsensitive types. Therefore, as CPE removes the CPE-sensitive claudins selectively from TJ strands, the barrier function of TJ strands containing larger amounts of CPE-sensitive claudins may be affected more strongly. Considering that the binding region for claudin-4/CPE-R can be narrowed down to the 30–amino acid sequence in the COOH-terminal half of CPE , it may be worthwhile searching for ∼30–amino acid oligopeptides that specifically bind to individual claudin species. Selective removal of specific claudin species from TJ strands with the combination of these oligopeptides would provide a new way to modulate the TJ barrier function in situ and to improve bioavailability of drugs to targeted organs. | Study | biomedical | en | 0.999993 |
10510079 | A DNA sequence containing the extracellular domain of T1/ST2 was PCR amplified and cloned into an expression vector containing the CD5 signal sequence and the hIgG1 constant region. COS cells were transiently transfected with T1/ST2-Ig cDNA, and the recombinant proteins were purified via affinity chromatography (protein A). The purity of T1/ST2-Ig was subsequently assessed by Coomassie-stained SDS-PAGE and determined to be >90%. The identify of the T1/ST2-Ig was further confirmed by mass spectrometry by comparing the trypsin peptides generated from the extracted gel band with a theoretical trypsin digest (peptide mass fingerprinting by matrix-associated laser desorption ionization time-of-flight [MALDI-TOF] analysis). We also PCR amplified a DNA sequence containing the extracellular domain of a novel Ig superfamily member identified in a murine brain cDNA library unique to a Millennium Proprietary Database. This gene, termed H1, was cloned into the identical vector as T1/ST2 containing the CD5 signal sequence. H1-Ig failed to bind to either T, B, or dendritic cells and unlike T1/ST2, was not detectable by PCR analysis in resting or activated Th1 or Th2 cells (data not shown); therefore, we used H1-Ig as an irrelevant control reagent in some experiments. The rat anti-T1/ST2 mAb (clone 3E10) was generated and characterized as described elsewhere 12 . Mice expressing the transgene for the DO11.10 α/β-TCR, which recognizes residues 323–339 of chicken OVA in association with I-A d , were provided by Dr. D. Loh (Washington University, St. Louis, MO 16 ). Naive TCR transgenic CD4 + T cells were isolated as described 12 and cultured in complete RPMI 1640 with OVA 323–339 (10 μg/ml) and mitomycin C–treated splenocytes in a 1:5 ratio. For Th1 phenotype development, recombinant murine IL-12 (10 ng/ml) and neutralizing anti–IL-4 mAb (11B11, 40 μg/ml; R&D Systems) were added, and for Th2 development, recombinant murine IL-4 (10 ng/ml) and neutralizing polyclonal anti–murine IL-12 (TOSH-2, 3 μg/ml; Endogen) were used. After 5–7 d, cells were washed and restimulated up to three times under identical polarizing conditions. Cells were stained after 5–7 d with digoxigenin-labeled 3E10, and the number of T1/ST2-positive cells was detected by antidigoxigenin Fab fragments (Boehringer Mannheim) conjugated to PE. Expression of T1/ST2 was analyzed on a FACSCalibur™ (Becton Dickinson). To determine the cytokine profile at each time point, cells were washed and a viable CD4 population was isolated over a ficoll gradient and activated (2 × 10 5 /well) in a 96-well plate for 24 h using plate-bound CD3 (2C11, 10 μg/ml; PharMingen). IL-4 and IFN-γ levels were measured in the supernatant by ELISA (Endogen). CD4 + T cells from DO11.10 α/β-TCR mice were activated as described above in the absence of exogenous cytokines (termed neutral conditions) or in the presence of IL-12 or IL-4, together with T1/ST2-Ig (100 μg/ml) or hIg as the appropriate isotype control. Cells were washed and replated in 96-well plates (5 × 10 4 /well) together with 10 5 splenocytes/well and restimulated with OVA peptide, and cytokines were measured 48 h later. To determine the effect of T1/ST2-Ig in effector cells, Th1 and Th2 cells were reactivated with OVA peptide in the presence of either hIg or T1/ST2-Ig. In some experiments, H1-Ig was used as a second control reagent for the specificity of T1/ST2-Ig. Recipient normal BALB/c mice were injected intravenously with 2 × 10 6 Th1 or Th2 effector cells. 24 h later, mice were exposed to an aerosol of OVA (50 mg/ml) for 20 min on two consecutive days. 1 h before allergen exposure, mice were injected intravenously with either 20 or 100 μg of mAb against T1/ST2 or 100 μg of rat IgG1. 24 h later, the trachea was cannulated, a bronchoalveolar lavage (BAL) was performed as described 17 , and cytokine levels in the lavage fluid were measured by ELISA. A second series of experiments was also performed using T1/ST2-Ig (100 μg i.v.) or hIg as the appropriate isotype control. Cytospin preparations were prepared (Shandon), stained with Giemsa reagent, and a total of 200 cells were counted differentially. Lungs were then removed, inflated with 10% neutral buffered formalin, and paraffin embedded. 4-μm sections were stained for cyanide-resistant peroxidase and counterstained with hematoxylin using standard techniques. Airway inflammation was determined by semiquantitative scoring using an arbitrary system where a score of +1 represents one small focus of cells and +5 indicates widespread infiltrates. All scoring was performed by an investigator (C. Lloyd) unaware of the treatment. Airway responsiveness was measured in Th2 recipient mice 24 h after the last aerosol challenge by recording respiratory pressure curves by whole body plethysmography (Buxco; EMKA Technologies) in response to inhaled methacholine (Sigma Chemical Co.) at concentrations of 2.5–20 mg/ml for 1 min. Airway responsiveness was expressed in enhanced pause ( P enh ), a calculated value, which correlates with measurement of airway resistance, impedance, and intrapleural pressure in the same mouse: P enh = ( t e / t r1 ) × Pef/Pif ( t e = expiration time, t r = relaxation time, Pef = peak expiratory flow, Pif = peak inspiratory flow) 18 . Male BALB/c mice (15–20 g) were immunized intraperitoneally with 7.5 μg of OVA and 1.5 mg AI(OH) 3 in saline on days 0 and 7. On days 14 and 21, the mice were challenged with aerosolized OVA (10 mg/ml) for 1 h. Control mice were challenged with PBS instead of OVA. 1 h before antigen sensitization and challenge, the mice were injected with 100 μg of mAb against T1/ST2 or 100 μg of rat IgG1. 24 h after the second challenge, a BAL was performed and IL-5 levels in the BAL fluid were measured. Serum OVA-specific IgE was determined by specific ELISA. The percentage of T1/ST2-positive cells increased under Th2 polarizing conditions from 5.2% after primary restimulation to 41% after tertiary restimulation , and correlated with an enhanced capacity of cells to secrete IL-4 upon restimulation (data not shown). Naive cells and Th1 effector cells fail to express T1/ST2. These results extend our previous observations that the majority of IL-4– and IL-5–producing cells either under bulk culture conditions 12 or ex vivo from Th2-dominated immune responses 19 are contained within the T1/ST2-positive cell populations. Taken together, these data suggest that T1/ST2 is a useful surface marker for identifying IL-4– and IL-5–producing cells in vitro and in vivo. To determine whether T1/ST2 plays a critical role as a signaling molecule required for Th2 function, experiments were performed using a T1/ST2-Ig fusion protein. Under neutral conditions, cells acquired the capacity to secrete high levels of IL-4 (≈1,700 ng/ml), IL-5 (≈1,600 ng/ml), and IFN-γ (≈2,500 pg/ml) upon restimulation. T1/ST2-Ig treatment inhibited IL-4 and IL-5 secretion by >70% and resulted in a 10-fold augmentation in IFN-γ production. Under Th2 polarizing conditions, cells produced equivalent amounts of IL-4 and IL-5 as in neutral conditions, but produced only ≈300 pg/ml of IFN-γ. In the presence of T1/ST2-Ig, Th2 cytokine production was also reduced, and a modest but reproducible increase in IFN-γ secretion (≈1,500 pg/ml) was observed. In contrast to these observations, when cells were cultured in the presence of IL-12, inhibition of T1/ST2 failed to modify IFN-γ production. These results are in some respects similar to recent data generated using either CTLA-4–Ig fusion protein to inhibit CD28/B7 interactions 20 or B7-deficient APCs 21 . However, in contrast to CD28-mediated costimulation, which is required for optimal secretion of both IL-4 and IFN-γ when cells were cultured under neutral conditions 21 , inhibition of T1/ST2 resulted in skewing of the immune response from a Th2 to a Th1 phenotype. Moreover, while the absence of CD28 costimulation results in an attenuation of IFN-γ and IL-4 secretion when cells are cultured under either Th1 or Th2 polarizing conditions, respectively, inhibition of T1/ST2 signaling selectively inhibited cytokine secretion from Th2 cells without modifying IFN-γ secretion from Th1 cells . These data suggest that T1/ST2 delivers an important signal instructing naive cells to switch to Th2 cytokine production. To determine the requirement of T1/ST2 signaling for activation of effector cells, Thp cells were differentiated for two rounds of polarization to Th1 or Th2 effector populations. Effector populations were then activated with peptide and APCs in the presence of different concentrations of T1/ST2-Ig. Under these circumstances, blockade of T1/ST2 signaling reduced cytokine production from Th2, but not Th1 effector cells in a dose-dependent manner . The specificity of the T1/ST2-Ig protein is further supported by experiments with the control H1-Ig protein. These studies are in marked contrast to recent data generated using B7-deficient APCs demonstrating that cytokine production from Th1 and Th2 effector cells, respectively, is largely independent of CD28/B7-mediated costimulation 21 . Taken together, our data suggest that signaling through T1/ST2 can account, at least in part, for CD28/B7-independent activation of Th2 but not Th1 effector cells. We next determined whether T1/ST2 contributes to a nascent Th2-dominated immune response in vivo. Mice were immunized systemically with antigen in adjuvant before allergen provocation, and the cellular and humoral responses were evaluated. Anti-T1/ST2 mAb was effective in inhibiting allergen-induced lung eosinophilic inflammation, IL-5 production, and the induction of OVA-specific IgE . Taken together, our data demonstrate that T1/ST2 is a critical regulatory molecule for both cellular and humoral allergic inflammation in mice in vivo. While the above data demonstrate an important role for T1/ST2 in a Th2-dominated response induced by antigen and adjuvant, it is possible that the observed effects on airway inflammation are not mediated via suppression of Th2 cells, as other cell types, including mast cells, also express T1/ST2 22 . To address this issue, we next used a model of Th1 and Th2 cell adoptive transfer 23 . Aeroallergen provocation of Th1 or Th2 effector cell recipient mice resulted in either a neutrophilic or eosinophilic lung mucosal inflammatory response, respectively 23 . Inhibition of T1/ST2 in OVA-exposed Th2 recipient mice with either anti-T1/ST2 mAb or T1/ST2-Ig ( Table ) inhibited the secretion of IL-4, IL-5, IL-6, and IL-13 in the BAL fluid by >90%. Intriguingly, IL-10 secretion was independent of T1/ST2, suggesting either that the majority of IL-10–producing cells are from a population distinct from cells that produce other Th2 cytokines or that the mechanisms of IL-10 secretion are regulated differently from other Th2 cytokines. Administration of anti-T1/ST2 mAb or T1/ST2-Ig also markedly suppressed eosinophilic inflammation of the airways as assessed both histologically and by analysis of the number of eosinophils in the BAL fluid . In contrast to the effects of anti-T1/ST2 mAb in Th2-mediated inflammation, inhibition of T1/ST2 did not modify Th1 effector responses as revealed either by IFN-γ secretion or Th1-mediated neutrophilic lung inflammation . Likewise, using whole body plethysmography 18 , both anti-T1/ST2 mAb and T1/ST2-Ig treatment suppressed the development of airway hyperresponsiveness induced by OVA challenge in Th2 recipient mice or in the active immunization model (data not shown). However, whether the ability of T1/ST2 to suppress airway hyperresponsiveness is secondary to attenuated eosinophilic inflammation or is via the suppression of other key effector molecules such as IL-13 24 25 remains to be determined. In conclusion, our data suggest that T1/ST2 is more than a useful marker for detecting Th2 cells, but plays a crucial role in the differentiation to and activation of Th2, but not Th1 cells. These in vitro observations are supported by in vivo data that inhibition of T1/ST2 signaling attenuates Th2-mediated inflammatory responses without affecting Th1-mediated inflammation. Our data add to the increasing appreciation of IL-1 receptor superfamily members as central regulators of a number of key events in both innate and adaptive immunity 10 13 14 15 . | Study | biomedical | en | 0.999997 |
10510080 | V H 12 Tg mice (6-1) were previously generated 10 and maintained in our pathogen-free animal facility at the University of North Carolina (UNC) by backcrossing to C.B17 mice. Offspring carrying the transgene were identified by PCR of tail genomic DNA as previously described 10 . Mice homozygous for the deletion of the κ locus were provided by GenPharm International 21 and bred to 6-1 mice to obtain 6-1/κ −/− mice. The κ L chain repertoire was determined for subsets of PtC-binding and -nonbinding lymphocytes from 6-1 mice. Splenic lymphocytes were stained with anti-B220–PE antibodies and liposome-encapsulated carboxyfluorescein, and the B220 + cells that were PtC bri , PtC int , and PtC neg were sorted on a MoFlo high speed sorter (Cytomation, Inc.). Total RNA was extracted from ∼10 6 sorted cells using TRIzol (Life Technologies, Inc.) according to the manufacturer's protocol. The RNA was subjected to RT-PCR using 5′ RACE (rapid amplification of cDNA ends; version 2.0; Life Technologies, Inc.). For reverse transcription, we used a GSP1 custom primer, GGGGTAGAAGTTGTT, that anneals to the Cκ encoding sequence ∼80 bases 3′ of the J–Cκ junction. For the PCR, we used the 5′ RACE anchor primer and a custom GSP2 primer, CAUCAUCAUCAUCTGAGGCACCTCCAGATGTTA, that anneals to a region of the Cκ sequence 50 bases from the J–C junction. 40 cycles of amplification were performed. The conditions for the PCR were 94°C for 1 min, annealing at 55°C for 1 min, and primer extension at 72°C for 1.5 min. The final extension was at 72°C for 5 min. The amplification product was cloned into the pAMP1 vector using the CloneAmp ® pAMP1system (Life Technologies, Inc.) according to the manufacturer's protocol. Plasmid DNA was isolated from randomly picked colonies and sequenced using the Sequenase II kit (Stratagene, Inc.) or the UNC Automated DNA Sequence Facility. The oligonucleotide used to prime sequencing was GGCTCTGACTAGATCTGCAAGAGAT. Sequence comparisons and analysis used DNASIS 2.5 software and the BLAST (Basic Local Alignment Search Tool) sequence search facility (GenBank). For the sorting of B cells for adoptive transfer, spleen cells from 6-1 mice were stained for B220 and CD23 and sorted for B220 + CD23 − cells. Sorting was done using the MoFlo high speed sorter (Cytomation, Inc.). Approximately 3–5 × 10 6 cells were transferred intravenously to unirradiated C.B17 mice. Spleens were taken after 24 h and after 7 d for sectioning and analysis by immunofluorescence microscopy as described below. The antibodies used for flow cytometry and the method for staining were as previously described 10 18 . The cells were analyzed using a FACScan™ (Becton Dickinson) with hardware interface and acquisition and analysis software from Cytomation, Inc. All data represent cells that fall within the lymphocyte gate determined by forward and 90° light scatter. All contour plots are 5% probability. For immunofluorescence microscopy, spleens were imbedded in TBS compound (Triangle Biomedical Sciences) and flash frozen in liquid nitrogen and 2-methylbutane. Frozen sections were air dried and fixed in acetone for 2 min and subsequently washed with 1× PBS. Blocking was performed for 1 h at room temperature with normal rat and mouse serum. The first step staining was performed for 1 h at room temperature with anti-IgM a (or -IgM b )–FITC and anti-CD23–biotin. After incubation, sections were washed two times with 1× PBS and then stained with anti-CD3–PE and streptavidin–Cy5, washed with PBS, and mounted in Fluoromount G (Southern Biotechnology Associates, Inc.). Slides were examined with a Leica TCS-NT confocal laser scanning microscope (Leica Inc.) equipped with argon and helium-neon lasers. Photomultiplier tube voltages and laser powers were set to eliminate the background signal given by the IgG isotype and streptavidin–Cy5-only controls. Images from three different fluorescent channels were recorded simultaneously. Image processing was performed with the Leica TCS-NT proprietary software and Adobe Photoshop (Adobe Systems, Inc.). Gene transfections were done as described previously 22 . In brief, μ expression vectors were transfected into L chain–only hybridoma cell lines. These hybridoma lines were J558L (λ), 4A9 (VκRF), 2-12 (Vκ31), 1E5 (Vκ8), D35.2 (Vκ8), and CH12.2b4 (Vκ10) 22 . An H chain loss mutant of the anti-Sm hybridoma 1-8C2 23 , provided by M. Borrero (UNC), was used to test association with Vκ1A L chains. Vκ4/5H and Vκ21C L chain–only cell lines were not available. Therefore, we cotransfected into P3-X63-Ag8.653 myeloma cells with the 10/G4 or 2-12 expression constructs with L chain expression vectors containing Vκ4/5H or Vκ21C rearrangements as described 22 . To test whether a complete Ig molecule was formed, supernatant was subjected to ELISA using microtiter plates coated with polyclonal goat anti–mouse μ (Southern Biotechnology Associates, Inc.) and alkaline phosphatase–labeled polyclonal goat anti–mouse κ (Southern Biotechnology Associates, Inc.) to develop the reaction. In those cases where Ig secretion was not detected, the production of H and L chains was confirmed by ELISA using cell lysates and the polyclonal goat anti–mouse μ– or polyclonal goat anti–mouse κ-coated plates as above. The former were developed with phosphatase-labeled polyclonal goat anti–mouse μ to detect H chain, and the latter were developed with phosphatase-labeled goat anti–mouse κ to detect L chain. OD readings were determined with an automated plate reader (Emax; Molecular Devices). We have previously described the PtC-specific B-1 cells of V H 12 Tg (6-1) mice 10 . As a result of antigen-driven clonal expansion, the number of these cells is considerable, comprising 60–90% of the splenic B cell population in adults 10 . They can be detected by staining with a liposome probe that contains PtC as a membrane component and that encapsulates carboxyfluorescein 8 . These cells stain brightly with liposomes (PtC bri ) and, as described previously 10 18 , are B220 low IgM high CD23 − , and most express CD5 and CD43 . Based on the liposome staining, there are at least two other B cell populations in 6-1 mice , cells that do not stain with liposomes (PtC neg ) and cells that have an intermediate level of liposome staining (PtC int ). The PtC neg and PtC int populations are about equal in size and together account for 10–30% of splenic B cells. Most cells of both populations are B-0, i.e., CD23 + CD5 − CD43 − B220 high . Some cells of both populations are CD23 − . This population is likely to include immature B cells but may also include B-1 cells, as the PtC neg and PtC int populations appear to include cells that express CD43. Inasmuch as there are essentially no 6-1 splenic B cells that express IgM b 10 , we attribute the differences in liposome binding and segregation to the B-0 or B-1 subsets to differences in the L chain. The Vκ repertoire of 6-1 splenic B cells was determined independently for sorted PtC neg , PtC int , and PtC bri B cell populations. mRNA was isolated from each population and used in a κ-specific RT-PCR. cDNA clones were generated, and randomly selected clones were sequenced to identify the Vκ and Jκ gene segments. Sequence analysis indicates remarkable similarities in the Vκ repertoire among these populations. All but two of the cDNA clones from the PtC bri B-1 cells and three of the clones from the PtC int B-0 cells are of the identical Vκ gene (designated Vκ4/5H; reference 24 ) . This is the same Vκ gene expressed by PtC-specific lymphomas and peritoneal B-1 cell hybridomas 19 . In contrast, the Vκ genes cloned from the PtC neg population are more heterogeneous . 57% of the repertoire consists of non-Vκ4/5 genes from 12 Vκ groups. The remaining 43% are Vκ4/5 genes, and of these, 68% are Vκ4/5H . Combining the PtC neg and PtC int B-0 populations, we estimate that 66% of B-0 cells use a member of the Vκ4/5 group and 59% use Vκ4/5H. Such a bias among B-0 cells is not evident in non-Tg littermate mice, as only two (6%) of the B-0 sequences express a Vκ4/5 gene, neither of which are Vκ4/5H . Thus, Vκ4/5H dominates both the B-0 (PtC neg and PtC int ) and B-1 subsets in 6-1 mice as a consequence of V H 12 expression. These three populations of B cells in 6-1 mice are distinct in Jκ use. The PtC bri B-1 cells use predominantly Jκ2 and Jκ4 , as is true of PtC-specific lymphomas and hybridomas 19 . This bias undoubtedly reflects selection for this specificity and expansion in the B-1 subset. PtC int B-0 cells use Jκ2 almost exclusively. We attribute this to the fact that a tyrosine at the Vκ–Jκ junction, which is encoded by Jκ2, results in PtC int binding. Vκ4/5H-expressing PtC neg B-0 cells use Jκ2, -4, and -5, with Jκ5 used at almost twice the frequency as Jκ2 . Notably, almost none of the Vκ4/5H-expressing B cells from any of these populations use Jκ1. This contrasts with the predominant use of Jκ1 and Jκ2 by non-Tg B-0 cells and even by non-Vκ4/5 genes from the PtC neg B-0 cells . The Vκ4/5H L chains of each population have a characteristic CDR3 sequence. Most (67%) of the Vκ4/5H rearrangements from PtC bri B-1 cells encode a charged amino acid (primarily arginine) at the Vκ–Jκ junction, position 96 (R96) 25 , although several have the nonpolar residues leucine and phenylalanine at this position . PtC-specific hybridoma and lymphoma antibodies have R96 or L96 19 , confirming that anti-PtC B cells use these junctions. PtC int rearrangements are the most homogeneous in their encoded CDR3. Nearly all (∼90%) encode Y96. Jκ2 is the only Jκ that encodes Y96. That this residue can confer binding to PtC is indicated by the anti-PtC lymphoma CH32 that has Y96 19 . The presence of Y96, along with the lower IgM levels inherent to B-0 cells, offers an explanation for the weak liposome staining by these cells. The non-Vκ4/5H CDR3 sequences of the PtC neg population are quite heterogeneous in length and sequence (data not shown), reflecting the fact that most of the L chain CDR3 is encoded by Vκ. The CDR3 regions of Vκ4/5H L chains commonly have a nonpolar, noncharged amino acid at position 96 and in some cases are one or two amino acids shorter than the majority of those in the PtC int and PtC bri populations. Paradoxically, many Vκ4/5H junctional sequences from PtC neg cells are identical to sequences from PtC bri cells. Four sequences encode L96 (and are nine amino acids in length), seven encode F96, and four encode R96. The presence of rearrangements compatible with PtC binding in this population are unlikely to be due to a contamination from other populations during the sort for the following reasons: sort contamination could come from two sources, the inability to resolve cells from neighboring populations and machine error, in which an incorrect cell not necessarily from a neighboring population is sorted. In the case of the former, PtC int cells would most likely contaminate PtC neg cells. But this is not the case, as the potential contaminants in the PtC neg population do not encode Y96. In the case of machine error, the most common contaminant would be from the PtC bri population, as it is the largest population. We rule this out as an explanation for two reasons. First, by this scenario, both the PtC neg and PtC int populations should be contaminated with PtC bri cells, but the PtC int population is almost completely lacking in sequences that could derive from the PtC bri population. Second, the potential PtC-specific sequences in the PtC neg population are not representative of the PtC bri population; the former are predominantly L96 and F96, whereas the latter are predominantly R96. Thus, the potential PtC-specific sequences in the PtC neg population are not contaminants. We calculate from these data that ∼18% of the PtC neg repertoire, which would amount to 12% of the total B-0 repertoire, could be PtC binding. One possible explanation for the restricted Vκ repertoire expressed by V H 12 B cells is that V H 12 is unable to associate with all L chains. To test this possibility, we transfected a 10/G4 V H 12–D–J H 1 expression construct into several L chain–only-expressing cell lines, or cotransfected it with different L chain expression constructs into a nonexpressing cell line, and assayed for secretion of IgM. This rearrangement is identical to that used to generate 6-1 mice. As shown in Table , we were unable to detect secreted antibody with λ1 and 5 of the 8 κ chains. In those cases in which secreted antibody was not detected, we could detect cytoplasmic H and L chains. Thus, most L chains failed to associate with V H 12 and were considered nonpermissive. The inability of V H 12 to associate with λ1 chains was confirmed in vivo by generating 6-1 mice that lacked an intact κ locus (6-1/κ −/− ). The B cells in these mice can only use λ chains. As seen in Fig. 6B cells are rare in both the spleens and bone marrow of these mice, indicating that λ1 is nonpermissive in vivo, as are probably most other λ chains, leading to cell death in the bone marrow. The few B cells present in the spleen use λ L chains and appear to be immature, i.e., CD23 − CD5 − CD43 − (data not shown ). Thus, the limited ability of V H 12 to associate with L chains affects B cell production. 6-1 B-0 and B-1 cells are distinct in specificity and Vκ gene use. To determine whether there is also a histological distinction between B-0 and B-1 cells within the spleen, we prepared splenic sections from 6-1 and control mice for histological comparison. Spleen morphology in 6-1 mice, as shown by hematoxylin and eosin staining, does not appear to be different from non-Tg control littermates (data not shown). In the white pulp, follicular structures are associated with periarteriolar lymphatic sheaths (PALS) and are separated from the red pulp by lymphatic sinuses. Considering that 60–70% of the splenic B cells in 6-1 mice are B-1, it is likely that most if not all B-1 cells are in follicles. To determine if B-1 cells occupy splenic follicles, we analyzed spleen sections from 6-1 and non-Tg littermate mice by confocal scanning laser microscopy. Sections were stained with antibodies specific for IgM a or IgM b and CD3 to identify B and T cells, respectively. To discriminate between B-0 and B-1 cells, we used antibodies to CD23, as CD23 is present on B-0 cells but not B-1 cells. As can be seen in Fig. 7 B, 6-1 mice have T cell–rich PALS that are adjacent to B cell–rich follicles. In addition, these follicles have identifiable marginal zones. Most follicles have B cells that do not stain for CD23 , indicating that they contain B-1 cells, whereas follicular B cells from control non-Tg spleen sections stained simultaneously demonstrate unambiguous expression of CD23 . Thus, B-1 cells occupy splenic follicles in 6-1 mice. CD23 + B-0 cells can be seen in splenic sections of 6-1 mice, but, surprisingly, they are concentrated to a subset of follicles rather than intermixed with B-1 cells in all follicles . The ability of B cells to occupy follicles can be affected by the presence of B cells of diverse specificity 26 . To determine whether PtC-specific B-1 cells enter follicles in the presence of a majority of heterogeneous B-0 cells, we adoptively transferred sorted B220 + CD23 − B-1 cells from 6-1 mice to non-Tg littermates. At 24 h and 7 d after transfer, mice were killed and spleens taken for histological and flow cytometry analyses. At the time mice were killed, essentially all of the recovered transferred cells were PtC bri B-1 cells ( CD23 − CD5 + CD43 + ) (data not shown) . As can be seen in Fig. 7D and Fig. E , IgM a B cells are visible in follicles and PALS at both time points. On day 7, numerous IgM a bright cells showing cytoplasmic IgM staining are present in the red pulp . Similar cells are seen in 6-1 mice . These cells were not seen at 24 h, suggesting that some B-1 cells have differentiated to plasmablasts by 7 d after transfer and have migrated to the red pulp. Many of these cells are present in clusters of two to five cells , suggesting recent cell division in the red pulp. The role played by the Ig receptor specificity in the induction of the B cell developmental program is a well established immunological paradigm. Our data show here that the specificity of the cell surface Ig receptor is not only important in the developmental steps taken among conventional B-0 cells but also in the segregation of cells to the B-0 and B-1 subsets. Our previous analysis of anti-PtC Tg mice supports the idea of an antigen-driven differentiation of cells from B-0 to B-1. This was suggested by the observation that segregation of PtC-specific B cells to the B-1 subset is intact in 6-1 mice and in 6-1/Vκ4/5 double-Tg mice in which essentially all developing B cells are PtC specific 10 . These data argue that the mechanism of segregation operates after Ig gene rearrangement. The differentiation of B-1 cells was more directly addressed by combining the V H 12 and Vκ4 transgenes with the xid mutation 20 . The xid mutation is a loss of function mutation in the gene for Bruton's tyrosine kinase 27 28 29 30 that causes a disruption in BCR signaling. Among other deficiencies, xid mice have few B-1 cells 1 . V H 12/Vκ4 double-Tg mice with the xid mutation exhibit a significant deficiency in B-1 cell development as expected 20 . However, the majority of splenic PtC-specific B cells are B-0, not B-1, revealing the existence of a differentiative pathway from B-0 to B-1 that is dependent on signals initiated by the BCR. We have recently demonstrated this differentiative pathway in anti-PtC Tg, non- xid mice by manipulation of PtC-specific cells that are at intermediate differentiative stages in this pathway (Arnold, L.W., S.K. McCray, C. Tatu, and S.H. Clarke, manuscript submitted for publication). Viewed in this context, we interpret the segregation of 6-1 B cells to be based on their ability to bind PtC. All newly differentiated B cells from the adult bone marrow are B-0. However, those that bind PtC with high affinity (PtC bri ) are induced to become B-1, whereas those that bind PtC weakly or not at all (PtC int and PtC neg , respectively) are not signaled sufficiently and remain B-0. Among 6-1 cells that express a 10/G4 V H 12 H chain and Vκ4/5H L chain, the ability to bind PtC is dependent on the amino acid at the Vκ–Jκ junction, position 96. These data therefore disprove any notion that V H or Vκ gene expression plays a role in segregation and demonstrate that the level of PtC binding determines differentiation to B-1. This is further evidence that segregation to B-1 occurs after Ig gene rearrangement. An unexpected finding from this analysis was the presence of Vκ4/5H rearrangements in the PtC neg population that are identical to some in the PtC bri B-1 population. Because IgM − cells exist among the PtC neg population, it is plausible that these rearrangements derive from PtC bri cells that have lost surface IgM and are therefore sorted with the PtC neg population. IgM − cells are ∼20% of the PtC neg population, similar to the 18% estimate made from the sequence analysis. Loss of surface Ig can occur in B cells undergoing cell division. For example, rapidly dividing germinal center centroblasts do not express surface Ig 31 . A similar downregulation may occur in dividing B-1 cells or in cells differentiating to B-1. Alternatively, these cells could be plasmablasts that have lost surface IgM, such as the cells seen in the red pulp in 6-1 mice and in normal mice after adoptive transfer of PtC bri B-1 cells . Upon differentiation in 6-1 mice, PtC-specific B-1 cells reside in splenic follicles and in fact occupy most splenic follicles, as they are in the majority. However, it is interesting that B-0 and B-1 cells segregate to different follicles. Whether this occurs in non-Tg mice is unknown. B-1 cells are not excluded from entry into a follicle composed mostly of B-0 cells as are other autoreactive B cells 26 , indicating that exclusion from B-0 follicles is not the basis for segregation. Perhaps B-0 cells are excluded from B-1 follicles, or this segregation reflects competition between B-0 and B-1 cells during the time of follicle formation. Some adoptively transferred B-1 cells have moved into the red pulp by 7 d after transfer and differentiated to plasmablasts. That not all B-1 cells differentiate to plasmablasts in 6-1 mice or in non-Tg mice that have received B-1 cell transfers suggests that migration to the red pulp and differentiation to a plasmablast are regulated independently from differentiation to B-1, although both presumably require antigen. This transition could be a controlling checkpoint for the secretion of natural IgM, as B-1 cells are considered the major source of this antibody 1 9 12 . Although it was anticipated that Vκ4/5H would dominate the B-1 PtC bri population, the dominance of Vκ4/5H in the B-0 subset was a surprise. As many as 59% of B-0 cells in these mice use Vκ4/5H. Even though some Vκ4/5H-expressing B cells may be PtC-specific B-1 cells that have lost surface Ig, this only decreases the proportion to 51%. Thus, extraordinarily high frequencies of B-0 cells express Vκ4/5H, indicating that the bias in Vκ expression is independent of PtC binding. As the segregation to B-1 occurs after expression of H and L chains 10 , this bias must also precede segregation to the B-1 subset. The inability of V H 12 to associate with many L chains is no doubt a contributing factor, as it limits Vκ repertoire expressed by V H 12 B cells. The fact that most L chains are nonpermissive in 6-1 mice , provides an explanation for why 6-1 mice develop 10% as many B-0 cells as non-Tg littermates 18 . The basis for the inability of V H 12 to associate with most κ and λ chains is unknown. It is well established that V region structures influence L chain association 32 33 34 and that this involves both framework regions and CDRs 32 33 34 35 36 . A second V H 12 H chain, differing only in CDR3 from the 10/G4 V H 12 used here, is similarly deficient in ability to associate with L chains (Ye, J., H. Wang, L.W. Arnold, and S.H. Clarke, manuscript in preparation), implicating the V H -encoded segment rather than CDR3 in this inability. As this V H is unmutated, this characteristic must be evolutionarily selected, possibly to limit the diversity of V H 12 B cells. Although the large number of nonpermissive L chains would account for the small B-0 population in 6-1 mice, it would not by itself account for the dominance of Vκ4/5H in B-0, as there are multiple permissive L chains . Favor for Vκ4/5H could be achieved by a higher rearrangement frequency for this gene than for others. Although there is evidence that Vκ4/5 genes preferentially rearrange 37 , there is no apparent Vκ4/5 bias among B-0 cells from non-Tg littermates. We therefore propose that V H 12 B cells that express Vκ4/5H are favored over others for survival or clonal expansion. In this context, we have recently observed that the B cells in 6-1/Vκ1A double-Tg mice are predominantly immature, suggesting that not all V H 12 B cells expressing a permissive L chain have an equal ability to contribute to the long-lived mature repertoire. We are currently testing this hypothesis. The pattern of Jκ use among B-0 cells in 6-1 mice provides clues to the mechanism behind Vκ4/5H dominance. In normal mice, ∼80% of Vκ rearrangements in B-0 cells are to Jκ1 and Jκ2 38 , as seen in our non-Tg control mice . But 6-1 B-0 cells show a different pattern of Jκ use depending on whether or not they express Vκ4/5H. Vκ4/5H rearrangements are skewed to downstream Jκ gene segments and are rarely to Jκ1 . In contrast, non-Vκ4/5 rearrangements are significantly less biased to downstream Jκs, and ∼35% are to Jκ1 . The absence of rearrangements to Jκ1 in Vκ4/5H-expressing B-0 cells could be due to an inability of this gene to efficiently rearrange to Jκ1. Although we cannot exclude this possibility, we think it unlikely because we identified several rearrangements to Jκ1 , and we know of no precedent for such a molecular defect. An alternative possibility is that Vκ4/5H-Jκ1 rearrangements cannot associate with V H 12. Junctional amino acids affect association 35 , and W96, unique to Jκ1, is not seen among the few Vκ4/5H-Jκ1 rearrangements identified and is present in only one Vκ4/5 (non-H) rearrangement . However, rearrangements that delete the first codon of Jκ1 are common and would yield Jκ regions identical to those encoded by Jκ2, as Jκ1 and Jκ2 are identical in amino acid sequence after the first amino acid. In fact, many of the rearrangements with R96 are the result of a rearrangement to Jκ2 that includes the last codon of Vκ4/5H and deletes the first codon of Jκ2 19 . The identical L chain could be generated by rearrangement to Jκ1. Thus, Jκ1 use should be seen among the PtC bri B-1 population, if not the PtC neg B-0 population, even if W96 disrupts association with V H 12. That it is not argues against the idea that Jκ1 rearrangements are not represented because they disrupt association with V H 12. The more likely explanation is that Vκ4/5H expression is the result of secondary rearrangement. The occurrence of secondary rearrangement to delete a primary rearrangement is well established and would result in a bias for the use of downstream Jκ gene segments 39 40 41 42 . Secondary rearrangement could occur in V H 12 pre-B cells to replace primary rearrangements that encode nonpermissive L chains. As a majority of L chains appear to be nonpermissive with V H 12 H chains, secondary rearrangement may occur in a high proportion of these cells. Unless replaced, expression of a nonpermissive L chain will result in cell death, as seen in 6-1/κ −/− mice . A prediction of this hypothesis is that nonpermissive L chains are unable to mediate allelic exclusion. This was argued by others based on analyses of murine plasmacytomas that produce multiple L chains, of which only one was able to pair with the expressed H chain 43 44 , and based on an analysis of κ Tg mice 45 . We therefore suggest that Vκ4/5H rearrangements are predominantly secondary to primary rearrangements that encode nonpermissive L chains. Thus, secondary rearrangement probably contributes to the dominance of Vκ4/5H by V H 12 B-0 cells. As secondary rearrangement occurs in the bone marrow before commitment to B-1, the anti-PtC B-1 repertoire must be dependent on secondary Vκ4/5H rearrangements as well, accounting for the absence of Jκ1 rearrangements from the B-1 repertoire. Why Vκ4/5H rearrangements are more skewed to downstream Jκs than non-Vκ4/5 rearrangements, implying that the former are more often secondary rearrangements than the latter, is still unresolved. This may be related to the suspected advantage that Vκ4/5H-expressing B cells have in entry into the long-lived mature B-0 repertoire and may involve additional Vκ rearrangement in transition to a mature B cell. We are currently testing this possibility. These data reveal yet another checkpoint in B cell development that imposes a stringent limitation on V H 12 B cell repertoire diversity. The first occurs at the transition from pre-BI to pre-BII, where the length and sequence of V H 12 CDR3 are selected 20 . Pre-B cells with 10/G4 rearrangements support pre-B cell differentiation, whereas most non-10/G4 rearrangements cannot, resulting in an enrichment of pre-B cells with 10/G4 rearrangements 20 . The data reported here document a checkpoint after L chain gene rearrangement, during the transition to an immature B cell. The Vκ repertoire is limited at this stage due to an inability of most L chains to associate with V H 12, resulting in a much smaller than normal B-0 subset. Because this cannot account for the dominance of Vκ4/5H among B-0 cells, we suggest an additional selective mechanism operating at a later stage that favors V H 12/Vκ4/5H-expressing cells, possibly during the transition from an immature to a mature B cell. This process generates a pool of B-0 cells from which cells with the PtC bri phenotype are selected by antigen for entry into the B-1 repertoire and clonal expansion. As 10/G4 V H 12 H chains and Vκ4/5H L chains are critical for the PtC bri phenotype, we suggest that there has been evolutionary pressure to develop a V H 12 B-0 repertoire enriched in PtC bri cells. This would promote the production of a large number of PtC-specific B-1 cells. The evolutionary selection for the development of anti-PtC B cells complements an earlier finding by Booker and Haughton 46 that the V H 12 and V H 11 (also encoding anti-PtC antibodies) genes are evolutionarily more conserved than other V H genes. In spite of sustained research efforts, the function of B-1 lymphocytes remains elusive. Their characterization in many vertebrate species suggests a strong phylogenetic conservation and a fundamental homeostatic or protective role. A limited repertoire of antigen specificities and the expression of low-affinity Ig receptors could indicate that these cells are a “first line” immune defense against bacterial organisms 47 . A recent report by Boes et al. 48 showing that anti-PtC antibodies are protective in acute peritonitis provides direct evidence that anti-PtC antibodies are important in immediate protection against bacterial infections. Perhaps the physiological target of these antibodies is unlikely to change over time and is shared by a large number of organisms, and therefore the development of a response to this antigen could be evolutionarily selected to provide immediate protection before a T cell–dependent high-affinity response can develop. For the same reasons, B-1 cells could also have a role in the “scavenging” of senescent or apoptotic cells resulting from physiological or pathological events; these cells can express self-antigens on their surfaces (e.g., PtC, nuclear antigens, DNA) 49 50 , toward which the B-1 Ig repertoire is oriented. As these autoantigens do not vary their epitopes in time, mechanisms to eliminate them could also be evolutionary selected. Such a strong survival value could have the 10/G4 V H 12 H chain gene and its L chain partner, Vκ4/5H, expressed as a sine qua non component of the B-1 repertoire in mice and other species. | Study | biomedical | en | 0.999999 |
10510081 | The polymeric Ig receptor locus (PIGR) was isolated from a genomic sv129 library in EMBL4 by probing with radiolabeled oligonucleotides based on the rat pIgR cDNA sequence. The targeting vector included (5′ to 3′): a 1.9-kb upstream arm; a neo R cassette inserted at the PmlI site in exon 3, disrupting the noncovalent pIg-binding site; a 7.5-kb downstream arm; and a herpes simplex virus thymidine kinase gene for negative selection of nonhomologous recombinants. E14.1 embryonic stem cells were electroporated with SalI linearized vector, and homozygous mutant mice were generated as described previously 11 . 129/OLa × C57BL/6 black mixed males, 5–6 mo old, were kept in accordance with institutional guidelines and used for all analyses. Southern blots were performed with 10 μg of embryonic stem cell DNA or tail biopsy DNA digested with HindIII, separated by agarose gel electrophoresis, and probed with a 1.4-kb genomic NcoI fragment adjacent to the targeting construct. RNA was isolated from the small intestine with RNAesy kit (QIAGEN, Inc.), and 10 μg was separated on a formaldehyde agarose gel, blotted, and hybridized to radiolabeled murine pIgR cDNA 12 (gift from C.S. Kaetzel, University of Kentucky, Lexington, KY). For reverse transcription PCR, 500 ng of RNA was primed with oligo dT. PCR was performed with pigr-e2 for 5′-GCTCTACTTGTTCACGCTC versus pigr-e4.rev 5′-TTTCTGCCTATGTCCTTTG. The products were sequenced directly with a cycle sequencing kit (Amersham International PLC). Excised organs were washed briefly in ice cold PBS, fixed overnight in cold 70% ethanol, and paraffin embedded (56–57°C, 3–4 h) after graded dehydration. Primary rabbit antibody reagents against mouse IgA and mouse IgG were obtained commercially as fluorescein (Zymed Labs., Inc.) and Texas Red (Jackson ImmunoResearch Labs., Inc.) conjugates, respectively. Rabbit polyclonal antibody to murine SC (gift from B. Corthesy, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland) was used with a secondary rhodamine-labeled donkey IgG anti–rabbit conjugate (Jackson ImmunoResearch Labs., Inc.). Optimal working concentrations of all immune reagents were determined by performance testing on relevant tissue substrates. Peripheral blood, whole saliva, extract of small intestinal wick-retrieved mucus, and extract of feces were sampled and processed as described 13 . ELISA was used to determine IgA, IgG 13 , and albumin (Bethyl Labs.) concentrations. ELISA was also used to measure serum IgG antibodies to formalin-inactivated murine Escherichia coli and Lactobacillus isolates (courtesy of T. Midtvedt, Karolinska Institutet, Stockholm, Sweden) and to wheat gluten (Sigma Chemical Co.). For Western blots, the indicated amount of sample was separated by nonreducing SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal rabbit antiserum against murine IgA (DAKO Corp.) or murine SC. Secondary antibody was horseradish peroxidase–conjugated goat anti–rabbit IgG used at 1:3,000 followed by enhanced chemiluminescence revealing reaction (ECL; Amersham Corp.). All incubations were in PBS with 0.05% Tween. A targeting vector with a disruption in exon 3 that encodes the ligand-binding extracellular receptor domain 1 (D1) was used to knock out the pIgR gene (locus PIGR) in mice . Wild-type and mutant chromosomes were distinguished by Southern blots . To test expression of the mutant allele, we performed Northern blots with small intestinal RNA from wild-type and pIgR −/− mice ; the latter were expected to encode mRNA 1.7 kb larger than wild type, but mutant pIgR mRNA was in fact smaller and less abundant. Cloning and sequencing of pIgR cDNAs from pIgR −/− mice revealed two alternatively spliced mRNA forms (one in frame and one out of frame) that both deleted pIgR D1. Thus, there was a possibility that a truncated receptor lacking D1 might be produced, but this variant would not bind IgA. Sections of small intestinal mucosa from pIgR −/− and wild-type mice were immunostained for pIgR/SC, IgA, and IgG. The wild-type mice had relatively less interstitial IgA in their lamina propria than the pIgR −/− mice . Conversely, the epithelium was IgA positive only in the wild-type mice; the staining was intensified at the apical face, indicating active external transport of pIgA. Thus, the pIgR −/− mice showed no evidence of intracellular IgA transport despite increased concentration of subepithelial IgA. Lack of epithelial transport was also evident for IgM (not shown). Likewise, immunostaining for SC demonstrated abundant cytoplasmic expression both in crypt and surface epithelium of wild-type mice, whereas only occasional faint crypt staining was seen in the pIgR −/− mice with a polyclonal reagent directed against D1–D5 . This inconsistent expression might be spurious but could reflect expression of mutant pIgR lacking D1 and therefore not represent functional receptor protein. Immunofluorescence staining revealed a similar picture in colon mucosa and also confirmed that there was no vesicular IgA transport through hepatocytes in pIgR −/− mice in contrast to wild-type mice (not shown). Thus, the hepatic pIgR-mediated “IgA pump” was lacking in pIgR −/− mice. Similarly, the hepatic IgA transport was reported to be absent in J chain knockout mice because pIgA and pentameric IgM require this peptide for binding to pIgR 7 14 . To measure the effect of pIgR mutation on total transfer of IgA to secretions compared with that of IgG and albumin (which are not actively transported), we tested serum, whole saliva, small intestinal secretions, and fecal extracts from pIgR −/− and wild-type mice by ELISA . As expected, pIgR −/− animals had grossly elevated levels of IgA in serum due to lack of the hepatic IgA pump operating in rodents 15 . Western blot analysis confirmed that this IgA was mostly polymeric . More surprisingly, pIgR −/− animals also had increased levels of serum IgG. This elevation could be caused by less efficient IgG degradation due to catabolic competition with the elevated serum IgA. Alternatively, increased serum IgG in the knockout mice might reflect enhanced triggering of their systemic immune systems. We found that the serum IgG elevation was due in part to increased levels of antibodies to E . coli , whereas there was no difference in the IgG antibody response to Lactobacillus or gluten (their main dietary protein). This finding provided strong support for a selective stimulation of systemic IgG response to E . coli in the absence of SIgA (and SIgM). The wide scatter of the IgG response among the knockout mice was not surprising in view of similar variability of intestinal immunopathology induced by gut bacteria in different cytokine-deficient and transgenic rodents 16 17 . There was no statistical difference in the salivary IgA levels of wild-type and pIgR animals, whereas both IgG and albumin levels were elevated in saliva from the knockout mice, suggesting epithelial leakage of serum-derived proteins . Each pIgR −/− mouse had higher levels of salivary IgG than IgA, whereas each wild-type mouse had more IgA than IgG. Furthermore, there was good correlation between albumin and both IgA and IgG levels in saliva of the knockout mice ( r = 0.94, P = 0.02 for both), supporting the idea of increased external bulk diffusion of serum proteins. In wild-type mice, however, there was only a significant correlation between the much lower levels of albumin and IgG ( r = 0.89, P = 0.03), presumably reflecting that their salivary IgA did not depend on leakage of serum IgA but rather on generation of SIgA. At the mucosal surface in the small intestine, IgA concentrations were lower in pIgR −/− than in wild-type mice, despite elevated serum (and lamina propria) levels in the former. This contrasted with findings in J chain–deficient mice that showed no difference in IgA levels at this site compared with wild-type animals 18 , although their fecal and biliary levels of IgA were reported to be significantly repressed 14 . This disparity in the J chain knockout mice was proposed to be due to a putative alternative active epithelial transport mechanism for monomeric IgA and pIgA operating selectively at certain secretory sites 18 . However, we found that small intestinal IgG levels were about twofold higher in the pIgR −/− than in the wild-type mice, clearly reflecting the difference in their serum IgG levels . The high levels of albumin in the same samples also suggested a significant bulk leakage of serum proteins across the epithelium. We believe that the harsh physical manipulation of the small intestine performed to collect secretions at the luminal surface, used in both this study and the J chain knockout study 18 , considerably increased the leakage of proteins from the lamina propria. Thus, although the use of wicks to retrieve secretions may be appropriate for the determination of changes in specific production of antibodies along the intestinal tract, it may not be suitable to differentiate between luminal IgA derived via active or passive (in vivo or in vitro) external transfer. This is in good agreement with the documented liability of mucosal epithelia to allow profuse external bulk transfer of serum proteins after local irritation 19 . The IgA levels of fecal extracts were significantly lower in pIgR −/− than in wild-type mice, which probably was explained both by abrogated active transport and reduced stability of the paracellularly diffused IgA (compared with actively transported SIgA) in the knockout mice (see below). A similar disparity between small intestinal mucus and fecal extracts was reported in J chain knockout mice compared with wild-type mice 14 18 . Importantly, the fecal levels of albumin were much higher in the pIgR −/− than in the wild-type mice, suggesting increased epithelial protein leakage even in the untouched large bowel, as was also noted for saliva . We performed Western blots to assess the molecular form of IgA collected from various compartments . Both monomeric and polymeric forms of IgA were present in serum from wild-type mice, whereas their salivary IgA was mainly polymeric . By contrast, serum from pIgR −/− mice contained almost exclusively polymers of IgA, and their salivary IgA was of a similar molecular form. Fecal and small intestinal IgA from both wild-type and pIgR −/− mice migrated as a smear, suggesting some degradation, and the largest forms showed a size consistent with intact SIgA only in the wild-type mice. This was confirmed by blotting with an antibody to SC . Together, these results demonstrated that only exocrine IgA from wild-type mice contained SIgA, whereas IgA in the secretions of pIgR −/− mice appeared to be derived from serum and interstitial fluid by passive external diffusion. As alluded to above, it is well known that such paracellular bulk transfer of proteins is greatly enhanced when mucous membranes are irritated 19 , which was unavoidable in the small intestinal sampling procedure. The functional basis of the now internationally accepted common model for receptor-mediated epithelial transport of pIgA and pentameric IgM into external secretions was proposed by this laboratory 25 years ago 8 20 21 . It stated that J chain–expressing local plasma cells produce pIgA and pentameric IgM that, by virtue of their J chain incorporation, contain a noncovalent binding site for pIgR/SC. Mostov et al. first demonstrated in 1980 that free SC could be derived from a transmembrane precursor by endoproteolytic receptor cleavage 22 . Subsequent cDNA cloning of transmembrane rabbit SC 23 and its functional expression in polarized Madin-Darby canine kidney cells 24 demonstrated that SC indeed operated as pIgR in being capable of external pIgA transport in vitro. Thus, SIgA and SIgM are generated by pIgR-mediated ligand transport across secretory epithelial cells and subsequent release into secretions after receptor cleavage. Secondary covalent bonding of SC to pIgA makes SIgA the most stable antibody operating in external secretions 6 25 . The relatively minor differences observed between pIgR −/− and wild-type mice in small intestinal mucus and salivary IgA levels showed that pIgR function is not essential for external IgA transfer. Thus, our results documented that pIgA, like IgG and monomeric IgA, can reach secretions by paracellular leakage and that this transfer is increased by epithelial irritation. External defense resulting from such passive luminal supply of antibodies probably explained that the intestinal mucosae of the knockout mice generally exhibited no histological signs of inflammation and that the animals were of normal size and fertility (data not shown). Nevertheless, their defective epithelial barrier function, as revealed by increased albumin levels at untouched mucosal surfaces (that is, in saliva and feces) and increased IgG anti– E . coli levels in serum, supported the notion that pIgR normally executes an important function in maintaining mucosal homeostasis. Although not shown here, it is possible that increased bacterial colonization and related irritation of epithelial surfaces explained the “leaky phenotype” of our knockout mice. This possibility would harmonize with the striking role shown for SIgA from breast milk in inhibiting translocation of E . coli from the gut lumen to mesenteric lymph nodes in neonatal rabbits 26 . Furthermore, the markedly reduced fecal IgA levels in the knockout mice confirmed a role of bound SC to stabilize pIgA in secretions 25 . Also, apical recycling of pIgR–pIgA complexes has been reported 10 , and the strong apical IgA staining of wild-type intestinal epithelium suggested that pIgA is normally associated with the epithelial cells for some time, in keeping with its ability to neutralize intracellular virus during pIgR-mediated transcytosis 4 27 28 29 . Thus, in addition to mediating active external antibody transport, the function of pIgR/SC is most likely important at various other critical points of mucosal defense 5 . A reduced epithelial barrier function leading to abrogation of immunological tolerance against normally innocuous antigens, including dietary proteins and components of the indigenous bacterial flora, appears to be a significant immunopathogenic mechanism in several important mucosal disorders such as gluten-sensitive enteropathy (celiac disease) 30 and IBD 31 32 . One marker of this untoward development is lack of mucosal integrity 33 and increased production of IgA and IgG antibodies to normally encountered luminal antigens, particularly against gluten in celiac disease 30 and E . coli in IBD 31 34 . Complete lack of pIgR has not been convincingly identified in humans, perhaps reflecting evolutionary avoidance of such a deficiency 35 . However, our results showed that this receptor is not essential for the health of mice in a conventional laboratory animal facility. This might reflect redundancy of SIgA in terms of local immunity under such conditions, but its presence might nevertheless protect against development of mucosal immunopathology over time. Thus, individuals with selective IgA deficiency show an increased frequency of mucosal disorders such as celiac disease 36 and IBD, particularly Crohn's disease (Hammarström, L., personal communication), despite generally eliciting compensatory SIgM responses 35 . Also, increased mucosal leakiness is observed in patients with AIDS who have defective production of intestinal IgA antibodies against the infecting virus 37 38 . In conclusion, this study provides the first direct evidence that generation of SIgA and SIgM has a basic impact on the epithelial barrier function. It may therefore be an important factor for sustenance of mucosal homeostasis and thus a variable in local defense and immunopathology. Importantly, pIgR −/− mice present a unique opportunity to explore the relative contribution of secretory antibodies versus systemic immunity in protection against mucosal pathogens and maintenance of immune tolerance. Such studies may promote a rational development of efficient mucosal vaccines. | Study | biomedical | en | 0.999996 |
10510082 | Age-matched wild-type and CD11b/CD18-deficient mice 12 of the pure 129Sv strain were bred and maintained in a virus antibody-free facility at the Longwood Medical Research Center, Harvard Medical School. GM-CSF–deficient mice of the 129Sv/C57Bl6 mixed background and their age-matched wild-type counterparts 18 were provided by Dr. Lloyd Old (Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY). The GM-CSF–deficient mice used in our study were 6–8-wk-old males and showed no pulmonary pathology 18 or elevated PBN counts. These mice were housed in the specific pathogen-free facility at the Memorial Sloan-Kettering Cancer Center until they were shipped to the quarantine facility of Harvard Medical School, where they were housed briefly. Cytokine-induced meningitis was performed essentially as described by Tang et al. 17 . Mice were injected with IL-1β/TNF-α by lumbar puncture at vertebrate level L-1 or L-2 and, after 4 h, exsanguinated, and CSF was retrieved from the posterior fossa by aspiration into glass capillaries, as described by Griffin 19 . CSF from each mouse was placed directly into PolyHEME (Sigma Chemical Co.)-coated wells containing IMDM and 5% FCS, since in our experience these cells rapidly adhere to plastic or glass. 2 × 10 5 cells/well from each mouse were plated in duplicate and incubated at 37°C, 5% CO 2 . Peripheral blood samples were collected by retroorbital puncture in tubes containing a final concentration of 5 mM EDTA. PBNs were isolated from the anticoagulated blood pooled from two to three animals by density centrifugation using NIM2 gradients (Cardinal Associates), as described previously 12 , and were >95% pure. Alternatively, 300 μl of whole blood from individual mice was subjected to two rounds of RBC lysis with 0.2% NaCl and resuspended in IMDM, 5% FCS. 2 × 10 5 cells/well were incubated at 37°C. Neutrophil apoptosis was assessed by morphological criteria, and viability was assessed by trypan blue exclusion. To determine if GM-CSF was present in the CSF of mice, CSF was isolated from untreated and cytokine-injected mice. The CSF was harvested 2 h after injection of IL-1β/TNF-α because very few leukocytes have transmigrated into the CSF at this time point 17 . The retrieved CSF was centrifuged to remove any cells present, and the concentration of GM-CSF was determined by ELISA (Endogen). Human umbilical vein endothelial cells (HUVECs) were used at confluence on transwell inserts coated with human fibronectin (Becton Dickinson Labware). The bottom chamber was coated with polyheme to prevent adherence of PMNs to the plastic 12 . Endothelial cells were treated with human recombinant IL-1β (10 U/ml; Genzyme) and TNF-α (50 ng/ml; Genzyme). The monolayer was washed four times with white cell medium (WCM: HBSS without Ca 2+ or Mg 2+ , 0.5% BSA, 25 mM Hepes, pH 7.35). Human PBNs, isolated from normal donors, were resuspended in WCM at 5 × 10 6 PBNs/ml. 2.5 × 10 6 PBNs were added to the top chamber of the transmigration chamber, and the chemoattractants FMLP (Sigma Chemical Co.) or leukotriene B 4 (LTB 4 ; Biomol Research Labs, Inc.) or buffer alone was added to the bottom chamber. Transmigration of neutrophils was allowed to proceed for 1 h at 37°C. Control samples contained neutrophils in WCM or WCM plus the chemoattractant used in the transmigration assay, and were placed in polyheme-coated wells for 1 h at 37°C. Neutrophils that had transmigrated into the lower chamber and control samples were collected, spun down, resuspended in IMDM plus 5% FCS, plated in polyheme-coated wells at 2 × 10 5 PBNs/well, and placed at 37°C, 4% CO 2 (time 0 h). All samples were plated in duplicate, and apoptosis was assessed (see below) after 6 and 8 h in culture. HUVECs were plated in 24-well plates coated with gelatin and were used at confluence 48 h after plating. Actinomycin D (Calbiochem) and cycloheximide (Sigma Chemical Co.) were used at 12 μM and 50 μg/ml, respectively. The proteasome inhibitors MG132 and lactacystin (Calbiochem) were used at 20 and 40 μM, respectively. Calpain Inhibitor II (Calbiochem) was used as a negative control peptide in these experiments. The HUVEC monolayer was pretreated with these reagents for 1 h before the addition of cytokines, and then washed four times with WCM. The endothelial cells were then incubated with 5 × 10 6 isolated neutrophils in WCM or with WCM alone to collect factors released into the medium. In the latter case, the conditioned medium was removed and incubated with fresh 2.5 × 10 6 neutrophils for an additional 1 h. Neutrophils were collected, spun down, and resuspended in IMDM, 5% FCS. In experiments where neutrophils were cultured in the continuous presence of conditioned medium, the medium was harvested in IMDM. The conditioned medium was then supplemented with 5% FCS and incubated with neutrophils at 37°C from 6 to 24 h. In all cases, 2.0 × 10 5 cells in 500 μl medium were plated in polyheme-coated wells, and 6 or 18 h after plating aliquots were assessed for apoptosis, as described below. Small aliquots of each sample (100 μl) were removed and cytospun onto slides. Cytospin preparations were fixed in methanol, stained with Wright-Giemsa, and examined by oil immersion light microscopy at a final magnification of 1,000×. The percentage of apoptotic neutrophils was determined by counting the number of cells showing features associated with apoptosis (chromatin condensation and fragmented nuclei). At least 200 cells per slide were counted without prior knowledge of the sample. We and others have previously demonstrated that morphological assessment of neutrophil apoptosis closely correlates with results obtained using other methods to assay apoptosis, such as propidium iodide staining, annexin V binding, and decreased surface CD16 expression 12 20 . The data were tabulated as percentage of control apoptosis (percentage of apoptosis occurring in PMNs that had been incubated with endothelial cells, divided by the percentage of apoptosis in control samples). Control refers to samples which were subjected to the same experimental treatment but without any contact with the endothelial cell or conditioned medium from activated endothelial cells. We found this necessary because the rate of spontaneous neutrophil apoptosis varies from donor to donor (10–60% at 6 h). Apoptosis was also assessed by examining the percentage of unfixed cells able to bind annexin V and exclude propidium iodide (Apoptosis Detection kit from R&D Systems, Inc.). Assays were performed as outlined in the manufacturer's instructions and analyzed by flow cytometry using a FACScan™ (Becton Dickinson). Phagocytosis of complement-opsonized Oil-red-O was measured as we have described previously 12 . Serum opsonization of Oil-red-O particles was performed as described previously 21 . In brief, 4 × 10 6 cells were incubated with 100 μl of the serum-opsonized particles in a total volume of 500 μl of Dulbecco's PBS, with or without 1 mM N-ethyl-maleimide (NEM), an inhibitor of phagocytosis. Cells were washed, and the red dye was extracted using dioxane and quantified at OD 525 . The rate of phagocytosis was calculated as micrograms of Oil-red-O per 10 6 PMNs per minute. HUVECs were activated with IL-1β/TNF-α for 90 min, the cells were washed four times to remove the cytokines, and conditioned medium was collected after an additional 90 min. A neutralizing polyclonal antibody directed against human GM-CSF (Genzyme) bound to Sepharose–protein A was added to the conditioned medium at 100 μg/ml. A rabbit IgG, similarly coupled to Sepharose, was used as a negative control. The samples were incubated overnight at 4°C. The antibody-bound Sepharose was removed from the supernatant by centrifugation at 12,000 g for 10 min. Human PMNs were incubated in the medium for 18 h, and the percentage of apoptotic PMNs was assessed. The concentration of GM-CSF present in conditioned medium was measured by ELISA (Genzyme) before and after immunoadsorption. Human recombinant GM-CSF (Genzyme) was used at concentrations of 20–500 pg/ml. Data are presented as average ± SEM. Statistical significance was assessed by unpaired Student's t test. We have previously developed a model of acute, cytokine-induced meningitis in mice. In this model, significant accumulation of leukocytes (>95% neutrophils) in the CSF occurs 4 h after introduction of human IL-1β/TNF-α into the subarachnoid space by lumbar puncture. Accumulation of leukocytes is dependent on neutrophil–endothelial interaction, as mice deficient in P- and E-selectins displayed an almost complete inhibition in CSF leukocyte influx 17 . We used this model to assess apoptosis of neutrophils during inflammation in vivo. Neutrophils that had accumulated in the CSF 4 h after lumbar injection of IL-1β/TNF-α were harvested, and PBNs were purified by density centrifugation from whole blood obtained from the same animals at the same time point. The neutrophils were placed in culture, and apoptosis was assessed at subsequent times. Apoptosis of PBNs in mice with cytokine-induced meningitis was significantly delayed compared with those isolated from untreated mice. Furthermore, in mice with meningitis, there was a significant reduction in the percentage of apoptotic neutrophils in the CSF compared with PBNs from the same animals . Thus, cytokine-induced inflammation in the central nervous system leads to a delay in apoptosis of circulating neutrophils and a further delay in neutrophils that have migrated across the endothelial–blood interface into an inflammatory site. In these experiments, we routinely used density gradient centrifugation to isolate neutrophils from murine whole blood. To rule out the possibility that this procedure might induce apoptosis or select for a subset of neutrophils, we compared apoptosis in blood neutrophils isolated either by our standard density centrifugation procedure or by two cycles of hypotonic lysis of RBCs in samples of anticoagulated whole blood. A 0.3-ml blood sample obtained from a mouse with meningitis yielded >80% neutrophils compared with >95% in neutrophils isolated by density gradient centrifugation. PBNs from mice with meningitis, isolated by either method, had similar levels of apoptosis after 5 h in culture (data not shown). Previous studies have suggested that ligation of CD11b/CD18 with antibodies will modulate apoptosis in neutrophils 15 22 . To more definitively examine the potential role of CD11b/CD18-dependent adhesion in our model system, we subjected CD11b/CD18-deficient mice and their wild-type counterparts to cytokine-induced meningitis. Mice deficient in CD11b/CD18 showed no significant difference in the numbers of neutrophils that accumulated in the CSF during cytokine-induced meningitis (Tang, T., and T.N. Mayadas, unpublished data). Moreover, there was no significant difference in the percentage of spontaneous apoptotic neutrophils in samples harvested from the CSF of wild-type and CD11b/CD18-deficient mice and subsequently cultured for 10 h (wild-type, 2.25 ± 0.6%; and CD11b/CD18 −/− , 1.2 ± 0.34%; P = 0.15). Similarly, neutrophil apoptosis in PBNs was comparable between wild-type and CD11b/CD18-deficient mice (wild-type, 12.83 ± 2.13%; and CD11b/CD18 −/− , 12.4 ± 2.67%; P = 0.91). Thus CD11b/CD18-dependent adhesion does not appear to play a role in the delayed apoptosis observed in extravasated neutrophils, or PBNs, during this model of cytokine-induced meningitis. To determine whether antiapoptotic factor(s) are released into the extravascular compartment in vivo, mouse PBNs were incubated with medium alone, CSF harvested from untreated mice, or CSF from mice 2 h after cytokine induction of meningitis. The 2-h time point was chosen because very few neutrophils are present in the CSF at this time point 17 , thus diminishing the potential contribution of neutrophil-derived factors to the measured antiapoptotic activity. CSF from mice with meningitis caused a significant reduction in the spontaneous apoptosis of isolated PBNs in culture, whereas CSF from untreated mice had little effect . These studies indicate that the normal constituents of the CSF do not delay apoptosis, but rather that cytokine activation leads to an accumulation of antiapoptotic factors into the CSF. The IL-1β/TNF-α injected into the CSF by lumbar puncture was not responsible for the observed delay in neutrophil apoptosis, since direct IL-1β/TNF-α treatment of PBNs in vitro led to slightly elevated levels of apoptosis compared with PBNs cultured in medium alone (data not shown). Endothelial cells are uniquely situated to regulate apoptosis of circulating neutrophils in the blood, as well as those transmigrated into the extravascular space. Therefore, we examined the ability of cytokine-activated endothelial cells to modulate neutrophil apoptosis in an in vitro model of transmigration. Ca 2+ and Mg 2+ are omitted from the buffers so as to preserve the integrity of the activated endothelial cell monolayer during coculture with neutrophils 23 24 . Neutrophils were allowed to transmigrate across untreated or IL-1β/TNF-α–treated HUVECs plated in transwells, in the presence or absence of chemoattractants in the bottom chamber. Transmigrated neutrophils were removed, placed in fresh medium, and after 6 h incubation, apoptosis was assessed. Neutrophils that had transmigrated across cytokine-activated endothelial cells had a significant reduction in apoptosis compared with control neutrophils which were similarly treated but were not exposed to endothelial cells . Transmigration across unactivated HUVECs in response to FMLP attenuated apoptosis to a much lesser extent than cytokine-activated HUVECs, suggesting that the endothelial cell–derived antiapoptotic activity is cytokine inducible. To determine if transmigration was necessary for this effect or if contact with the endothelial cells was sufficient, neutrophils were cocultured for 1 h with HUVEC monolayers in tissue culture dishes, and then recovered and cultured for 6 h, as above. Neutrophils incubated with unactivated endothelial cells had a slight delay in apoptosis, whereas IL-1β/TNF-α–treated endothelial cells resulted in a much greater delay in apoptosis . A significant delay in neutrophil apoptosis was detectable after as little as 0.5 h of IL-1β/TNF-α activation and was manifested over an 18-h period of cytokine stimulation. If endothelial cells were cytokine treated for 2 h, followed by a 4-h “chase incubation” without cytokines, antiapoptotic activity was no longer detectable. Therefore, these endothelial-derived “antiapoptotic” factor(s) appear to be rapidly downregulated after cytokine withdrawal. Cytokine-stimulated HUVECs pretreated with actinomycin D, an inhibitor of transcription, no longer delayed the apoptosis of neutrophils during coculture experiments. In addition, pretreating cytokine-stimulated HUVECs with cycloheximide, an inhibitor of protein synthesis, also attenuated the delay . Therefore, the expression of the antiapoptotic factor(s) requires the transcriptional activation of one or more endothelial genes. In cytokine-activated endothelial cells, nuclear factor κB (NF-κB) plays a prominent role in the upregulation of genes encoding adhesion molecules and other proinflammatory products such as cytokines and growth factors 25 . To determine the role of NF-κB in the expression of the endothelial-derived antiapoptotic factor(s), we used two structurally unrelated inhibitors of NF-κB, lactacystin and the peptide aldehyde MG132 26 27 . Both of these inhibitors prevented translocation of NF-κB to the nucleus at the concentrations used, as assessed by gel shift analysis (data not shown). Treatment of IL-1β/TNF-α–stimulated endothelial cells with 40 μM lactacystin decreased the delay in neutrophil apoptosis to levels that were closer to those seen with unstimulated endothelial cells (IL-1β/TNF-α + lactacystin, 45.9 ± 4.5%; P < 0.005 compared with IL-1β/TNF-α alone, 10.5 ± 5%; unstimulated, 76.5 ± 12.5%). Similarly, treatment with MG132 attenuated the antiapoptotic affect but to a lesser extent than lactacystin (data not shown). These data suggest that NF-κB–dependent gene regulation may be involved in the elaboration of the antiapoptotic activity of cytokine-treated endothelial cells. The production of this antiapoptotic activity also depends on the particular stimulus used to activate HUVECs ( Table ). For example, IL-1β treatment alone was not as effective as TNF-α alone, and treatment with both together had a synergistic affect in delaying neutrophil cell death. IFN-γ treatment of endothelial cells resulted in a delay of apoptosis comparable to that seen with IL-1β alone. Of the stimuli used to activate endothelial cells, a combination of TNF-α and IL-1β appeared to be most effective in inducing expression of the antiapoptotic activity. To determine if the inhibition of neutrophil apoptosis was dependent on contact with the endothelial cells, or if the antiapoptotic factor(s) were released by the cells, conditioned medium was harvested from untreated and IL-1β/TNF-α–treated endothelial cells. Conditioned medium from IL-1β/TNF-α–treated endothelial cells, but not untreated cells, significantly delayed neutrophil apoptosis to a level comparable to that observed after coincubating neutrophils with endothelial cells. Again, if cytokine-activated endothelial cells were pretreated with actinomycin D or cycloheximide, the observed antiapoptotic effect was attenuated; pretreatment with the NF-κB inhibitors, MG132 or lactacystin, also attenuated the delay in apoptosis to a similar extent, as seen in Fig. 4 B (data not shown). These data suggest that the antiapoptotic gene products are secreted and regulated at the level of transcription, and that this is partly due to activation of NF-κB. Experiments described so far were designed to mimic the brief exposure of neutrophils to the endothelium during transmigration in vivo. That is, transmigration assays or coincubations were done for 1 h, after which the neutrophils were cultured in fresh medium and therefore removed from the endothelial cell–derived antiapoptotic stimuli. Under these conditions, neutrophil apoptosis was significantly delayed after 6 h, as shown in Fig. 2 . However, after 8 h in culture the delay in apoptosis was not as striking as at 6 h (data not shown), suggesting that the endothelial cell–derived antiapoptotic effect was transient. To assess whether the continuous incubation of neutrophils with conditioned medium from activated endothelial cells would delay neutrophil apoptosis over a prolonged period, neutrophils were incubated with conditioned medium for 18 h, a time period when >90% of untreated neutrophils typically are apoptotic. Apoptosis was assessed by morphological criteria as in previous experiments, as well as by FACS ® analysis of neutrophils stained with FITC–annexin V. Annexin V recognizes phosphatidylserine, which is increased on the surface of apoptotic cells . After 18 h, neutrophils incubated with conditioned medium from IL-1β/TNF-α–stimulated endothelial cells had a dramatic delay in apoptosis compared with cells that were incubated with conditioned medium from untreated endothelial cells or fresh medium . Since apoptotic neutrophils lose their ability to phagocytose 3 , we assessed whether the endothelial cell–mediated delay in apoptosis leads to a retention of this function. We assessed the ability of neutrophils to phagocytose complement-opsonized particles after they were incubated in the presence or absence of endothelial cell–derived conditioned medium for 18–21 h. Neutrophils incubated with conditioned medium derived from cytokine-treated endothelial cells had a similar level of phagocytosis as freshly isolated neutrophils, whereas neutrophils that were incubated with conditioned medium from untreated HUVECs or medium alone had lost the ability to phagocytose opsonized particles ( Table ). GM-CSF has been previously shown to inhibit neutrophil apoptosis 5 29 and is released by several different cell types, including cytokine-activated endothelial cells. GM-CSF has been reported in the conditioned medium of HUVECs after 4 h, with peak activity after 24 h of IL-1β stimulation 30 . To test the hypothesis that GM-CSF was contributing to the observed delay in neutrophil apoptosis, we first assessed GM-CSF levels in the medium of endothelial cells under conditions that led to the expression of antiapoptotic activity ( Table ). These studies revealed that the presence of GM-CSF correlated with the endothelial cell–derived antiapoptotic activity, with the exception of IL-3, which delayed neutrophil apoptosis but had no detectable GM-CSF, suggesting that antiapoptotic factor(s) other than GM-CSF were operative under this condition. GM-CSF was detected as early as 0.5 h after IL-1β/TNF-α treatment, a time point at which significant antiapoptotic activity was observed. In addition, removal of the cytokines for 4 h before collection of conditioned medium from 2-h cytokine-treated endothelial cells led to a loss of GM-CSF expression and loss of antiapoptotic activity. Furthermore, combined treatment of endothelial cells with IL-1β and TNF-α, which led to the highest expression of antiapoptotic activity, also led to the greatest release of GM-CSF activity. To directly determine whether GM-CSF in the endothelial-conditioned medium was contributing to the antiapoptotic activity, the conditioned medium from activated endothelial cells was immunodepleted using a polyclonal antibody to GM-CSF. Purified human PMNs were then incubated in the medium for 18 h, and apoptosis was assessed. Antiapoptotic activity was greatly diminished in conditioned medium that was immunodepleted of GM-CSF, as assessed by ELISA, whereas similar incubations with an isotype control IgG had no effect on GM-CSF levels or apoptosis . Immunodepletion of GM-CSF with an mAb (BVD2-23B6) yielded similar results (data not shown). Together, these studies indicate that GM-CSF is present in the conditioned medium of activated endothelial cells under conditions that lead to the expression of antiapoptotic activity, and that removal of GM-CSF by immunodepletion leads to loss of this activity. Therefore, we conclude that GM-CSF is primarily responsible for the delayed apoptosis observed in our in vitro assay system. To critically examine the contribution of GM-CSF to neutrophil apoptosis in vivo, we used GM-CSF–deficient mice and their wild-type counterparts. We first isolated PBNs from untreated animals of both genotypes and found no significant difference in the levels of apoptosis after culture (data not shown). We then performed cytokine-induced meningitis in these mice. GM-CSF–deficient mice had normal peripheral blood counts ( 18 ; data not shown), and mice of both genotypes responded with significant accumulation of neutrophils in the CSF (wild-type, 1.1 ± 2.7 × 10 6 ; GM-CSF −/− , 2.5 ± 5.47 × 10 6 cells recovered in CSF). Significantly, there was a 30–40% increase in the level of apoptosis in PBNs isolated from GM-CSF–deficient mice compared with wild-type mice . Therefore, GM-CSF plays an important role in the regulation of neutrophil apoptosis in the peripheral blood only during inflammation. We observed no difference in the percentage of apoptotic neutrophils retrieved from the CSF of GM-CSF–deficient and wild-type mice, despite the fact that GM-CSF was detected in the CSF of wild-type animals (naive mice, 0 pg/ml; mice subjected to meningitis for 4 h, 135.1 ± 42.2 pg/ml). Thus, soluble factors other than GM-CSF are responsible for increasing neutrophil survival in the extravascular compartment. The localized accumulation of functional neutrophils at sites of inflammation is pivotal in the host's defense against infection, and the orderly elimination of neutrophils is equally important in resolution of the inflammatory response. It is well recognized that the endothelium, upon activation by various proinflammatory stimuli including cytokines such as IL-1β and TNF-α, plays an active role in recruiting leukocytes via the expression of adhesion molecules and chemoattractants 31 . The results reported here establish a previously unrecognized role for the activated endothelium in prolonging neutrophil survival and retaining neutrophil functional capabilities in the context of inflammation, one that may have important implications for the regulation of the acute inflammatory response. Our observations suggest two levels at which neutrophil apoptosis is regulated during inflammation. The first is a delayed apoptosis in neutrophils in the peripheral circulation, and the second is a more substantial delay in apoptosis of neutrophils that have extravasated into a site of inflammation. The endothelium is positioned to directly affect the apoptosis of circulating neutrophils. It may also affect the apoptosis of extravasated neutrophils, although the role of other cell types in the tissue cannot be ruled out. Our in vitro studies demonstrate that the antiapoptotic activity of endothelial cells is rapidly inducible and that it is sustained over 24 h in the presence of cytokines. However, the activity decays in the absence of ongoing stimuli. Thus, the antiapoptotic activity from endothelial cells may function to influence the survival of neutrophils during the earliest periods of their recruitment. Furthermore, only under conditions where neutrophil recruitment and inflammation were ongoing would neutrophil survival be extended, since the elaboration of the antiapoptotic factors is dependent on cytokine stimulation of the endothelial cells. NF-κB, which is a pleiotropic regulator of the proinflammatory activities of endothelial cells such as leukocyte adhesion receptor expression 25 , is important in the expression of endothelial antiapoptotic activity. Thus, mechanisms regulating the recruitment of neutrophils to the endothelium also play a role in the survival of those neutrophils. CD11b/CD18-mediated adhesion is not required for the delay in neutrophil apoptosis in vivo, although another study in vitro suggested that this may be the case since antibody cross-linking of CD11b/CD18 or CD11a/CD18 attenuated neutrophil cell death 15 . We show that the antiapoptotic activity is secreted and that it is extremely potent. The number of neutrophils that are apoptotic after a 24-h period in culture is reduced by 80% in neutrophils that are incubated with conditioned medium from endothelial cells compared with those incubated in medium alone. The enhanced neutrophil survival in the presence of endothelial-derived factors is associated with a retention in the ability of the neutrophil to phagocytose, a principal function of these phagocytes at sites of inflammation. Thus, endothelial cells promote the survival of neutrophils and their function in response to cytokine activation, thereby potentially enhancing the accumulation of functional neutrophils at the site of inflammation. Many recombinant forms of inflammatory mediators can inhibit apoptosis and prolong neutrophil survival in vitro 3 5 . Some of these, GM-CSF, IL-6, and IL-1β, are known to be released by the activated endothelium, although the role of these cytokines in a physiological context is not clear. Here, we demonstrate that GM-CSF released from cytokine-activated endothelial cells in vitro is responsible for the antiapoptotic activity. Two aspects of the regulation of GM-CSF expression observed in this study have not been previously described 30 32 33 . GM-CSF was released into the medium of endothelial cells as early as 0.5 h after cytokine activation, and IL-1β and TNF-α had a synergistic effect in stimulating GM-CSF production in endothelial cells. The role of GM-CSF in neutrophil apoptosis in vivo was critically examined by assessing apoptosis of neutrophils retrieved from both untreated GM-CSF knockout and wild-type mice, and mice subjected to cytokine-induced meningitis. These studies suggested that GM-CSF has no effect on the spontaneous programmed cell death of PBNs in untreated mice, but is responsible for delaying the spontaneous apoptosis of PBNs during an inflammatory response. On the other hand, the greater delay in apoptosis of extravasated neutrophils was found to be regulated by soluble factors other than GM-CSF. Thus, neutrophil apoptosis is differentially regulated in the peripheral blood and extravascular tissue, not only in terms of the extent of the delayed apoptosis but also in the soluble mediator(s) involved. Data from this study, together with previous data demonstrating that phagocytosis promotes neutrophil apoptosis 12 13 , have led us to propose the following model for the regulation of neutrophil apoptosis during an inflammatory response. The vascular endothelium actively recruits PBNs to sites of inflammation via expression of adhesion molecules and chemoattractant cytokines. The constitutive cell death program in the recruited neutrophils is delayed by antiapoptotic factors released from the endothelium, including GM-CSF. This delay in the programmed cell death of neutrophils during inflammation allows the neutrophil to retain its functional capabilities. Once neutrophils have completed their principal function, which is the phagocytosis of bacteria and cellular debris, the death program is reengaged. It follows that defects in the regulation of neutrophil apoptosis could have severe consequences for the host. Dysfunctional upregulation of the endothelial antiapoptotic factor(s) could result in increased accumulation of PMNs in the extravascular tissue and prolong the inflammatory response, culminating in tissue damage. Alternatively, a reduction in the antiapoptotic activity could lead to premature apoptosis of neutrophils in the tissue, thereby compromising host defense. Further studies of the regulation of expression of the key effectors of neutrophil apoptosis secreted by activated vascular endothelium, including GM-CSF and other as yet undefined mediators, should add to an understanding of the control potential of the inflammatory response and the disease-related consequences. | Study | biomedical | en | 0.999999 |
10510083 | The following individuals provided reagents: T.K. Kishimoto (DREG56 anti–L-selectin mAb), John Lowe (FTVII cDNA), Minoru Fukuda (core 2 β-1,6- N -acetylglucosminyltransferase [C2GnT] cDNA), G.S. Kansas (300.19 B cells transfected with E-selectin), and Arthur Weiss (Jurkat cells). Cells were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 5% heat-inactivated FCS (Hyclone), with 100 μM 2-ME added to the 300.19 cells. Human PBLs were isolated by Ficoll-Hypaque sedimentation. For generation of GlyCAM-1/IgG fusion proteins, COS-7 cells (80% confluent in 10-cm dishes) were transfected with plasmids encoding C2GnT (pCDNA1.1, 1 μg), FTVII (pCDM8, 1 μg), GlyCAM-1/IgG (pIG1, 2 μg), and KSGal6ST (pCDNA3.1/ Myc -His, 0.5/1/2 μg), huGlcNAc6ST (pCDNA3.1, 0.5/1/2 μg), HEC-GlcNA c6ST (pCDNA1.1, 0.5/1/2 μg), or the empty vector (pCDNA3.1, 1 μg) using Lipofectamine (Life Technologies) and were cultured for several days. For radiolabeling, cells were grown for 24 h in Opti-MEM supplemented with 0.1 mCi of Na 2 35 SO 4 (1,400 Ci/mmol; ICN Biomedicals, Inc.). The GlyCAM-1/IgG chimeras were purified on protein A–agarose and were exhaustively exchanged into PBS. In two independent experiments, the following specific activities (cpm/μg protein) were measured (mean ± range): no exogenous sulfotransferase (2,367 ± 142); KSGal6ST (19,262 ± 1,824); huGlcNAc6ST (9,624 ± 1,804); and HEC-GlcNAc6ST (10,068 ± 1,347). To equalize coating densities of the GlyCAM-1/IgG chimeras, ELISAs were performed. Proteins were coated onto 96-well polystyrene plates (Costar Corp.) in Tris-buffered saline, pH 9, at 4°C. After blocking with BSA, the chimeras were detected with biotinylated anti–GlyCAM-1 peptide Ab 5 or biotinylated anti–human IgG (Fc specific) and streptavidin-conjugated alkaline phosphatase. For flow experiments, polystyrene dishes coated similarly with the chimeras as in the ELISA assays were incorporated as the lower wall of a parallel plate flow chamber 17 18 . Cells were perfused through the flow chamber at 1–2 × 10 6 cells/ml. For inhibition studies, cells were pretreated with 5 μg/ml DREG56 or 10 μg/ml fucoidin (Sigma Chemical Co.; 10 min, 22°C) or resuspended in Ca 2+ , Mg 2+ -free HBSS with 5 mM EDTA. For sialidase experiments, coated substrates were incubated with 5 mU/ml Vibrio cholera sialidase (Oxford Glycosystems) for 30 min in 50 mM sodium acetate, 4 mM CaCl 2 , and 0.1% BSA, pH 5.5, or with buffer alone for the control. For the initial comparison of rolling behavior on the different substrates, cells were perfused for 2 min through the flow chamber, after which equilibrium was reached and counts of rolling cells were taken. For analysis of tethering, cells were perfused through the chamber over a range of shear stresses (3–0.2 dyn/cm 2 ). The fraction of cells that came into close proximity with the substrate and tethered stably (rolling for >1 s after initial attachment) was determined. For velocity and detachment determinations, cells were infused for 2 min at 1 dyn/cm 2 , after which shear stress was increased in 1.5–2-fold increments up to 35 dyn/cm 2 at intervals of 5 s. Cell displacement was followed for 1–3 s to determine rolling velocities. For single-cell analysis, ≥4 randomly chosen cells were tracked at 1 dyn/cm 2 over a period of 4 s in successive video frames (1/30 s) on each substrate, and duration of pauses and the velocity between pauses was determined. In detachment assays, the number of rolling cells was determined at each shear stress and calculated as the percentage of the peak value. Optimal equilibrium binding of L-selectin to its HEV ligands requires sialylation, fucosylation, and sulfation 3 4 19 . To determine the contribution of sulfation to ligand activity under flow conditions, we examined the rolling of PBLs or Jurkat cells on recombinant GlyCAM-1/IgG that was immobilized on the bottom plate of a flow chamber. To produce the different forms of GlyCAM-1/IgG, COS cells were cotransfected with cDNAs for GlyCAM-1/IgG, FTVII 19 , and C2GnT 20 and a cDNA encoding a sulfotransferase (KSGal6ST, huGlcNAc6ST, or HEC-GlcNAc6ST). The sialylation requirement for ligand activity was met by endogenous sialyltransferases within the COS cells. FTVII was included to satisfy the known fucosylation requirement 19 , and C2GnT was added because it elaborates a core structure for O-linked glycans that allows the formation of sLe x capping groups 9 . The activity of the three sulfotransferases was verified by direct measurement of [ 35 S]sulfate incorporation into the recombinant proteins. Transfection with the individual sulfotransferase cDNAs increased sulfate incorporation four- to eightfold over background (see Materials and Methods). The recombinant proteins were purified and coated onto the flow chamber polystyrene plate at equal site densities, as determined by ELISA using similar conditions and polystyrene ELISA plates. Rolling of Jurkat cells and PBLs on GlyCAM-1/IgG required fucosylation, as there was no rolling on GlyCAM-1/IgG produced without FTVII transfection, with or without the inclusion of sulfotransferase cDNAs . The addition of FTVII cDNA without a sulfotransferase cDNA resulted in a very low number of rolling PBLs and Jurkat cells . The number of rolling PBLs and Jurkat cells was markedly increased on KSGal6ST-, HEC-GlcNAc6ST–, or huGlcNAc6ST-modified GlyCAM-1 when 1 μg of each sulfotransferase cDNA was used for transfection. The use of 0.5 or 2.0 μg sulfotransferase cDNA yielded smaller or comparable effects (not shown). An anti–L-selectin mAb (DREG56) abrogated the interaction of PBLs and Jurkat cells with the substrates . EDTA or fucoidin also blocked rolling completely , as expected for L-selectin–mediated binding 3 . Treatment of the substrates with sialidase completely prevented tethering and rolling of Jurkat cells, consistent with previous observations made with native HEV ligands 3 . We measured the ability of Jurkat cells to tether on the different GlyCAM-1/IgG substrates as a function of shear stress. We confirmed the shear threshold phenomenon that has been previously documented 21 . Thus, stable tethering of Jurkat cells occurred at 0.4–0.6 dyn/cm 2 , below which little or none was observed . Although a maximum tethering rate of 9% was found for the interaction of cells with fucosylated GlyCAM-1/IgG (FT), the frequency of tethered cells was increased threefold upon sulfation of C-6 on GlcNAc (FT plus huGlcNAc6ST; FT plus HEC-GlcNAc6ST) and increased sixfold upon sulfation on C-6 of Gal (FT plus KSGal6ST). The latter modification also resulted in a shift of the threshold toward lower shear stresses . Sulfation on C-6 of GlcNAc or Gal significantly reduced the overall rolling velocity of Jurkat cells relative to that measured on fucosylated GlyCAM-1/IgG over a wide range of sheer stresses . The same result was obtained with PBLs (not shown). To determine if these differences in tethering and rolling velocity were maintained at different site densities of coated ligand, we reduced the coating concentrations of GlyCAM-1/IgG in steps of threefold dilutions. As coating concentrations were decreased, tethering rates decreased and rolling velocities increased at 1 dyn/cm 2 , consistent with a reduced coating density of immobilized ligand. GlyCAM-1/IgG modified by FT plus KSGal6ST yielded higher tethering rates and slower rolling velocities than GlyCAM-1/IgG modified by FT or FT plus HEC-GlcNAc6ST at all site densities tested. Notably, cells still tethered and rolled on FT plus KSGal6ST–modified GlyCAM-1/IgG at a very low site density (27-fold diluted), whereas no interactions were observed with the other substrates. For HEC-GlcNAc6ST plus FT–modified GlyCAM-1/IgG as compared with the FT-modified ligand, we observed a significant increase in the frequency of tethering and a decrease of rolling velocity at the two highest coating concentrations, whereas there were not significant differences at lower concentrations . As others have observed 17 22 , we found that L-selectin–mediated rolling was “jerky,” with periods of smooth movement interrupted by pauses. Analysis of the behavior of individual cells revealed that sulfation did not affect the duration of pauses but that each modification significantly increased the frequency of pauses. Sulfation of C-6 on Gal significantly reduced the rolling velocity of cells between pauses, whereas sulfation on C-6 of GlcNAc caused a marginal reduction ( Table ). These effects resulted in a net decrease in the rolling velocity measured over long distances . Sulfation of GlyCAM-1/IgG also resulted in enhanced adhesive strength, as measured by an increase in the resistance of rolling Jurkat cells to shear-induced detachment . We recently demonstrated that coexpression of two sulfotransferases (KSGal6ST and HEC-GlcNAc6ST) in CHO/FTVII/CD34 cells resulted in much better binding of an L-selectin/IgM chimera than expression of the individual sulfotransferases 11 . However, in several independent experiments, we were not able to demonstrate synergistic or even additive effects when we compared rolling on GlyCAM-1/IgG modified with individual sulfotransferases to that on GlyCAM-1/IgG modified with a combination of two sulfotransferases. For example, when GlyCAM-1/IgG was produced with FT plus 1 μg KSGal6ST cDNA, 69 ± 3% of cells tethered stably onto the substrate and rolled at a velocity of 71 ± 3 μm/s at 1 dyn/cm 2 . The combination of KSGal6ST and HEC-GlcNAc6ST (0.5 μg of each cDNA) plus FT resulted in a decrease of tethering frequency (43 ± 5% at 1 dyn/cm 2 ) and an increase of velocity (103 ± 6 μm/s). Furthermore, we failed to observe synergistic effects of the sulfotransferases when a wide range of coating densities was tested (not shown). E-selectin is capable of binding to and supporting the rolling of transfectants on L-selectin ligands 22 , although this interaction is of questionable functional significance. Because E-selectin has a C-type lectin domain highly homologous to that of L-selectin, we wanted to determine the effects of ligand sulfation on E-selectin–mediated rolling. To address this issue, we allowed 300.19 cells transfected with E-selectin to interact with the various forms of GlyCAM-1/IgG under flow conditions. Whereas there was no rolling of the parental 300.19 cells (not shown), the E-selectin transfectants rolled on fucosylated GlyCAM-1/IgG ( Table ). Consistent with previous results 17 22 , E-selectin–mediated rolling was significantly slower than L-selectin–mediated rolling. Also, the shear resistance of rolling adhesions was much greater for the E-selectin interactions . However, sulfation of the substrates produced no significant effects on tethering frequency, rolling velocity, or the strength of rolling adhesions ( Table ). The comparable rolling behavior suggests similar degrees of fucosylation and sialylation in all of the substrates. Sulfation has been well established as a key modification of L-selectin ligands in HEVs of lymphoid organs and HEV-like vessels that are induced at sites of chronic inflammation 1 . The molecular cloning of a novel family of sulfotransferases 11 12 13 allows the detailed study of the functional impact of Gal-6-sulfate and GlcNAc-6-sulfate modifications on the interactions between L-selectin and its physiological ligands. Previously, native GlyCAM-1 has been shown to be an excellent ligand for supporting L-selectin–dependent rolling in a parallel plate flow chamber 23 . For our purposes, we generated a set of recombinant GlyCAM-1/IgG chimeras in which we could control fucosylation and sulfation by transfection of appropriate enzyme cDNAs. We evaluated the recombinant chimeras as adhesive substrates for lymphocytes under physiological flow conditions. As anticipated, L-selectin interactions with the sulfated ligands were calcium dependent and required both fucosylation and sialylation of the ligand. The sulfation modifications had a pronounced effect on L-selectin–dependent tethering and rolling and the strength of rolling adhesions. In agreement with previous data indicating that E-selectin interactions with its ligands are sulfation independent 24 , we observed that E-selectin–mediated rolling on the chimeras was not affected by sulfation, further demonstrating a difference in the nature of the recognition determinants for these two selectins. Cell tethering and rolling through L-selectin is a complex phenomenon that depends on the kinetic rate constants for the selectin–ligand bond 17 25 as well as various cellular factors 22 26 27 . Our detailed tracking of individual cells indicated that the dominant contributing factor to decreased rolling velocities on the sulfated substrates was an increase in the frequency of pauses. There was no significant effect of sulfation on pause duration. These results are consistent with the possibility that sulfation increases the intrinsic on-rate of bond formation rather than decreasing the off-rate 17 . The enhanced tethering rates and strengths of rolling adhesion that we observed with sulfated ligands in this study and the increased equilibrium binding observed previously 11 are also compatible with this explanation. However, because our rolling studies were performed on a relatively high ligand density, we cannot draw firm conclusions regarding the effects of sulfation on single bond properties. The application of biophysical techniques, such as surface plasmon resonance 28 , will be required to address these questions. In the assays described herein and in the CHO cell transfection experiments reported previously 11 , sulfation on C-6 of Gal or GlcNAc within GlyCAM-1 and CD34 enhanced L-selectin interactions. At this point, we cannot judge the relative importance of each modification, because we were able to measure only overall sulfation levels without knowing how much of the incorporated sulfate actually participated in L-selectin binding. An interesting possibility is that the optimal recognition determinant for L-selectin involves both sulfation modifications. However, in experiments to date, we have not been able to demonstrate synergistic effects on the velocities or tethering rates of lymphocytes when GlyCAM-1/Ig was modified by both sulfotransferases. These results contrast with those of our previous study 11 , in which we measured the binding of an L-selectin/IgM probe to CHO cells transfected with CD34, FTVII, C2GnT, and combinations of the sulfotransferases. Further experiments are needed to reconcile these equilibrium binding observations with measurements of dynamic parameters in the flow chamber reported herein. Lymphocyte homing to lymph nodes and other secondary lymphoid organs is a multistep process in which lymphocytes must tether and roll on HEVs before firmly arresting via chemokine-induced integrin activation 1 2 . The findings of this study indicate that sulfation of ligands could promote the overall process at several levels: (a) by increasing the extent of tethering, (b) by increasing the strength of rolling adhesions, and (c) by decreasing overall rolling velocity and thereby facilitating the effects of integrin-activating stimuli. The family of newly cloned sulfotransferases will allow further analysis of the functional contribution of the different sulfation modifications to these dynamic processes in lymphoid organs and sites of inflammation. | Study | biomedical | en | 0.999995 |
10510084 | Thymi were obtained from adult or fetal C57Bl/6 mice. Intact lobes were placed in a small volume of DMEM without NaHCO 3 and phenol red. The tonicity of the basal medium was adjusted to 310 mOsm with ∼1.6 g of NaCl per liter and supplemented with 10 mM Hepes buffer, 5% heat-inactivated fetal bovine serum, and 1 mM l -glutamine. Thymocytes were released by gently tearing open the capsule with small forceps. Noncellular material was removed by filtering this preparation through 70 mM nylon mesh. The filtered preparation was maintained at room temperature, and aliquots were used for calcium measurements without further manipulation. Murine thymocytes were loaded with the cell-permeant calcium indicator fura-2 acetoxymethyl ester (AM, 3.0 μM; Molecular Probes, Inc.) in DMEM for 10 min at room temperature (25°C), placed into the recording chamber on an inverted fluorescent microscope (Nikon Inc.), and allowed to adhere to Poly-D-lysine (100 μg/ml; Sigma Chemical Co.) treated coverslips for 5 min. Ca 2+ i was measured at room temperature unless indicated otherwise. We observed no measurable difference in the calcium signaling response at 25 vs. 37°C. mAbs to relevant surface receptors were added to thymocyte suspensions during the final 10 min of fura-2 loading. Excess extracellular fura-2 AM and unbound antibody was washed away by perfusing the microscope recording chamber with extracellular bath solution for 5 min. The bath solution consisted of 155 mM NaCl, 4.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose, and 10 mM Hepes and was adjusted to pH 7.4. Discrete bandwidth excitation light (340 ± 10 nm, 380 ± 10 nm) from a xenon source coupled to a computer-controlled monochromator (TILL; Applied Scientific Imaging) was directed to the epifluorescence attachment of the microscope through a small quartz fiber-optic guide. Excitation light was directed to the fluorescence objective (100×; Nikon Inc.) via a dichroic mirror. The emitted fluorescence from the fura-2–loaded cells passed through a 470-nm-long pass filter, and images were obtained with an intensified charge–coupled video camera connected to the side port of the inverted microscope. Four fluorescent video images were averaged and digitized (0.5 Hz) with a video frame grabber (Matrox) using Metafluor acquisition and analysis software (Universal Imaging Corp.). Stored images were analyzed off-line using the Metafluor package. Within cursor-defined areas of interest, paired 340/380 images were background subtracted and the ratio was calculated. The absolute ratio values in the cursor-defined areas were exported into Microsoft Excel and converted to [Ca 2+ ] 18 . In situ calibration factors, R max (340/380 ratio obtained in presence of 5 μM ionomycin and 10 mM Ca 2+ ), R min (340/380 ratio in presence of 5 μM ionomycin and excess EGTA), and F380 max , and F380 min (maximum and minimum 510 nm emission with 380 nm excitation) were determined. The values obtained for R min , R max , F380 min , and F380 max were 0.25, 20.0, 40, and 100, respectively, and a dissociation constant ( K d ) of 224 was used for fura-2. Biotin-conjugated mAbs specific for CD4 (RM4-5, PharMingen), TCR (H57-597), CD28 (37.51, PharMingen), CD5 (53-7.3, PharMingen), CD3 (145-2C11, PharMingen), and CD28 (37.51, PharMingen) were used to activate thymocytes. Calcium signaling responses were initiated by aggregating antibody-bound surface receptors with streptavidin, which was added to the recording chamber. For routine measurements, streptavidin was added to the bath at a final concentration of 0.25–0.5 μg/ml. In several calcium experiments, polystyrene microspheres (5 μM diameter; Interfacial Dynamics Corp.) were used to aggregate surface receptors. Cells were first treated with biotin-conjugated antibodies against relevant receptors (see Results), and these antibody-bound receptors were aggregated with streptavidin-coated (0.5–1.0 μg/ml) polystyrene microspheres (Interfacial Dynamics Corp.). Microspheres (10 6 ) were prepared by incubation (1.5 h, 37°C) with αCD28 (5 μg/ml) and/or αCD3 (1 μg/ml) and αCD4 (1μg/ml), and were rinsed three times with PBS containing 1% BSA. Antibody-treated microspheres were cultured at a 1:1 ratio with CD4 + CD8 + thymocytes. The functional responses were determined after 16–20 h of incubation. In some experiments, after measuring Ca 2+ i , surface CD4, CD8, and CD5 expression levels were determined for each cell. Antibodies (PharMingen) against CD8 (53-6.7, FITC), CD5 (53-7.3, PE) and CD4 (RM4-5, Cy-Chrome) were added directly to the microscope chamber (15 min, 25°C), and unbound antibody was removed by perfusion with bath solution (5 min). Digital images were collected sequentially for each fluorochrome. Three separate filter cubes (Chroma Technologies) were used to image FITC, PE, and Cy-Chrome fluorescence. These digital images were first used to determine the CD4/CD8 phenotype of each cell. The activation/maturation state of CD4 + CD8 + thymocytes was further refined based upon the level of CD5 expression. A value for the CD5 fluorescence intensity was obtained at an intermediate focal plane for each cell in the image field to identify CD5 low , CD5 int , and CD5 high cells. Thymocytes were grouped simply according to average percentages obtained for each population from flow cytometric analysis. Thus, 30% of cells with the lowest CD5 intensities were designated as CD5 low cells, 55% of cells as CD5 int. , and 15% of cells as CD5 high . To determine whether distinct TCR-mediated stimuli evoke distinct calcium responses in immature T cells, we measured the intracellular free calcium concentration ([Ca 2+ ] i ) in individual thymocytes after coengagement of surface molecules that induce either maturation (CD4 and TCR or CD3; reference 5) or apoptosis (CD28 and TCR or CD3; reference 7). Each of these costimulatory receptors had a distinct effect on TCR/CD3-mediated Ca 2+ signaling within individual thymocytes . Maturation stimuli (TCR/CD4 or CD3/CD4) evoked two different patterns of calcium signaling. Responding cells (∼75%) typically exhibited an initial rapid [Ca 2+ ] i elevation, which peaked within 30 s and decayed . After the initial rise, [Ca 2+ ] i either decreased to an elevated steady state (biphasic response) or exhibited repetitive two- to fourfold elevations (spikes) above resting levels. In spiking cells, the frequency was similar for both CD3/CD4 (0.013 Hz)- and TCR/CD4 (0.010 Hz)-stimulated thymocytes . Apoptotic stimuli (CD3 and CD28 coengagement) evoked a limited [Ca 2+ ] i response in a small proportion (∼25%) of thymocytes. Although [Ca 2+ i ] spikes were observed in a few CD3/CD28-stimulated cells (∼10%), none of these thymocytes exhibited a large initial [Ca 2+ ] i transient . Moreover, the response to CD3/CD28 was indistinguishable from the response to CD3 alone . We were interested in examining the basis for the different patterns of CD3/CD4-mediated Ca 2+ signaling and reasoned that they may reflect the responses of cells in different maturational states. We tested this hypothesis first by comparing [Ca 2+ ] i signaling responses initiated by CD3/CD4 coengagement in two isolated populations representative of the most immature and mature TCR + cells . Immature thymocytes isolated from the day 17 fetal thymus contained no mature subpopulations and displayed a markedly homogeneous spiking [Ca 2+ ] i response . Conversely, all responding mature peripheral CD4 + T lymphocytes exhibited a biphasic [Ca 2+ ] i response after CD3/CD4 coengagement, and [Ca 2+ ] i spikes were never observed . These results suggest that the TCR/CD3-mediated calcium response is determined, at least in part, by the maturation state of each lymphocyte. We next examined CD3/CD4-mediated signaling in each major thymocyte subpopulation to identify the precise maturational stage at which the [Ca 2+ ] i signaling response changes from a spiking to a biphasic pattern. Rather than purify cells of each maturational state, we recorded CD3/CD4-initiated calcium responses within freshly isolated unfractionated thymocytes and subsequently determined the maturation phenotype of each cell. We were able to distinguish multiple thymocyte subpopulations at distinct developmental stages by staining for surface CD4, CD8, and CD5 . As expected, the most immature thymocytes (double negative, CD4 − CD8 − CD5 low ) did not respond to CD3/CD4 stimulation, presumably because they do not express CD3. Two subpopulations of CD4 + CD8 + thymocytes were clearly distinguishable by differences in surface levels of the maturation/activation marker, CD5 19 . Immature DP (CD4 + CD8 + CD5 low/int ) thymocytes, the bulk of immature T cells in the thymus, responded with an initial transient [Ca 2+ ] i elevation followed by Ca 2+ spikes. These [Ca 2+ ] i spikes often originated from and returned to the resting baseline level and continued for more than 20 min. Mature CD4 + CD8 + CD5 high thymocytes typically exhibited a biphasic [Ca 2+ ] i change with little spiking activity, whereas the most mature (CD4 + CD8 − CD5 high ) thymocytes exhibited a pure biphasic response. These results suggest that the capacity of thymocytes to generate calcium spikes is developmentally restricted in the mouse to the immature CD4 + CD8 + thymocyte stage. The molecular basis for different TCR/CD3-mediated developmental responses of CD4 + CD8 + thymocytes is unknown but clearly governed by the strength of TCR engagement and could arise from differences in the proximal signals generated, including calcium. We therefore examined the relationship between the [Ca 2+ ] i response pattern and the strength or avidity of the TCR signal. We quantitatively varied the signal strength in our system by modulating the extent of CD3/CD4 coaggregation. To do this, we systematically altered the concentration of streptavidin used to cross-link the stimulating (biotinylated) antibodies, thereby altering the degree of receptor aggregation. We reasoned that at high concentrations, streptavidin would tend to form monovalent complexes with biotin, whereas at limiting concentrations, the tetravalent streptavidin would form large complexes with multiple biotin molecules, resulting in aggregation of biotinylated antibodies and their ligands . To verify this strategy, we evaluated the extent of receptor aggregation by assessing the presence of unbound biotin molecules on antibody and unoccupied biotin-binding sites on streptavidin. We detected unbound biotin molecules and unoccupied biotin-binding sites by treating cells prepared for stimulation with R670-conjugated streptavidin (R670–SA) or PE-conjugated biotin (PE–biotin) . Consistent with the notion that maximal receptor aggregation occurs at low streptavidin concentrations, PE–biotin bound minimally at limiting concentrations of aggregating streptavidin, indicating that biotin-binding sites were fully occupied . Conversely, PE–biotin bound maximally at high streptavidin concentrations, indicating that biotin-binding sites on streptavidin were minimally occupied. We also measured R670–SA binding to unbound, antibody-associated biotin to precisely determine the concentrations of aggregating streptavidin at which all antibody-associated biotin was maximally engaged. R670–SA binding decreased progressively with increasing concentrations of aggregating streptavidin , indicating that antibody-associated biotin was maximally engaged by aggregating streptavidin at concentrations exceeding 1.0 μg/ml. These data suggest that maximal antibody (and by inference, receptor) aggregation will occur at a streptavidin concentration at which R670–SA and PE–biotin fluorescence are both minimal . This occurs near the intersection of the curves . Thus, optimal aggregation of CD3 and CD4 can be achieved at 0.25–0.75 μg/ml of aggregating streptavidin, whereas minimal aggregation will occur at relatively high streptavidin concentrations (5 μg/ml), when biotin-binding sites on streptavidin exceed available biotin. The model presents an idealized interpretation of how streptavidin can be used to vary the extent of receptor–antibody aggregation. It should be noted that primary antibodies used in these experiments are bivalent and could themselves cause a limited degree of cross-linking. Also, it is likely that even at very high concentrations, streptavidin would cross-link some antibody-bound receptors. Thus, we use the term minimal aggregation to describe this experimental condition. To evaluate the relationship between receptor aggregation and calcium signaling, surface CD3 and CD4 were maximally (0.5 μg/ml streptavidin) or minimally (5.0 μg/ml streptavidin) aggregated, and calcium responses of individual thymocytes were grouped according to the level of CD5 expression, a sensitive measure of maturation status 19 . The majority of immature, CD5 low (CD4 − CD8 − and some CD4 + CD8 + ) thymocytes were relatively nonresponsive to receptor aggregation. A few, presumably DP CD5 low cells, exhibited calcium spikes . The pattern of calcium signaling among these CD5 low responders was converted to a monophasic pattern under conditions of minimal receptor aggregation . The Ca 2+ i signaling pattern in immature, CD5 int (CD4 + CD8 + ) thymocytes was also markedly affected by the extent of receptor/coreceptor aggregation. Maximal receptor aggregation (0.5 μg/ml streptavidin) induced a relatively rapid initial elevation of [Ca 2+ ] i , similar to that observed in mature cells. However after ∼1 min, [Ca 2+ ] i decayed from its peak level and a spiking (0.019 Hz) signaling response persisted in most (26/45) responding cells . Minimal aggregation of CD3 and CD4 receptors (5 μg/ml streptavidin) on immature DP thymocytes evoked a biphasic [Ca 2+ ] i increase in all responding cells. The initial mean [Ca 2+ ] i peak was higher than under maximally aggregating conditions; however, the [Ca 2+ ] i decayed to an elevated steady state , similar to nonspiking mature peripheral cells . Aggregation of CD3 and CD4 evoked a biphasic calcium signaling response in mature CD5 high thymocytes that was largely independent of the receptor aggregation status. . The only significant effect of aggregation on mature cells was a higher mean peak [Ca 2+ ] i induced under conditions of maximal receptor engagement but minimal receptor aggregation. Mature peripheral CD4 + lymph node lymphocytes responded similarly to mature thymocytes. Peripheral CD4 + cells exhibited a biphasic calcium signaling response regardless of the streptavidin concentration used for stimulation . Thus, only DP CD5 low and CD5 int thymocytes have the capacity to vary their calcium responses to changes in TCR avidity. For most thymocytes, CD4 is functionally dissociated from the TCR complex due to its engagement by MHC class II expressed on thymic epithelium. This dissociation inhibits the participation of CD4-associated tyrosine kinase Lck in TCR signaling 20 . Thus, freshly isolated immature CD4 + CD8 + thymocytes do not respond to isolated TCR/CD3 signals and require CD4 costimulation to induce a calcium response . Coengagement of TCR/CD3 and coactivators such as CD4 allow Lck to participate in TCR signaling . CD28 does not cooperate with the TCR to induce a calcium response in freshly isolated lymphocytes because it does not recruit Lck to the complex 22 . To determine whether CD28 signaling could modulate the TCR-mediated [Ca 2+ ] i response when Lck was available, we examined the calcium response to coengagement of CD3, CD4, and CD28. The amplitude of the initial calcium rise was significantly higher in the presence of CD28 costimulation than in its absence (solid lines). This was true both at maximally and minimally aggregating concentrations of streptavidin. Interestingly, CD28 significantly enhanced the spiking activity of thymocytes, even at high concentrations of streptavidin , when spikes are not ordinarily observed. Thus, although CD28 alone cannot cooperate with CD3/TCR to generate calcium signals in immature thymocytes, it does modify the CD3/CD4 response. In fact, CD28 costimulation appears to mimic the effect of increased receptor aggregation on calcium signaling, even under conditions of minimal aggregation. The observed correlation between receptor avidity/aggregation and calcium signaling and the ability of CD28 to convert biphasic to spiking responses is consistent with an underlying role for calcium in thymocyte fate determination. Because the in vitro induction of thymocyte death and maturation requires immobilized antibody, we used microspheres to cross-link the TCR complex and coreceptors in parallel calcium signaling and thymocyte maturation experiments. Using this approach, coaggregation of CD3 and CD4 evoked a biphasic calcium response similar to that evoked by high concentrations of soluble streptavidin . Coengagement of CD3, CD4, and CD28 resulted in a spiking calcium response within many cells and a greater elevation in the mean peak and plateau [Ca 2+ ] i compared with CD3/CD4 stimulation . This effect of CD28 was similar to the CD28-mediated augmentation of the calcium signal after aggregation with soluble streptavidin . We next examined the effect of CD3, CD4, and CD28 stimulation on CD4 + CD8 + thymocyte fate in vitro. Consistent with previous studies using plate-bound antibody, microspheres treated with CD3 and CD4 induced activation of all thymocytes but not death . CD28 coengagement caused a marked and specific increase in thymocyte death. . In this report, we demonstrate that TCR/CD3-mediated Ca 2+ signaling patterns differ in developing thymocytes depending on (a) the maturation stage, (b) the extent of TCR aggregation, and (c) the participation of costimulatory or coactivating molecules. Notably, immature CD4 + CD8 + thymocytes, whose developmental responses are known to vary with the nature of TCR signals, have the unique capacity to decode distinct TCR signals by generating distinct calcium responses. Our data demonstrate that low avidity TCR engagement of CD4 + CD8 + thymocytes initiated a single biphasic Ca 2+ signal, whereas high avidity TCR engagement initiated an oscillatory or spiking Ca 2+ signaling pattern. We observed markedly heterogeneous Ca 2+ signaling patterns within thymocytes after CD3/CD4 aggregation and found that these signaling differences correlated with the thymocyte developmental stage. As one might expect, the most immature thymocytes (CD4 − CD8 − CD5 low ), which express little if any TCR on their surfaces, did not respond to TCR stimulation. Mature thymocyte populations (CD4 + CD8 − CD5 high and CD4 + CD8 + CD5 high ) exhibited a biphasic Ca 2+ signaling response that was also typical of mature peripheral T cells. In contrast, immature CD4 + CD8 + cells (CD4 + CD8 + CD5 low and CD4 + CD8 + CD5 int ), the targets of most selection events that occur within the thymus, could vary their Ca 2+ signaling responses. Immature DP thymocytes typically exhibited an initial transient elevation of [Ca 2+ ] i after receptor aggregation that subsequently decayed to an elevated steady state or oscillated depending upon the degree of receptor aggregation. Within a homogeneously immature population of fetal thymocytes, CD3/CD4 evoked only Ca 2+ i spikes in responding cells. In contrast, [Ca 2+ ] i spikes could not be evoked in mature, peripheral murine T cells regardless of the extent of TCR/CD3 aggregation. Together, these data indicate that the capacity to generate Ca 2+ spikes is restricted to immature CD4 + CD8 + thymocytes. The ability to generate biphasic or oscillatory Ca 2+ i signaling responses based upon the extent of TCR/CD3 aggregation is a unique characteristic of immature CD4 + CD8 + thymocytes, which suggests that Ca 2+ could play a role in determining the developmental fate of these cells. CD4 + CD8 + thymocytes are the targets of both negative and positive selection and will die or differentiate depending on the nature of the TCR signal they receive. Although the precise mechanism by which TCR engagement can specify negative and positive developmental responses is unknown, data clearly suggest that differences in TCR affinity and aggregation, collectively referred to as avidity, are key 1 4 . Receptor avidity and the maturational response are modulated by subtle structural alterations in peptide ligands. This ability of similar peptide ligands to induce positive or negative selection also correlates with the mean amplitude of the calcium response evoked by each ligand 14 23 . By varying the concentration of our receptor-engaging reagent, streptavidin, we have been able to modulate the extent of TCR/CD3 aggregation and, hence, mimic differences in antigen–TCR avidity. We find that avidity differences can be translated into distinct patterns of Ca 2+ signaling within thymocytes. High avidity TCR interactions, such as those associated with negative selection of DP thymocytes, evoked [Ca 2+ ] i spikes and an elevation in the mean [Ca 2+ ] i amplitude, whereas low avidity interactions, which are associated with positive selection, initiated a biphasic calcium signal. These findings imply that signals that induce Ca 2+ spikes will induce thymocyte apoptosis, whereas those that generate a single biphasic response will initiate maturation. It should be noted that semimature CD4 + HSA high cells can also undergo negative selection 24 25 . We did not look specifically at the calcium responses of this population; however, we did not observe spiking responses in CD4 + single-positive cells. Thus, negative selection of CD4 + HSA high thymocytes likely occurs by a distinct mechanism from immature DP thymocytes. Interestingly, the biphasic Ca 2+ signal generated by low avidity CD3 and CD4 engagement was converted to a spiking pattern by CD28, which provides an apoptotic signal in thymocytes 7 8 9 . CD28 also increased the peak amplitude of Ca 2+ i oscillations and the mean [Ca 2+ ] i after high avidity engagement of CD3/CD4. These data are consistent with the observation that peptides capable of triggering negative selection cause higher mean amplitude calcium elevations than those that trigger positive selection 14 . The mechanism underlying this effect of CD28 may be related to a recent observation that CD28 engagement amplifies TCR-mediated signaling 26 by promoting the redistribution and clustering of activation molecules, possibly within kinase-rich microdomains 27 , at the site of TCR engagement. Thus CD28-mediated amplification of calcium spiking activity in thymocytes may reflect its ability to change the structure and makeup of the TCR signaling complex. Our data are consistent with previous studies indicating that TCR/CD3 engagement with CD2 or CD4 induces maturation to the CD4 lineage in vitro 5 . In fact, TCR engagement without aggregation is associated with positive selection 28 . In these previous studies, stimulation was provided by saturating levels of plate-bound rather than soluble antibody. In our system, positive selecting conditions induced by high levels of soluble streptavidin (maximal receptor engagement but minimal receptor aggregation) are associated with biphasic calcium signals. We also duplicated conditions that induce thymocyte activation versus death using antibody fixed to microspheres. These experiments demonstrated that a biphasic calcium signaling response is associated with cell activation and repetitive spikes are associated with thymocyte apoptosis. The significance of these findings is underscored by recent studies indicating that calcium alone can specify different patterns of gene transcription in lymphocytes and that specificity is encoded in the amplitude and/or frequency of cytoplasmic Ca 2+ changes 15 16 29 . A variety of calcium-regulated protein kinases and phosphatases expressed by lymphocytes, including calcineurin, calmodulin-dependent kinase II (CaMKII) and protein kinase Cγ, could translate different Ca 2+ i signaling patterns 30 31 . Each is expressed in thymocytes and is responsive to TCR signals 32 33 34 . Notably, CaMKII activity is modulated by the [Ca 2+ ] i oscillation frequency 30 . These or other calcium-sensitive enzymes, which can distinguish between calcium amplitude– or frequency–encoded receptor signals, may initiate different patterns of gene expression and specify distinct developmental fates. The molecular basis for Ca 2+ spikes has not been defined in murine or human thymocytes, although calcium oscillations have been extensively studied in human peripheral lymphocytes 35 36 . It is generally accepted that [Ca 2+ ] i initially increases due to inositol 1,4,5-trisphosphate–mediated release from intracellular stores and is sustained by extracellular influx. In primary human T cells, [Ca 2+ ] i oscillations are initiated by transient feedback inhibition of rising calcium on its own influx 37 38 , and [Ca 2+ ] i levels are reduced by uptake into mitochondria and reuptake into the endoplasmic reticulum 39 40 . As Ca 2+ i falls, inhibition of its own influx is relieved. The net result is that [Ca 2+ ] i increases and decreases due to the sequential and repetitive activation and inactivation of influx under conditions of constant TCR stimulation 36 . In thymocytes, we found that both calcium spikes and steady state elevations in [Ca 2+ ] i were inhibited by removal of extracellular calcium, suggesting that extracellular [Ca 2+ ] plays a role in both signaling responses (data not shown). Several additional mechanisms may determine the pattern of calcium signaling. For example, in human T lymphocytes the plasma membrane potential strongly influences the magnitude of calcium influx and, consequently, the pattern of signaling. Sequential activation and inactivation of voltage-dependent and calcium-activated potassium channels has been shown to rapidly modulate the membrane potential and the driving force for calcium influx 41 . Other studies have demonstrated that ryanodine receptors play a role in the generation of calcium oscillations in human T cells 42 43 . Finally, in human B lymphocytes, [Ca 2+ ] i oscillations result from repetitive calcium release and reuptake into the endoplasmic reticulum, whereas sustained calcium elevations require extracellular calcium influx 44 . In summary, our data provide evidence that Ca 2+ i , which is one of the most proximal molecular reflections of TCR signaling, underlies the ability of immature CD4 + CD8 + thymocytes to distinguish between low and high avidity TCR signals. The unique ability of CD4 + CD8 + thymocytes to generate distinct Ca 2+ i signaling responses (depending upon the avidity of the TCR signal) strongly suggests that calcium plays a central role in regulating thymic selection. | Study | biomedical | en | 0.999997 |
10510085 | Mice doubly deficient in both the TNFR p55 and p75 genes (TNFR KO; from Dr. J. Peschon, Immunex Corp., Seattle, WA) were generated as described 18 and maintained as random C57BL/6 × 129 hybrids at the University of Manchester. Age- and sex-matched C57BL/6 × 129 F 2 mice were bred from F 1 littermates (Harlan Olac) and used as wild-type (WT) controls. BALB/c IL-4 KO mice were generated by Noben-Trauth et al. 19 and bred at the University of Manchester. Age- and sex-matched C57BL/6 and BALB/c mice were purchased from Harlan Olac. In all experiments, mice were infected when 6–9 wk old, and experimental groups contained four to six animals. All experiments were performed under the regulations of the Home Office Scientific Procedures Act . The maintenance, infection, and recovery of T . muris were as described previously 20 . Mice were infected on day 0 with ∼200 embryonated eggs, and numbers of larvae were counted on day 10 postinfection (p.i.) to ensure equivalent establishment of infection in different groups. Worm burdens were assessed on various days p.i. as described previously 21 . T . muris excretory/secretory antigen (ES Ag) was prepared as detailed previously 7 . In vivo depletion of TNF-α was carried out using purified rat IgG1 mAb XT22 (neutralizing TNF-α; from Dr. R. Coffman, DNAX Research Institute, Palo Alto, CA) injected intraperitoneally as detailed in the text. Control groups were treated with either isotype-matched control (GL113; from Dr. F. Finkelman, University of Cincinnati, Cincinnati, OH) or purified rat IgG (Sigma Chemical Co.). Recombinant TNF-α (Dr. G. Luheshi, University of Manchester) was delivered intraperitoneally as described in the text. Mesenteric lymph node cells were removed from uninfected and infected mice and resuspended in RPMI 1640 supplemented with 10% FCS, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all from GIBCO BRL), and 60 μM monothioglycerol (Sigma Chemical Co.). Cultures were stimulated with a predetermined optimum concentration of T . muris ES Ag (50 μg/ml) at 37°C and 5% CO 2 . Anti–IL-4R (M1) mAb (5 μg/ml; from Dr. C. Maliszewski, Immunex Corp., Seattle, WA) was added to cultures to increase detection of IL-4. Cell-free supernatants were harvested after 24 h and stored at −20°C. Cytokine analysis was carried out by sandwich ELISA using paired mAbs to detect IL-4 (BVC4-1D11 and BVD6-24G2.3; PharMingen), IL-5 (TRFK.5 and TRFK.4; PharMingen), IL-9 (249.2 and biotinylated 1C10/2C12; from Dr. J. van Snick, Ludwig Institute of Cancer Research, Brussels, Belgium), IFN-γ (R46A2 and XMG.2; PharMingen), and IL-12 (C15.6 and C17.8; from Dr. G. Trinchieri, Wistar Institute, Philadelphia, PA). TNF-α and IL-13 production was assessed using an R&D Systems ELISA kit. Quantification of cytokines was determined by reference to commercially available recombinant murine standards, and the sensitivity of the assays was determined by taking the mean + 3 SD of 16 control wells containing medium alone. Analysis of parasite-specific IgG1 and IgG2a production was carried out by capture ELISA as described 22 . In brief, Immulon IV plates (Dynatech) were coated with T . muris ES Ag (5 μg/ml) in carbonate/bicarbonate buffer, pH 9.6, overnight at 4°C. After blocking (3% BSA in PBS, 0.05% Tween), eight serial 2-fold dilutions of sera (from an initial 20-fold dilution) were added to the plates. Parasite-specific antibody was detected using biotinylated rat anti–mouse IgG1 (Serotec Ltd.) and biotinylated rat anti–mouse IgG2a (PharMingen). Significant differences ( P < 0.05) between experimental groups were determined using the Mann-Whitney U test. We have previously demonstrated that C57BL/6 mice mount Th2 responses and expel T . muris 7 . The role of TNF-α in regulating this host protection was investigated by administering 2 mg of anti–TNF-α or control antibody (GL113) intraperitoneally every 3 d between days 7 and 19 p.i. Blockade of TNF-α significantly prevented worm expulsion at day 21 p.i. compared with control treated groups . With the exception of depressing IL-9 production, anti–TNF-α treatment did not significantly alter the magnitude of the Th2 response generated at day 21 p.i. . Prevention of worm expulsion is transient, and cessation of anti–TNF-α treatment resulted in the clearance of infection in all treatment groups by day 35 p.i. . As the cytokine response has diminished by day 35 p.i. (data not shown), this suggests that a sufficient Th2 response was generated in the mesenteric lymph nodes of anti–TNF-α treated mice to induce worm expulsion . Therefore, although depletion of TNF-α has no demonstrable effect on the initiation of Th2 responses, it appears to be critical in the regulation of Th2 cell effector function in the intestine. This hypothesis is reinforced by the parasite-specific IgG isotype response at day 35 p.i. Expulsion of T . muris occurs in the absence of antibody 23 ; however, assessment of IgG isotype production provides an indication of the polarization of the Th response after infection. IgG1 production is known to be under the control of Th2 cytokines 24 , and there was no significant difference in this response between treatment groups , substantiating the fact that there is no reduction in the magnitude of the Th2 response after anti–TNF-α treatment. The elevated IgG2a response (under the control of IFN-γ 25 ) observed after blockade of TNF-α may reflect elevated IFN-γ production in these mice resulting from protracted exposure to infection. As anti–TNF-α treatment blocked protection in C57BL/6 mice, we investigated the kinetics of infection in mice completely deficient in TNFR signaling. We consistently found that TNFR KO mice were highly susceptible to T . muris and unable to expel worms by day 35 p.i. . Previous data have shown that mature worms (day 32 p.i.) can promote their own survival and can only be expelled through drug intervention after this time point 4 . WT mice began worm expulsion between days 10 and 18 p.i., and had completely cleared infection by day 22 . Surprisingly, analysis of Ag-specific cytokine production after restimulation of mesenteric lymph node cells showed that TNFR KO mice made little or no Th2 cytokines, whereas WT mice made a strong Th2 response (characteristic of resistant strains ) with high levels of IL-4, IL-5, IL-9, and IL-13 at day 18 p.i. . However, KO mice produced higher levels of Ag-specific IFN-γ (KO, 221.32 ± 121.17 U/ml; WT, 153.5 ± 50.0 U/ml) and IL-12 (KO, 585.51 ± 139.09 ng/ml; WT, 123.03 ± 103.38 ng/ml) after restimulation of mesenteric lymph node cells. Parasite-specific IgG isotype responses at day 35 p.i. reflected the resistant and susceptible phenotype of WT and TNFR KO mice and the polarized Th response observed. WT mice made high levels of IgG1 and relatively low levels of IgG2a typical of resistant mice. TNFR KO mice made lower IgG1 responses and high levels of IgG2a . The higher IgG2a response generated in KO mice supported the elevated Th1 cytokine levels observed in the absence of TNFR signaling. This is in accordance with previous observations in other susceptible strains that produced predominantly Th1 cytokines and high levels of IgG2a 4 5 6 7 . We have previously shown that IL-13 is directly involved in expulsion of T . muris , as IL-13 KO mice were unable to clear infection despite generating equivalent parasite-specific Th2 responses to WT mice at day 21 p.i. 7 . Subsequent studies have also found that expulsion of T . muris in female BALB/c IL-4 KO mice is IL-13 dependent. In the absence of IL-4, protection is almost completely abrogated by blockade of IL-13 using a soluble IL-13R fusion protein (Bancroft, A.J., manuscript in preparation). To investigate whether this IL-13–mediated expulsion is also dependent on TNF-α, infected female BALB/c IL-4 KO mice were treated with 2 mg of anti–TNF-α mAb or rat IgG intraperitoneally every 3 d between days 7 and 28 p.i. Blockade of TNF-α in female BALB/c IL-4 KO mice significantly prevented worm expulsion . In accordance with previously unpublished data, in control treated IL-4 KO mice worm expulsion was initiated around day 22 p.i. , and approximately 60% of worms were cleared by day 35 p.i. . Ag-specific cytokine production by mesenteric lymph node cells at day 18 p.i. showed comparable Th2 responses in the different treatment groups , although IL-9 and IL-13 production was lower in anti–TNF-α treated mice than in the control Ig group . No difference in the IgG1 response was observed between anti–TNF-α and control treated groups (data not shown), supporting the hypothesis that TNF-α blockade did not alter the generation of Th2 responses, but may be critical at the effector stage in the intestine. Comparable and relatively high IgG2a responses were observed in all groups (data not shown), reflecting the production of Th1 cytokines as previously reported in T . muris –infected C57BL/6 IL-4 KO mice 7 . Previous experiments have revealed a sex difference in resistance to T . muris in BALB/c IL-4 KO mice (Bancroft, A.J., and D. Artis, unpublished observations). We have now confirmed these data demonstrating that female mice produce higher parasite-specific Th2 cytokines (IL-5, IL-9, and IL-13) than males and elevated IgG1 responses during infection (data not shown). Therefore, female IL-4 KO mice expel worms around day 35 p.i., whereas males are unable to do so. To investigate whether elevation of TNF-α levels in vivo could promote a resistant phenotype in male BALB/c IL-4 KO mice, 2 μg of TNF-α was administered intraperitoneally to infected mice daily between days 10 and 24 p.i., and infection outcome was monitored at days 21 and 35 p.i. Administration of TNF-α to male BALB/c IL-4 KO mice considerably increased worm expulsion by day 35 p.i., with 43% of worms cleared in TNF-α–treated mice compared with no reduction in worm burden in the control group at day 35 p.i. ( Table ; P < 0.05). (TNF-α treatment did not have any obvious detrimental effects on the general health of the animals.) Interestingly, treatment with TNF-α did not alter the production of IL-5, IL-9, or IL-13 after in vitro restimulation with ES Ag ( Table ), suggesting again that TNF-α may potentiate the effects of Th2 cytokines (in this case IL-13) rather than enhance their production. In addition, no difference in IgG1 production was observed between TNF-α and control treated mice although higher IgG2a production was observed in TNF-α–treated mice, reflecting higher IFN-γ production (data not shown). Administration of TNF-α to male IL-4 KO mice conferred a similar rate of worm expulsion ( Table ) to that observed in female mice , suggesting that impaired protection in male mice may be due to reduced levels of TNF-α during infection. However, analysis of TNF-α production after Ag-specific in vitro restimulation of mesenteric lymph node cells showed no significant difference between male and female mice ( Table ). Therefore, impaired clearance of infection in male mice is more likely to be due to lower Th2 responses compared with females rather than a defect in TNF-α production. Thus, although there is no inherent difference between male and female IL-4 KO mice in the production of TNF-α, administration of this cytokine is able to induce a resistant phenotype in male mice without a significant elevation in parasite-specific Th2 responses. Previous studies have shown that immune-mediated expulsion of the intestinal helminth T . muris is dependent on IL-4 and IL-13 5 7 . The results presented here provide the first demonstration of a critical role for TNF-α in host protection against an intestinal helminth infection and extend our understanding of the role of TNF-α as an essential component of Th2 cell–mediated effector responses. TNF-α is known to be a key mediator of pathogenesis in a broad range of infectious, inflammatory, and autoimmune diseases (for a review, see reference 26 ), although few studies have investigated the role of this prototypic inflammatory cytokine in the regulation of immune responses dominated by Th2 cytokines. The role of TNF-α in the pathogenesis of predominantly Th1-mediated inflammation, including collagen-induced arthritis 27 , autoimmune encephalomyelitis 28 , and intestinal inflammation 29 30 , is well characterized. Our results extend these observations and identify a novel role for TNF-α in the regulation of Th2 cell–mediated protection during helminth infection. It is significant that immunodepletion of TNF-α in C57BL/6 and BALB/c IL-4 KO mice showed little or no reduction in the magnitude of Ag-specific Th2 cytokine production and IgG1 responses during infection, despite blocking worm expulsion. This demonstrated that blockade of TNF-α had little effect on the initiation of Th2 responses in the draining lymph node. Preliminary analysis of intestinal mast cell hyperplasia (which is under the control of Th2 cytokines) during infection in C57BL/6 mice showed a depressed mastocytosis after anti–TNF-α treatment (data not shown). This supports the hypothesis that although the magnitude of the Th2 response in the draining lymph node is unaltered, in the intestinal microenvironment the Th2 response is impaired, implicating a role for TNF-α in regulation of effector function. Previous experiments have shown that in vivo elevation of IL-9 enhances resistance to T . muris 31 , and after depletion of TNF-α in infected C57BL/6 and female IL-4 KO mice the depressed production of IL-9 may be important in preventing normal expulsion (although it is clear that mast cells are not important in host protection 32 ). TNF-α is known to regulate expression of a range of cell adhesion molecules on vascular endothelium 33 34 and leukocytes 35 and to control the expression of chemoattractant cytokines 36 . In addition, TNF-α has been shown to be necessary for homing of Th2 cells to the site of allergic inflammation 17 . Thus, ongoing studies are addressing leukocyte homing and recirculation to the intestine during infection in the presence and absence of TNF-α. TNF-α is a pleiotropic cytokine, and the mechanisms of regulating host protection to T . muris may not be through the recruitment of inflammatory cells, but rather through amplification of the existing Th2 response. Certainly, TNF-α has been shown to enhance Th2 cell–mediated phenomena in other systems, including pathogenesis of allergic inflammation in the gastric mucosa 37 , Schistosoma mansoni egg–induced granuloma formation 38 39 , and airway hyperresponsiveness 17 . A role for TNF-α in amplifying effector function of Th2 cytokines is recapitulated by our studies in which at least partial protection against T . muris can be conferred on normally susceptible male BALB/c IL-4 KO mice after administration of TNF-α. Comparison between the magnitude of the Th2 response mounted in male and female BALB/c IL-4 KO mice supports previously unpublished data demonstrating a Th2 bias in females and enhanced protection. Furthermore, it is clear that sex differences in host protection are not due to lower TNF-α production in male mice after infection. That enhancing in vivo levels of TNF-α in male IL-4 KO mice increased worm expulsion at day 35 p.i. to levels similar to those observed in female mice (without altering the levels of Th2 cytokines produced) supports the hypothesis that TNF-α is acting downstream of Th2 cell differentiation and is potentiating the protective effects of Th2 cytokines in male mice. Whether TNF-α administration can enhance IL-13–mediated phenomena in other systems such as airway inflammation 15 16 , S . mansoni egg–induced pulmonary granuloma 40 , and resistance to Nippostrongylus brasiliensis infection 41 remains to be determined. The development of chronic infection in mice deficient in TNFR p55 and p75 provides the first characterization of intestinal helminth infection in these mice. The inability of TNFR KO mice to expel T . muris may reflect a similar impairment of Th2 cell–mediated effector function as suggested above. However, we were unable to detect any significant Th2 cytokines after restimulation of mesenteric lymph node cells in these mice, suggesting that the absence of TNF-α function at the initiation of infection in TNFR KO mice had a profoundly different effect (i.e., impairing initiation of a Th2 response) than depletion of TNF-α later in infection (see above). Mice deficient in either or both p55 and p75 have been shown to be resistant to lethal shock induced by LPS 42 , have attenuated responses to cerebral malaria 43 , and were highly susceptible to intracellular bacterial 42 44 45 and protozoan infections 46 . TNFR KO mice have also revealed a novel role for these receptors in lymphoid tissue organogenesis and morphogenesis with the reported absence of B cell follicles, germinal center formation, and follicular dendritic cell networks (for reviews, see references 47 and 48 ). Our results demonstrate no impairment of immune responsiveness per se during T . muris infection, with polarized Ag-specific Th1 cytokine production and strong parasite-specific IgG isotype responses detected at day 35 p.i. It has been suggested that IgG isotype switching in TNFR KO mice is normal 49 , and given that expulsion of T . muris can occur in the absence of antibody 23 , we feel this is not the basis of susceptibility to infection in these mice. However, we cannot rule out impaired or altered Ag sampling, processing, and presentation in TNFR KO mice, given the reported absence of Peyer's patches in TNFR KO mice 50 and the suggested role for TNF-α in the maturation of dendritic cell function 51 52 and migration to the draining lymph node 53 . Such alterations in initiation of the response may result in the polarized Th1 response observed after infection, suggesting a potentially critical role for TNF-α in response induction. (LT-α is also known to bind TNFR with equal affinity and kinetics to TNF-α 54 , and may play a role in host protection.) In this context, there are several conflicting studies on the role of TNF-α in autoimmune diseases such as insulitis 55 56 with the outcome of TNF-α administration and blockade clearly dependent on the cytokine dose, duration of exposure, and site of expression 57 , and similar processes may be operating in our system. The data presented here support the hypothesis that TNF-α, when administered or blocked after the first week of infection (and coincident with the generation of a Th2 response), has a clear effect on host protection without altering the magnitude of the Th2 response, while the absence of TNF function from day 0 (in TNFR KO mice) inhibits the induction of Th2 responses. However, the exact molecular and cellular mechanisms through which TNF-α mediates its protective effects remain to be elucidated. For instance, does TNF-α act on lymphocyte populations to augment Th2 effector cell responses 58 , on APC function, or on multiple cell types derived from a nonlymphoid lineage? And what contribution do the independently regulated and functionally distinct p55 and p75 receptors make to host protection? That administration and blockade of TNF-α can modulate infection outcome without altering the production of Th2 cytokines suggests a role for TNF-α in regulating Th2 effector function. An attractive hypothesis is that TNF-α functions through regulation of IL-4 and IL-13 receptor expression on cells in the intestinal microenvironment, a function that has been observed in human endothelial cells 59 . However, it is clear that we have identified a novel role for TNF-α in regulating Th2 cytokine responses in the intestine, which has a significant effect on protective immunity to helminth infection. As such, these results extend our understanding of the complex interplay of the cytokine network and have significant implications for the design of rational therapies against helminth infection and allergic reactions in the gut and at other mucosal sites. | Study | biomedical | en | 0.999996 |
10510086 | C57BL/6, NOD/shi, and NOD/Scid mice were bred and maintained under specific pathogen-free housing conditions at the Scripps Institute Rodent Colony. Female 3-wk-old NOD mice were used as recipients in transfer experiments, and 8–10-wk-old female mice were used as donors and for in vitro experiments. Anti-CD3 mAb (clone 2C11, hamster Ig) for in vivo treatment was purified from tissue culture supernatants on a protein G column. Purified anti–TCR-β (clone H57-597), PE-conjugated hamster anti–mouse CD3, FITC-conjugated rat anti–mouse CD122 (IL-2 receptor β chain), and biotinylated rat anti–mouse Ly49A and streptavidin-cychrome to detect biotinylated antibodies were purchased from PharMingen. For ELISA assay, primary mAbs anti–IL-4 (clone 11B11) and anti–IFN-γ (clone R46A.2) and secondary biotinylated anti–IFN-γ (clone XMG1.2) were purified from ascites, and secondary biotinylated anti–IL-4 (clone BDV6-24G2) was purchased from PharMingen. To evaluate NKT cell homeostasis in vivo, a group of NOD and C57BL/6 mice were injected intravenously with purified anti-CD3∈ mAb (0.2 μg/mouse) in 200 μl of PBS. The mice were killed after 20 h, and their livers were collected for cell purification. Single cell suspension was prepared from liver, thymus, and spleen by passing them through nylon mesh. Splenocytes were treated with hypotonic solution to remove red blood cells. Total liver cells were resuspended in 40% Percoll solution (Sigma Chemical Co.) and underlaid with 80% isotonic Percoll solution. Mononuclear cells were then isolated at the 40/80% interface after centrifugation at 2,000 rpm for 20 min. Single cell suspensions were washed twice with PBS containing 20 mM Hepes solution to stabilize the pH at 7.3, incubated with PE–anti-CD3, FITC–anti-CD122, and biotinylated anti-Ly49A primary antibodies at 4°C for 30 min, and after two washes, with the secondary reagent, streptavidin-cychrome, at 4°C for 20 min. The cells were washed again twice and then analyzed on a FACScan™ using the CELLQuest™ program. Triple-positive (CD3 + , Ly49A + , and CD122 + ) T cells were sorted using a FACStar PLUS™ (Becton Dickinson). Simultaneously, CD3 + , Ly49A − , and CD122 − T cells were sorted. The sorted T cell populations were 99% pure and were used directly for cell culture or adoptive transfer experiments. 3 × 10 5 NKT cells purified by FACS ® sorting from total splenocytes of 8–10-wk-old NOD mice were injected in each 3-wk-old NOD recipient mouse. A control group was injected with the same number of CD3 + NK − T cells. A different group of mice received NKT cells that were stimulated in vitro in the presence of IL-7 (10 3 U/ml) for 3 d after the sorting. In different experiments, total splenocytes were completely deleted from NKT cells by FACS ® sorting and then injected in NOD/Scid mice either with or without 6 × 10 5 purified NKT cells. Blood glucose levels were measured weekly using Glucofilm blood glucose strips (Miles Diagnostics Division, Inc.). Mice were considered diabetic after two consecutive measurements of blood glucose level ≥250 mg/dl. NKT cells freshly sorted from total thymocyte or splenocyte populations were stimulated in vitro with plate-bound anti–TCR-β and grown in complete RPMI 1640 medium (GIBCO BRL) supplemented with 10% FBS, 50 μM 2-ME, 2 mM l -glutamine, 100 U/ml penicillin, 200 μg/ml streptomycin, 1 mM sodium pyruvate, and 100 μM nonessential amino acids. The cells were cultured at a density of 5 × 10 4 cells/ml in the presence of IL-2 (10 U/ml) alone, or with IL-7 (10 3 U/ml) or IL-12 (10ng/ml). IL-2 was added to the cultures every 3–4 d, and every week they were restimulated with plate-bound anti–TCR-β. 48 h after each restimulation, 100 μl of supernatants was collected from NKT cell cultures for cytokine measurement. The number of NKT cells/well was the same for NOD and C57BL/6 samples. We did not observe differences in NKT cell viability between the two groups after cytokine stimulation. 96-well plates were coated with anti–TCR-β mAb at 10 μg/ml for 2 h at 37°C and washed three times with cold PBS. Freshly sorted NKT cells were added to the wells at 10 4 cells/well in 200 μl of complete RPMI in duplicates in the presence of IL-2 alone or with IL-7 or IL-12. After a 2-d incubation at 37°C, 5% CO 2 , cells were pulsed with 1 μCi of [ 3 H]thymidine for 16 h, and radioactive thymidine incorporation was measured by a scintillation β-counter (Wallac). Supernatants of anti–TCR-β stimulated NKT cell cultures were analyzed for the presence of IL-4 or IFN-γ using an ELISA assay. In brief, ELISA 96-well plates (Nunc) were coated with primary mAb anti–IL-4 at 4 μg/ml or anti–IFN-γ at 2 μg/ml diluted in PBS overnight at 4°C. After washes with PBS containing 0.05% Tween and incubation with blocking solution (PBS/Tween containing 1% BSA and 10% FBS), the supernatant samples were added to the wells at serial fivefold dilutions and incubated overnight at 4°C. Recombinant murine IFN-γ and IL-4 (PharMingen) were used as standards. Biotinylated secondary antibody anti–IFN-γ or anti–IL-4 at 1 μg/ml and streptavidin-peroxidase plus H 2 O 2 –based developing system were used to detect the amount of cytokines. The concentrations of IFN-γ and IL-4 were interpolated using the Softmax program against the linear range on the standard curves (40–2,500 pg/ml). Differences in proliferation assay, cytokine secretion, and diabetes incidence were analyzed using the Student's t test. It has been reported that primary cytokine secretion upon anti-CD3 stimulation of thymic NKT cells is defective in NOD mice, but it is still unclear where the functional defect of peripheral NKT cells lies. We analyzed the ability of NKT cells from spleens of NOD mice to respond to anti-TCR–mediated activation. NKT cells were isolated by three-color staining with antibodies against markers of T cells (anti-CD3–PE) and NK cells (anti-Ly49A–cychrome and anti–IL-2 receptor β chain–FITC) followed by FACS ® sorting of triple-positive lymphocytes. Freshly sorted NKT cells of NOD mice were activated in vitro with plate-bound anti–TCR-β antibody. NKT cells from C57BL/6 mice were used as controls. Several cytokines known to act as growth factors for NKT cells were then added to the NKT cell cultures to determine their effect on the anti-TCR–mediated proliferation. As Fig. 1 A illustrates, the proliferative response of NKT cells from NOD mice either without cytokines or in the presence of IL-2 was significantly reduced compared with the same T cell population of C57BL/6 mice ( P < 0.05). Next, IL-7 was also added because it has been shown to induce in vitro maturation and growth of thymic NKT cells from NOD mice 39 . However, in our experiment IL-7 did not modify the defect in TCR-mediated stimulation in peripheral NKT cells of NOD mice. This defect became even more dramatic in the presence of IL-12, a critical cytokine for NKT cell activation and differentiation toward the IFN-γ–secreting phenotype. As expected, IL-12 added to like cultures from C57BL/6 mice enhanced TCR-mediated proliferation of NKT cells. Strikingly, IL-12 had no effect on NKT cells of NOD mice. In addition, we found that the expansion of the NKT cell population in response to anti-TCR plus IL-12 stimulation was also effected in NOD mice. In fact, after 3 d of culture in the presence of IL-12, anti-TCR–stimulated NKT cells of C57BL/6 mice showed a numerical increase of 50%. In contrast, there was no increase and even a slight decrease (5%) in the number of NKT cells from NOD mice, most likely related to cell death . It has been proposed that NKT cells may regulate autoimmunity by secreting cytokines such as IL-4 and IL-10 that are downmodulatory for the Th1 cytokine pathway. In addition, it has been reported that patients affected by IDDM have a defect selectively on IL-4 secretion by NKT cells 40 , although it recently emerged that the functional defect of NKT cells of IDDM patients does not involve only IL-4 secretion (Wilson, B., personal communication). Moreover, it is now clear that the function of peripheral NKT cells is related to their IFN-γ–secreting phenotype and inflammatory features 22 23 24 27 28 29 30 . Therefore, we investigated the ability of NKT cells from NOD mice to differentiate toward an IL-4– or IFN-γ–secreting phenotype to determine if they carry a defect in differentiation toward a specific cytokine pathway. To establish cytokine phenotype, NKT cells isolated from the thymi and spleens of NOD and C57BL/6 mice were examined by IL-4 and IFN-γ ELISA assays on supernatants of anti-TCR–stimulated NKT cells. We found that normal NKT cells from thymocytes of C57BL/6 mice had a predominant IL-4–secreting phenotype in accordance with the observation that NKT cells from the thymi of normal (BALB/c × NOD)F 1 mice protected NOD mice through secretion of IL-4 8 . NKT cells isolated from thymi of NOD mice not only secreted lower amounts of both cytokines but, in particular, were unable to secrete IL-4. This defect can be related to the lack of maturation of NOD NKT cells in the thymus. Several studies have now shown that NKT cells, once they reach secondary lymphoid tissue, acquire a strong IFN-γ–secreting phenotype. In fact, when we measured cytokine secretion of NKT cells isolated from spleens of C57BL/6, we observed a complete shift toward an IFN-γ–secreting phenotype . Strikingly, peripheral NKT cells from spleens of NOD mice showed no increase in cytokine secretion and did not differentiate toward the IFN-γ cytokine phenotype as did normal C57BL/6 NKT cells. Since IL-12 is considered a critical cytokine in maturation and differentiation of NKT cells 25 26 31 32 33 34 , we investigated the ability of IL-12 to drive peripheral NKT cells from NOD mice toward a strong IFN-γ–secreting phenotype. NKT cells purified from spleens of NOD and C57BL/6 mice were activated in vitro by anti-TCR stimulation in the presence of IL-7 or IL-12. Secretion of IL-4 and IFN-γ was then measured both during primary stimulation and after continuous stimulation in the presence of cytokines. As expected, peripheral NKT cells from C57BL/6 mice secreted higher amounts of IFN-γ than IL-4. In addition, IL-12 abolished IL-4 secretion while dramatically increasing IFN-γ secretion in the NKT cell cultures . On the other hand, in the NOD mice the secretion of IFN-γ in response to IL-12 stimulation was significantly lower . The lack of differentiation toward an IFN-γ phenotype was even more striking in NOD NKT cell cultures repetitively stimulated in the presence of IL-12 . It has been shown recently that IL-12– or anti-CD3–mediated activation in vivo induces pronounced cell death in peripheral NKT cells in <24 h, after which their number is rapidly restored by regeneration from bone marrow stem cells 41 . This uniquely rapid turnover seems necessary to prevent NKT cells from interfering with acquired immune responses. In other words, activated NKT cells with a biased IFN-γ–secreting phenotype must be rapidly removed once they clear pathogens, so as to halt inflammatory immune responses that are no longer required. To evaluate homeostasis of peripheral NKT cells in NOD mice, we analyzed the degree of CD3 + cell depletion in the liver, where ∼50% of the T cells are NKT cells. NOD and C57BL/6 mice were injected with anti-CD3 antibody, and the total number of CD3 + lymphocytes in the liver was measured by FACS ® analysis. Fig. 4 shows that CD3 + lymphocytes of C57BL/6 mice decrease by 50% at 20 h after anti-CD3 antibody injection. This CD3 + cell depletion could reflect the death of NKT cells, since previous studies have shown that other T cell populations in the liver are not affected by anti-CD3 administration. We confirmed that the depleted CD3 + T cell population in C57BL/6 mice carried markers of NKT cells such as NK1.1 (data not shown). Strikingly, in NOD mice the same CD3 + population was not at all affected by anti-CD3 stimulation. Our results suggest that peripheral NKT cells of NOD mice, once activated in vivo, did not undergo a rapid deletion, a phenomenon that can be ascribed to their defective TCR-mediated activation. Because NOD mice have fewer circulating NKT cells than nonautoimmune strains of mice, we questioned whether this quantitative defect is the only defining factor, or whether the functional defect we found in peripheral NKT cells of NOD mice could be crucial for the pathogenesis of IDDM. First, we transferred 3 × 10 5 purified NKT cells from adult NOD mice to markedly enlarge the NKT cell subset in young syngeneic recipients. NKT cells from splenocytes of 8–10-wk-old donors were stained with markers of T cells (anti-CD3) and NK cells (anti-Ly49A and anti–IL-2 receptor β chain). The triple-positive cells were isolated by FACS ® sorting and injected into 3-wk-old NOD mice. A control group received only phosphate buffer solution. The NOD transfer recipients, despite carrying an NKT cell repertoire double that of untreated NOD mice, were only partially protected against IDDM . However, the difference in diabetes incidence between the two groups of mice was not statistically significant ( P > 0.05) at any point in the age-related curve. Normal NKT cells in the periphery predominantly carry an IFN-γ–secreting phenotype, so the lack of protection from IDDM by a large number of peripheral NKT cells could be integrally ascribed to their functional defect in IFN-γ secretion. To further support this hypothesis, we isolated NKT cells from the spleens of NOD donors and stimulated them in vitro with IL-7, after which they were transferred into 3-wk-old recipients. This cytokine has been reported to be capable of stimulating IL-4 secretion by NOD NKT cells 39 . In fact, after 3 d in vitro stimulation with IL-7, NKT cells of NOD mice showed a normal IL-4 secretion but were weak in IFN-γ secretion . Although their IL-4 secretion was amplified, IL-7–stimulated NKT cells were still unable to protect against IDDM . Furthermore, the group of mice that received IL-7–stimulated NKT cells did not show the slight decrease in IDDM incidence that we found in NOD mice treated with freshly sorted, IFN-γ–secreting NKT cells. This demonstrates that the administration of Th2-differentiated NKT cells does not counterregulate, but rather worsens, IDDM pathogenesis. We found that the dramatic defect in proliferation and IFN-γ secretion in response to IL-12 rendered us unable us to induce NOD NKT cells toward the Th1 cytokine–secreting phenotype. We also tested the ability of NOD NKT cells to immunoregulate the effector phase of IDDM and downmodulate diabetogenic T cells. For that purpose, 10 7 total splenocytes were isolated from prediabetic NOD mice (14-wk-old), depleted of NKT cells by FACS ® sorting, and injected in NOD/Scid recipients. Another group of NOD/Scid mice received the same number of NKT-depleted splenocytes plus 6 × 10 5 purified NKT cells. The onset and incidence of IDDM in the two groups of mice were identical , indicating that a functional rather than numerical defect in peripheral NKT cells renders them unable to mediate immunoprotection against IDDM. The function of NKT cells in the immune system appears strongly related to their IFN-γ secretion and cytotoxic properties 22 23 24 27 28 29 30 . Our results clearly showed that peripheral NKT cells of NOD mice carry a pronounced defect in their TCR-mediated– and IL-12–induced activation and IFN-γ secretion. The lack of these inflammatory features associated with the inability to offset IDDM, which we found in NKT cells of NOD mice, suggests that the defect of their IFN-γ–secreting phenotype may be involved in T cell–mediated autoimmunity. The involvement of the NKT cells in modulation of autoimmunity is, in fact, proved by the observation that a defect of the NKT cell population is invariably associated with T cell–mediated autoimmune diseases. NOD mice which develop spontaneous IDDM, as well as other autoimmune-prone strains of mice, have a paucity of NKT cells 4 6 . In young NOD mice, the restoration of a normal NKT cell repertoire by transfer of NKT cells from non–autoimmune-prone mice conferred protection from IDDM 7 8 . However, we showed here that the restoration of a functional NKT cell population rather than simply an increase in cell number may be critical to offset IDDM. In fact, the transfer of NKT cells from adult, prediabetic NOD mice to young NOD recipients did not significantly affect the disease. Yet, transfer of diabetogenic T cells into NOD/Scid mice induced IDDM whether NKT cells were absent or coinjected in large numbers. These findings agree with a recent report showing that Vα14 TCR transgenic mice, despite an extremely large NKT cell population, developed IDDM, albeit at a reduced frequency 9 . Therefore, it is now clear that a functional rather than quantitative defect of NKT cell correlates with the pathogenesis of IDDM. However, the nature of this defect in NOD mice is still unclear. Previous studies in NOD mice and individuals affected by IDDM have shown that NKT cells are defective specifically in IL-4 secretion 6 40 . This finding, together with the observation that IL-4 is required for (BALB/c × NOD)F 1 NKT cells to mediate protection against IDDM 8 , had supported the hypothesis that the secretion of IL-4 is associated with NKT cell–mediated regulation of autoimmunity. However, this hypothesis is in sharp contrast to many studies showing that normal NKT cells in the periphery have a strong IFN-γ–secreting phenotype and do not secrete large amounts of IL-4. Therefore, we analyzed peripheral, IFN-γ–secreting NKT cells in NOD mice and found that their defect was more generalized, involved their IFN-γ secretion, and could relate to the pathogenesis of IDDM in distinct ways. The marked lack of proliferation and expansion of NKT cells in response to TCR-mediated activation that we reported could account for the typically limited number of these cells in NOD mice 6 . Moreover, the defect in TCR-mediated activation might dramatically compromise the ability of NKT cells to respond to their specific ligand, the CD1 molecule 42 . CD1 recognition in the thymus, and probably also in the periphery, is a critical signal for maturation of NKT cells 43 44 45 46 . In fact, CD1 knockout mice carry an NKT cell repertoire that is defective in cytokine secretion 19 , a defect similar to that which we found in NOD mice. The defect in NKT cell activation and response to IL-12 may also affect their homeostasis, i.e., turnover rate, in NOD mice. Like many cells belonging to the innate immune system, activated NKT cells, differentiated toward a strong IFN-γ–secreting phenotype, are required in the early stages of an immune response against pathogens, and then they need to be rapidly removed from the site of inflammation so as not to interfere with the secondary T cell responses. Both IL-12 and anti-CD3 activation induce a dramatic cell death among the NKT cell subset 40 . NKT cells may be refractory to activation through the IL-12 receptor or TCR–CD3 complex. The lack of rapid homeostasis of peripheral, IFN-γ–secreting NKT cells that we found in NOD mice could be the result of this defect, which predisposes them to sustain unnecessary Th1 inflammatory responses, including autoreactive responses, and ultimately poses the risk of T cell–mediated autoimmunity. An alternative hypothesis holds that peripheral NKT cells with a strong IFN-γ–secreting phenotype could be critical to directly dampen the destructive potential of autoreactive T cells. Contrary to the belief that NKT cells could prevent the onset of autoimmune diseases by secreting downmodulatory cytokines such as IL-4, we found that IL-4 secretion by peripheral NKT cells may be not so critical to offset IDDM. In fact, the release of IL-4 by peripheral NKT cells is minimal compared with IFN-γ secretion. In other words, normal NKT cells in the thymus secreted large amounts of IL-4 in our experiments, perhaps the route of IL-4–mediated protection from IDDM that NKT cells from thymocytes of nonautoimmune mice induced in NOD mice. However, our results showed that once normal NKT cells of C57BL/6 mice reach the periphery, they acquire a strongly biased IFN-γ–secreting phenotype. On the other hand, peripheral NKT cells of NOD mice failed to respond to activation and underwent IFN-γ phenotype differentiation induced by IL-12, thus suggesting that the defect in the IFN-γ–secreting phenotype of peripheral NKT cells may correlate with the pathogenesis of IDDM in the NOD mice. This hypothesis has been further supported by our finding that restoring a normal IL-4–secreting phenotype in NOD NKT cells before their transfer in young syngeneic recipients did not enhance their ability to offset the pathogenesis of IDDM. Several lines of evidence indicate that cytokines belonging to the Th1 pathway are ultimately needed to prevent pathogenic autoimmunity. Interestingly, the expression of IFN-γ in the pancreatic islets protects NOD mice from IDDM, and removal of Th1 cytokines such as IFN-γ exacerbated T cell–mediated autoimmune diseases 47 48 49 . IFN-γ has been proposed to play a direct role in downmodulation of T cell immunity 50 51 52 . In fact, IFN-γ is able to induce cell death on effector T cells that are activated by TCR cross-linking in the absence of costimulatory signals, a mechanism that seems critical for maintaining immune tolerance to self-antigens 53 . It has been previously shown that regulatory T cells such as TCR-specific CD4 + T cells protect from T cell–mediated autoimmunity through secretion of IFN-γ 54 . We believe that peripheral NKT cells with a strong IFN-γ–secreting phenotype could directly downmodulate Th1-type autoimmunity by releasing large amounts of IFN-γ in the microenvironment. Alternatively, they could directly eliminate Th1-autoreactive cells by Fas-mediated cytotoxicity 28 30 . NKT cells with a strongly biased IFN-γ–secreting phenotype and cytotoxic properties could participate in T cell–mediated immunity. At the same time, the burst of IFN-γ secretion induced by NKT cells in the microenvironment could be critical to turn off unnecessary T cell immune responses, including autoreactive responses. Our viewpoint is paradoxical with the earlier finding that administration of IL-12 leads to accelerated diabetes in the NOD mouse 55 . Indeed, widespread polarized Th1 responses are highly diabetogenic; however, the amplification of such responses may be thwarted by the NKT cell counterregulatory functions described here. We believe that the secretion of IL-4 by peripheral NKT cells is less critical for downregulation of T cell–mediated autoimmunity. In fact, the amount of IL-4 released by NKT cells in the periphery is minimal compared with the secretion of IFN-γ. Moreover, we found that the restoration of normal IL-4 secretion by NOD NKT cells before their transfer in young syngeneic recipients did not improve, but rather worsened, their ability to downregulate autoimmunity. There is now extensive evidence that a defect of the NKT cell subset is invariantly associated with T cell–mediated autoimmunity in mice as well as in patients affected by IDDM. We believe that the association between a lack of IFN-γ–secreting phenotype of NKT cells and IDDM in NOD mice suggests a critical homeostatic role for IFN-γ in regulation of T cell immunity. IFN-γ–secreting T cell subsets such as NKT cells that actively participate in innate immune responses against pathogens may also be critical for counterregulation of autoimmunity so that their functional defect leads, by default, to disease. | Study | biomedical | en | 0.999996 |
10510087 | G8 TCR-γ transgenic mice have been characterized previously 23 . C57BL/6 (B6), B6 IL-7Rα −/− , and B6 recombination activating gene (RAG)-1 −/− mice were purchased from The Jackson Laboratory. G8 TCR-γ transgenic mice that had been backcrossed to B6 mice at least three times were crossed to IL-7Rα −/− mice to generate TCR-γ transgenic + or transgenic − IL-7R −/− or IL-7R +/− littermates. Mice were typed by PCR using published primers and by flow cytometric analysis of peripheral blood lymphocytes. Mice were bred and maintained in specific pathogen-free facilities at the University of California, Berkeley. Anti-Vγ2 TCR (UC3-10A6), anti–TCR-δ (GL3), anti–TCR-β (H57), anti-CD5 (53-7.3), anti-NK1.1 (PK136), and anti–heat-stable antigen (anti-HSA; J11d) mAbs were purified and conjugated with FITC or biotin according to standard protocols. Anti-CD8α–Tricolor (53-6.7), anti-CD44–FITC (IM7.8.1), and anti-CD25–PE (PC61.5.3) mAbs were purchased from Caltag; anti-CD4–Red613 was from GIBCO BRL; and streptavidin-PE was from Molecular Probes. Red blood cell–lysed single cell suspensions of thymocytes and splenocytes were stained with the relevant antibodies at saturating concentrations, and 1–2 × 10 5 labeled cells (side and forward gated) were analyzed by four-color cytometry using an EPICS ® XL-MCL flow cytometer (Beckman Coulter). In some cases, splenocytes were passed through nylon wool to enrich for T cells before antibody staining. Cells were sorted using an ELITE ® cell sorter (Beckman Coulter). Flow cytometric profiles were analyzed using the WinMDI program (John Trotter, Salk Institute, San Diego, CA.). Induction of TCR-γ gene expression in mature α/β thymocytes was carried out by culturing sorted mature (HSA lo ) CD4 + or CD8 + α/β (TCR-β hi ) thymocytes along with γ/δ thymocytes in complete medium containing recombinant IL-2 (5 ng/ml; Chiron) with or without IL-7 (2–100 ng/ml recombinant murine IL-7 [Genzyme] or 1% IL-7 supernatant from J558 plasmacytoma transfected with an expression vector containing IL-7 cDNA 24 ) for 5 h to 4 d. Each sorting experiment used three to six B6 mice (4–8-wk-old) and involved depletion of HSA hi cells using a magnetic-activated cell sorter (MACS) before cell sorting. Genomic DNA (gDNA) isolation and Southern blotting were carried out as described 25 . 15 μg of gDNA was digested (except for the sample from RAG-1 −/− mice, which represents 2 μg of gDNA) with EcoRI, electrophoresed on 0.7% agarose gels, and blotted. The blot was hybridized with a Cγ1 probe that cross-hybridizes with Cγ2 and Cγ3 genes 26 . [α- 32 P]rUTP (Amersham Pharmacia Biotech)–labeled riboprobe specific for the TCR-γ transgene transcripts was generated from a linearized pKS Bluescript vector (Stratagene) containing the 273-bp KpnI-BsrI fragment that includes the V-J junction sequence of G8 transgene under the control of T7 promoter. Control riboprobe specific for γ-actin mRNA was generated using SP6 RNA polymerase. RNase protection assay was carried out essentially as described 25 using 5 μg of total RNA purified from sorted thymocyte populations (a pool of 9–15 IL-7Rα −/− mice per transgenic line was used in two independent sorting experiments). RNA was isolated using Ultraspec RNA solution (Biotecx). Each sample contained both probes to eliminate quantitative variations in input RNA. Densitometric analysis was performed using a PhosphorImager (Molecular Dynamics). Reverse transcription (RT) of 1–2 μg of total RNA from sorted (and cultured) thymocytes was performed using oligo-dT primers and avian reverse transcriptase (both from Boehringer Mannheim). Each sorting experiment used pooled thymocytes from four to eight IL-7Rα −/− mice for the analysis of transgene expression and pooled thymocytes from three to six B6 mice for the analysis of endogenous TCR-γ gene expression in α/β cells. cDNA was serially diluted 3- or 4-fold, and PCR was performed for 28 cycles with the addition of 1.0 μCi of [α- 32 P]dCTP (Amersham Pharmacia Biotech) per sample in a total reaction volume of 50 μl. Samples without the RT step were subjected to the same PCR protocol. Starting sample concentration of tubulin PCR reactions was three- or fourfold lower than the corresponding target gene PCRs. Products were resolved by gel electrophoresis on a 5% polyacrylamide gel and visualized by autoradiography. Densitometric analysis was performed using a PhosphorImager (Molecular Dynamics). For nonradioactive PCR, 35–39 cycles of amplification were performed. Products were subjected to gel electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. The following PCR primers were used: 5′ Vγ2, CTGGGAATTCAACCTGGCAGATGA; 3′ Jγ1, GGGAAGCTTACCAGAGGGAATTACTATGAG; 3′ G8 junction, GTGAAAACCTGAG-CTCCCCTCCC; 5′ tubulin, CAGGCTGGTCAATGTGGC-AACCAGATCGGT; 3′ tubulin, GGCGCCCTCTGTGTAG-TGGCCTTTGGCCCA. The TCR-γ gene rearrangement status in IL-7Rα −/− mice has been somewhat controversial. Southern blot analysis of TCR-γ gene rearrangement in thymocytes demonstrated that the rearranged TCR-γ genes are virtually undetectable in IL-7Rα −/− mice and that the pattern is very similar to that observed in RAG-1 −/− mice . This result supports previous Southern analysis 19 but is in contrast to the recent PCR data that indicated a significant level of TCR-γ gene rearrangement in IL-7Rα −/− mice 21 . This deficiency in TCR-γ gene rearrangement can be caused by a specific defect in TCR-γ gene rearrangement/transcription and/or a selective loss of cells that rearrange/transcribe TCR-γ genes. To investigate the role of IL-7R signaling in TCR-γ gene expression, we crossed TCR-γ transgenes onto an IL-7Rα −/− background. The use of a prerearranged transgene allowed us to examine TCR-γ gene expression independent of TCR-γ gene recombination. Two distinct α/β lineage developmental phenotypes of IL-7Rα −/− mice have been documented: in type I IL-7Rα −/− mice the thymus is composed exclusively of immature CD4 − CD8 − (double negative [DN]) thymocytes 5 , whereas in type II mice they exhibit near normal CD4/CD8 thymic composition 14 . The basis for the phenotypic variation is unknown. All IL-7Rα −/− mice lack γ/δ T cells and show a drastically decreased thymic cellularity. In our colony, the majority of the B6 IL-7Rα −/− mice (>80%) exhibit the type II phenotype. Mice harboring a productively rearranged TCR-γ (Vγ2-Jγ1-Cγ1) transgene 27 from the G8 cell line have been described previously 23 . It has been demonstrated that expression of this transgene correlates well with transgene copy number and is not subject to transgene position effects 23 28 . We compared 2 TCR-γ transgenic lines containing the identical transgene, but with different numbers of transgene copies (30 vs. 2 copies). Expression levels were determined in RNA samples from sorted (>99% pure) CD4 − CD8 − TCR-β − thymocytes, with an RNase protection assay using a V-J junction spanning probe specific for the transgene . The results were normalized with reference to an internal γ-actin control. In addition, semiquantitative RT-PCR was performed using transgene-specific primers . The low copy TCR-γ transgene is expressed in the normal thymus at similar levels as the endogenous TCR-γ genes in a γ/δ T cell hybridoma ( 29 ; Kang, J., and D.H. Raulet, unpublished results). Transgene transcript levels were reduced by >26-fold in CD4 − CD8 − TCR-β − thymocytes from the low copy transgenic IL-7Rα −/− mice compared with control transgenic IL-7Rα +/− thymocytes . The dramatic reduction in transgene transcription was confirmed in highly sensitive RT-PCR experiments. In two out of three such experiments, no transgene transcripts were detected in sorted CD4 − CD8 − TCR-β − thymocytes from transgenic IL-7Rα −/− mice, whereas abundant transcripts were detected in transgenic IL-7Rα +/− mice . In one experiment, transcripts were detected in sorted thymocytes from IL-7Rα −/− mice, but the levels were reduced by more than eightfold compared with IL-7Rα +/− mice . This level of transgene transcription was only detectable in one experiment, and only in the PCR assay. The high 30 copy transgene was expressed at much higher levels in transgenic IL-7Rα +/− thymocytes than the low copy transgene, as expected from the greater number of transgene copies . Nevertheless, transgene mRNA levels were substantially reduced in sorted thymocytes from IL-7Rα −/− transgenic mice, by a factor of at least five- to sevenfold . No significant difference was seen between samples from transgenic IL-7R +/+ and IL-7R +/− mice (data not shown). The less dramatic effects of IL-7R deficiency on transgene transcription in the high copy line may be due to cooperative effects of multiple tandem transgene copies, which may partly overcome the requirement for IL-7R–dependent factors. Taken together, the results demonstrate that the transcription of both high and low copy TCR-γ transgenes is highly dependent on IL-7Rα expression. Although optimal TCR-γ gene transcription depended on IL-7R signaling, the high copy TCR-γ transgene supported significant levels of transgene transcription. This persistent but reduced transgene transcription was likely due in part to the higher number of templates for transcription as well as to a partial reduction in IL-7R dependence due to tandem multimerization of the transgene. The significant expression of the transgene in the high copy line allowed us to address whether the absence of γ/δ cells in IL-7Rα −/− mice is due to the absence of TCR-γ chain synthesis. It was shown previously that in otherwise normal mice, TCR-γ transgene expression resulted in a two- to threefold enhancement in the development of γ/δ thymocytes. The resulting γ/δ cell population was >90% Vγ2 + compared with ∼40% Vγ2 + thymocytes in nontransgenic littermates . The increase in γ/δ cell numbers in the transgenic mice was independent of transgene copy number, but was dependent on the production of functional endogenous TCR-δ chains. γ/δ T cells were not detectable in IL-7Rα −/− mice 14 15 . The rearranged TCR-γ transgene, when present at 30 transgene copies, rescued γ/δ T cell (CD4 − , CD8 − , CD5 med , TCR-β − , NK1.1 − , HSA lo to hi ; data not shown) development in the thymus of IL-7Rα −/− mice . The proportion of DN thymocytes that expressed TCR-γ/δ in transgenic IL-7Rα −/− mice (20.6 ± 1.8%) was similar to that of transgenic littermates that were IL-7Rα +/− or IL-7Rα +/+ . Moreover, the proportion of the DN thymocytes that were HSA lo TCR-γ/δ + , a provisional phenotype for mature T cells, did not differ significantly between transgenic IL-7Rα −/− mice and wild-type mice (data not shown). The cells were stained with an anti–TCR-δ mAb, indicating that δ chain expression is independent of IL-7R signaling. Although the percentage of γ/δ thymocytes in transgenic IL-7Rα −/− mice was normal, the absolute number of these cells was reduced ∼14-fold at 4–5 wk of age compared with transgenic IL-7Rα +/− mice. This is to be expected, given the >20-fold reduction in the number of T lineage–committed progenitor cells (CD25 + CD44 + c-kit + CD4 − CD8 − CD3 − ) in these mice . Hence, the absolute number of γ/δ thymocytes generated per progenitor T cell in the transgenic IL-7Rα −/− mice was not substantially different from that in normal mice. Examination of DN developmental intermediates using CD25/CD44 expression revealed no significant alterations in the subset distribution between the transgenic and nontransgenic IL-7Rα −/− littermates, and a distinct CD25 + CD44 − DN pre-T cell subset was not evident even when the transgene was expressed . Peripheral γ/δ T cells were essentially undetectable in nontransgenic IL-7Rα −/− mice ( 14 15 ; Table ). Significant numbers of peripheral γ/δ T cells were detected in transgenic IL-7Rα −/− mice older than 3 wk, although they were reduced in absolute cell number by ∼13-fold compared with transgenic IL-7Rα +/− mice ( Table ), as might be expected considering the reduction in thymic progenitor cells in these mice. These observations suggest that IL-7R signaling is not absolutely required for γ/δ T cell maintenance in the periphery. Collectively, the results indicate that sufficient expression of a rearranged TCR-γ gene alone can lead to the development of γ/δ thymocytes in the absence of IL-7R–mediated signals. Production of endogenous TCR-δ chains was not dependent on IL-7R. However, it cannot be ruled out that a subtle difference in endogenous TCR-δ gene transcription exists in IL-7R −/− mice, although such a difference is not expected to affect functional TCR-δ chain generation. Importantly, the level of TCR-γ/δ receptors on the surface of the transgenic thymocytes was no higher than the receptor levels on γ/δ cells in normal mice , suggesting that γ/δ T cell development in the transgenic IL-7Rα −/− mice is not due to aberrantly enhanced TCR-γ/δ signaling. These results argue against the possibility that IL-7R–mediated signals are essential for the survival and/or maintenance of γ/δ T cells. Instead, the data suggest that an IL-7R-mediated signal(s) is necessary for the synthesis of TCR-γ chains. To determine whether the rescue of γ/δ T cells in IL-7Rα −/− mice is related to transgene copy number, the low 2 copy transgenic IL-7Rα −/− mice were analyzed. Flow cytometric analysis of DN thymocytes confirmed that the low copy transgene expression enhanced γ/δ T cell development in IL-7Rα +/− mice , similar to what was observed with the high copy line . However, unlike the high copy line, provision of the low copy transgene failed to rescue γ/δ cell development in IL-7Rα −/− mice . These data indicate that the impaired transcription of the rearranged transgene in low copy IL-7Rα −/− mice results in levels of TCR-γ chain synthesis that are too low to stimulate γ/δ T cell development. TCR-γ genes are rearranged in the majority of CD4 + and CD8 + α/β lineage cells, but they are normally maintained in a transcriptionally inactive state 26 29 30 . Rearranged Vγ2-Jγ1Cγ1 genes, in particular, are present in most α/β lineage cells but are completely repressed. To determine if the transcription of endogenous rearranged TCR-γ genes can also be influenced by IL-7R–mediated signals, we tested whether IL-7 can activate expression of endogenous TCR-γ genes in α/β lineage cells from normal mice. In contrast to immature DP thymocytes, the majority of CD4 + SP mature thymocytes from B6 mice express the IL-7Rα chain ( 31 ; and data not shown). A significant fraction of CD8 + SP mature thymocytes also express the IL-7Rα chain, but at a reduced level per cell compared with CD4 + thymocytes ( 31 ; and data not shown). Sorted CD4 + or CD8 + SP HSA lo TCR-α/β + as well as CD4 − CD8 − TCR-γ/δ + thymocytes (all >97% pure) from B6 mice were cultured in IL-2 for 2 d in the presence or absence of IL-7, and the level of TCR-γ gene transcription was measured by semiquantitative RT-PCR. The proportion of cells surviving after culture for all different conditions was similar in five independent experiments (data not shown). Fig. 5 A shows results from one representative RT-PCR experiment of four assays with identical results. Consistent with previous results, α/β T cells cultured without IL-7 failed to express the Vγ2-Jγ1 gene, despite the fact that a large fraction of α/β T cells harbor Vγ2-Jγ1 rearrangements 26 . Remarkably, however, IL-7 induced the expression of the endogenous Vγ2-Jγ1 TCR-γ gene in mature CD4 + SP thymocytes, to a level that was 1/3 to 1/2 the level seen in γ/δ thymocytes cultured with or without IL-7 . The induction of transcription occurred rapidly after only 5 h in culture, and persisted until the end of a 4-d culture . Treatment with IL-7 did not result in a significant induction of rag1/2 gene expression in these cultures (data not shown). These results demonstrate that IL-7R signals can induce transcription of the endogenous rearranged TCR-γ gene in CD4 + α/β lineage cells in vitro, and suggest an active role of the downstream mediators of IL-7R signaling in promoting transcriptional activities at the TCR-γ locus. In contrast to its effects on CD4 + SP thymocytes, IL-7 failed to induce Vγ2-Jγ1 TCR-γ gene transcription in mature CD8 SP thymocytes , perhaps due in part to the lower levels of IL-7R expression by these cells. The reason for the lack of transcription of rearranged endogenous TCR-γ gene in CD4 + SP thymocytes in vivo is unclear, since IL-7 is produced by some thymic stromal cells. It appears that CD4 + SP thymocytes do not receive sufficient IL-7R signaling in situ, possibly as a result of limiting local IL-7 concentration or antagonistic effects of other thymic inductive signals. At least two aspects of T cell precursor differentiation are regulated by IL-7R–mediated signals. IL-7R signals ensure survival by preventing programmed cell death of developing T cells 16 17 , and they directly and specifically regulate the production of TCR-γ chains. In IL-7Rα −/− mice, greatly reduced numbers of α/β thymocytes develop, but γ/δ thymocytes are completely absent. Based on the dramatically decreased levels of TCR-γ, but not -δ, -β, and -α, gene rearrangement observed in thymocytes of IL-7Rα −/− mice, it has been suggested that the IL-7R signals are necessary for activating the TCR-γ gene locus 19 . We show here that transcripts emanating from a prerearranged TCR-γ transgene are markedly reduced in IL-7Rα −/− mice, indicating that IL-7Rα signaling directly affects TCR-γ gene expression, independent of rearrangement. This conclusion is strikingly supported by the studies showing that IL-7 induces transcription of the endogenous rearranged Vγ2-Jγ1Cγ1 gene in isolated mature CD4 + SP thymocytes from normal mice. The downstream mediators of IL-7R have been previously shown to activate germline, sterile TCR-γ transcription in nonlymphocytes 32 , consistent with the proposed role of IL-7R in regulating TCR-γ gene transcription. Equally important, the present data indicate that the poor expression of TCR-γ genes in IL-7Rα −/− mice is mainly responsible for the selective deficit of γ/δ cells in these mice, since γ/δ cell development could be restored with a high copy rearranged TCR-γ transgene. The number of γ/δ T cells generated per pro-T cell was similar in the wild-type and high copy transgenic IL-7Rα −/− mice. Hence, it appears that the γ/δ lineage precursor cell generation, γ/δ lineage development, and maintenance are not strictly dependent on IL-7R signals as long as functional TCR-γ chains are present. Since no TCR-δ transgene was provided, these data also suggest that IL-7Rα signaling is not crucial for TCR-δ gene rearrangement and TCR-δ chain production, consistent with previous reports 19 20 . The residual level of transgene transcription in the high copy transgenic IL-7Rα −/− line was probably due to two factors. First, the high gene dosage provided a greater number of templates for γ gene transcription. Second, adjusting for gene dosage, it appeared that IL-7Rα deficiency had somewhat less effect on transcript levels in the high copy line compared with the low copy line, possibly reflecting a dysregulation that can occur in multiple tandemly integrated transgenes 33 . Nevertheless, transgene transcription was substantially reduced in thymocytes of these mice compared with IL-7Rα + mice. In the IL-7Rα −/− low copy γ transgenic mice, no restoration of γ/δ cell development was observed. Previous studies showed that the level of transgene-directed γ mRNA in IL-7Rα + thymocytes harboring the low copy γ transgene is comparable to the level of TCR-γ transcripts in a normal cloned γ/δ T cell line 29 . This level of expression is sufficient to increase the number of γ/δ cells in the thymus of IL-7R + mice to the same extent as that seen in the high copy IL-7R + transgenic mice 23 . The IL-7R mutation is not permissive for this normal level of expression, and the sharp reduction in expression is sufficient to completely prevent γ/δ cell development. This finding suggests that the deficiency in TCR-γ transcription in IL-7Rα −/− mice could account for the lack of γ/δ cells in these mice even if the locus underwent rearrangement normally. However, it is likely that TCR-γ gene rearrangement is also affected in IL-7Rα −/− mice. Although it might be proposed that the low levels of TCR-γ gene rearrangements observed in IL-7Rα −/− thymi are due to the rapid turnover of cells that have rearranged TCR-γ genes but do not survive as a result of the lack of IL-7R signaling, this is unlikely to be the complete explanation. Previous studies have shown that the majority of TCR-γ gene rearrangements in the normal thymus are nonproductive ones in α/β lineage CD4 + CD8 + DP thymocytes 34 . The IL-7Rα −/− mice we examined contained appreciable percentages of DP and SP αβ thymocytes, yet TCR-γ gene rearrangement was nevertheless sharply reduced. In IL-7Rα −/− mice, TCR-γ gene rearrangements are rare even in α/β lineage cells, which do not depend on TCR-γ/δ expression for survival. This consideration suggests that T cells of IL-7Rα −/− mice exhibit a specific deficiency in TCR-γ gene rearrangement. Many previous studies have correlated transcription of B and T cell receptor genes with rearrangement 35 . For example, enhancer and promoter elements that support transcription have been shown to play critical roles in rearrangement of the genes as well. Hence, the defective γ gene transcription in IL-7Rα −/− mice probably also results in reduced levels of γ gene rearrangement. Two previous reports have provided indirect evidence for a possible role of IL-7R signals in controlling rearranged TCR-γ gene transcription. First, during fetal ontogeny in γc −/− mice, rearrangement of some TCR-γ genes was evident, but fetal γ/δ thymocytes still did not develop 20 . It was not established that this absence of γ/δ cells was due to a defect in TCR-γ gene transcription. Second, in one study of fetal and adult IL-7Rα −/− mice, it appeared that TCR-γ gene rearrangements were not completely absent as assayed by PCR, but the corresponding transcripts were undetectable 21 . In our hands, IL-7Rα deficiency reduces the level of thymic γ gene rearrangements by at least 40-fold, as detected by genomic Southern blotting . In the aforementioned study, it was not clear whether the level of gene rearrangement detected was sufficient to result in a measurable quantity of mRNA. Furthermore, it was not established that the rearrangements that were detected were present in cells that are normally capable of expressing TCR-γ genes. It is known that γ gene expression is extinguished in certain thymic lineages, such as in CD4 + CD8 + DP cells 29 30 . By using a prerearranged transgene present in all thymocyte lineages, we were able to demonstrate directly that TCR-γ gene expression is impaired in the absence of IL-7Rα signaling. One other aspect of the data is notable. At 2–3 wk of age, the total thymocyte number in the high copy TCR-γ transgenic IL-7Rα −/− mice was only 36% of the number observed in nontransgenic IL-7Rα −/− counterparts ( Table ). Thus, the TCR-γ transgene expression depressed the number of thymocytes in IL-7Rα −/− mice. One possible explanation for this observation is that TCR-γ transgene expression in young mice enhances the rate of generation of thymocytes expressing TCR-γ/δ, resulting in reduced numbers of α/β lineage cells. Since thymic proliferative expansion occurs mainly in α/β but not γ/δ lineage cells, the reduction in α/β lineage cells would lead to a significant reduction in thymic cellularity. A similar effect of the TCR-γ transgene on thymic cellularity has been described previously in mice that generate α/β lineage cells suboptimally 23 . Our results with the G8 (Vγ2) TCR-γ transgenic IL-7Rα −/− mice differ somewhat from a study that employed a different TCR-γ transgene (T3.13.3, Vγ1.1) that was crossed into γc −/− mice 20 . The latter mice contained only 10% the number of splenic γ/δ T cells that we observed, although the difference in thymic γ/δ cell number was limited to two- to threefold. A plausible explanation for this difference in peripheral γ/δ T cell numbers is that γc-dependent cytokines other than IL-7 may be involved in the maintenance of mature γ/δ T cells. How does the IL-7R regulate TCR-γ gene expression? The TCR Cγ1 locus contains a downstream enhancer element, 3′E Cγ1 36 37 . We have recently identified another regulatory element, called 5′HsA, that cooperates with the 3′E Cγ1 to stimulate optimal TCR-γ gene rearrangement and transcription 28 . 5′HsA is a locus control region (LCR)-like element that does not exhibit enhancer activity as measured with transient transfection assays, but is active when integrated in the genome. It appears to play a role in rendering the TCR-γ locus accessible for gene rearrangement and expression in vivo. 3′E Cγ1 contains a consensus binding site for signal transducer and activator of transcription 5 (STAT5 38 ), a transcription factor that is activated by JAK1 and JAK3, which are in turn activated by the IL-7R. 5′HsA contains binding sites for other STATs. Reportedly, thymocytes of IL-7Rα −/− mice are devoid of nuclear STAT5 21 . Further studies will be necessary to assess the role, if any, that STATs play in TCR-γ gene transcription. | Study | biomedical | en | 0.999995 |
10510088 | Female B10.A mice (The Jackson Laboratory) were used at 8–10 wk of age. Invariant chain–deficient B10.BR mice were provided by Dr. D. Mathis (IGBMC, Strasbourg, France) and used at 6–12 wk of age. AND and AD10 transgenic mice containing pigeon cytochrome c (PCC)–specific TCRs were provided by Dr. S. Hedrick (University of California, San Diego, CA), as was the cytochrome c–specific T cell clone, AD10. The B cell lymphoma CH27, provided by Dr. G. Houghton (University of North Carolina, Chapel Hill, NC), was used as APC in some experiments. For IL-2 determinations, the IL-2–dependent T cell line CTLL-2 was used. BW5147α − β − cell line was used as fusion partner in the production of T cell hybridomas. pMCC (ANERADLIAYLKQATK) and the heteroclitic analogue (pMCC-A; AAAAAAAIAYAKQATK) were synthesized on a Rainin Symphony synthesizer (Peptide Technologies) as described previously 27 . Peptides were purified by reverse-phase HPLC to >95% purity. Identity of the peptides was substantiated by mass spectrometry. Tolerance induction and immunization were performed as described previously 26 . In brief, to induce tolerance, B10.A mice were injected intraperitoneally with 300 μg pMCC in IFA (Pierce Chemical Co.). 10 d later, tolerized mice were immunized with 20 μg pMCC or the heteroclitic antigen analogue, pMCC-A, in CFA (Difco) subcutaneously in the base of the tail. As controls, normal mice were immunized with 20 μg pMCC or pMCC-A in CFA. 10 d after immunization, draining lymph nodes were removed and single cell suspensions were made and used for proliferation and cytokine assays or to establish short-term T cell lines and hybridomas. Cells from draining lymph nodes (paraaortic and inguinal) were plated at 2.5 × 10 5 cells/well along with 2.5 × 10 5 irradiated (3,000 rad) syngeneic splenocytes and stimulated with the pMCC or pMCC-A peptide as indicated. After 72 h, the cultures were pulsed for an additional 18 h with 1 μCi of [ 3 H]thymidine and analyzed by beta-plate scintigraphy. In experiments to analyze relative immunogenicity of MCC analogues, proliferation assays were performed using the AD10 clone, as described previously 27 . For cytokine analysis, supernatants from the cultures that had been established to measure the proliferative response were removed after 24 h for IL-2 measurement, after 48 h for IL-4 and IL-10, or after 72 h for IFN-γ. IL-2 was measured by bioassay using the CTLL-2 cell line. IL-4, IL-10, and IFN-γ were assayed by ELISA according to the instructions provided by the manufacturer of the reagents (PharMingen). The sensitivities of the ELISA assays were as follows: IFN-γ, 100 pg/ml; IL-4, 50 pg/ml; and IL-10, 50 pg/ml. Peptides were analyzed for their ability to bind to purified I-E k molecules as described previously 28 . The binding capacity is reported as the concentration of peptide required to obtain 50% inhibition (IC 50 ) of binding of the radiolabeled ligand. CD4 + T cells were purified from the lymph node and spleen of pMCC-specific TCR transgenic mice (AND) by collecting nonadherent cells from a nylon wool column, followed by complement treatment of the cells that had previously been incubated with antibodies to CD8 (3.155), heat-stable antigen (JIID), class II MHC (M5/114 and CA-4.A12), macrophages (M1/70), and dendritic cells (33D1), and subsequently cross-linked with mouse anti–rat kappa antibody (MAR18.5). The percentage of CD4 + cells that were Vα11 + Vβ3 + T cells was determined by flow cytometry. This information was then used to calculate the total number of cells that had to be injected to achieve 2.5 × 10 6 Vα11 + Vβ3 + T cells/injection. Mice were injected intravenously with this number of cells in a vol of 0.4 ml of HBSS (GIBCO BRL) 21 . Recipient mice were unirradiated, invariant chain–deficient B10.Br mice. 3 d after cell transfer, the animals were injected intravenously with 300 μg of pMCC in a vol of 250 μl to induce tolerance. 10 d later, tolerized and nontolerized mice were immunized with 20 μg of pMCC or pMCC-A in CFA subcutaneously. After an additional 10 d, draining lymph nodes were removed and single cell suspensions were used for in vitro stimulation and cytokine analysis. Draining lymph node cells from immunized or tolerance-broken mice were restimulated in vitro with 0.5 μM pMCC or pMCC-A for 2 d and expanded with IL-2 (20 U/ml) for an additional 2 d. The lymphoblasts thus generated were fused with the BW5147α − β − cell line at ∼1:3 ratio, and hybridomas were selected in HAT medium and distributed in 96-well plates at ∼10 4 cells/well 29 . Wells were screened for reactivity to pMCC or pMCC-A by measuring IL-2 production. The positive wells were cloned at a concentration of 0.3 cells/well, and clones that produced IL-2 in response to antigen were expanded for further analysis. Cells were incubated on ice with anti-CD3–FITC, anti-Vα11–FITC, anti-Vβ3–PE (PharMingen), or anti-CD4–PE (Becton Dickinson) for 1 h and washed several times. Live cells were gated by forward and side scatter and analyzed by flow cytometry on a FACScan™ equipped with CELLQuest™ software (Becton Dickinson). Total RNA was isolated from T cell hybridomas using an RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Oligo dT–primed cDNA was synthesized using a cDNA Cycle kit (Invitrogen Corp.) following the manufacturer's protocol and then amplified by PCR for sequence analysis. The predicted size fragments from PCR amplification were further purified with a QIAquick Gel Extraction kit (QIAGEN) and sequenced using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems) on an Applied Biosystems 310 DNA Sequencer. The PCR and sequencing primers used were as follows: for α chain PCR: Cα, AAG TCG GTG AAC AGG CAG AG; for Vα11, TCA GGA ACA AAG GAG AAT GG; for α chain sequencing: Cα, AAC TGG TAC ACA GCA GGT TC; for β chain PCR primers: Cβ, AGC ACA CGA GGG TAG CCT T 30 ; for β-chain sequencing: GGA GTC ACA TTT CTC AGA TCC A. A set of Vβ primers was used for PCR as reported previously 31 . In a previous study to determine whether cross-reactive antigen analogues could break tolerance induced to the immunodominant T cell epitope from MCC, pMCC, it was found that certain heteroclitic antigens (as defined by their heightened antigenicity for a representative T cell clone) were capable of breaking tolerance 26 . Specifically, substitutions at the minor MHC contact residue L98 were effective in this regard. In this study, we have used a pMCC analogue with an L98A substitution that also had residues 88–94, which are outside the MHC peptide binding cleft, substituted with alanines. This poly-A MCC analogue (pMCC-A) had a similar binding capacity to I-E k as the native peptide, but was able to stimulate MCC-specific clones ∼10-fold more efficiently than the native pMCC peptide ( Table ). The ability of this analogue to break tolerance to pMCC is shown in Fig. 1 . Tolerized animals had a markedly reduced capacity to proliferate in response to stimulation with pMCC compared with the response of immunized controls and did not synthesize detectable amounts of IFN-γ or other cytokines such as IL-2, IL-4, or IL-10 (data not shown). In contrast, mice that were tolerized to pMCC and then immunized with the heteroclitic analogue, pMCC-A, mounted vigorous proliferative and cytokine responses, albeit requiring 10–100-fold more antigen than the normal immunized controls to achieve comparable responses. Because of reports that claim administration of antigen intraperitoneally in IFA leads to sequestration of the immune response in the spleen rather than tolerance induction, studies were carried out to evaluate this possibility. Cytokine analysis of spleen and draining lymph nodes after pMCC administration intraperitoneally in IFA followed by pMCC immunization subcutaneously in CFA indicated that tolerance was evident in both lymph node and spleen, with undetectable quantities of IFN-γ and IL-4 found in culture supernatants of both organs. Enzyme-linked immunospot (ELISPOT) analysis also indicated tolerance induction, although less dramatic than the supernatant analysis. IFN-γ–producing cells predominated in normal immune lymph node and spleen (600 and 500 cells/10 6 ), respectively, with a very small IL-4–producing population (<20 cells/10 6 ). In pMCC-tolerized and immunized animals, there was a 60 and 75% reduction in IFN-γ–producing cells in both spleen and lymph node, with no change or a slight increase in IL-4–producing cells (24 cells/10 6 in spleen). Thus, with respect to proliferation and the major cytokine, IFN-γ, tolerance was clearly established by the protocol used, although the small number of IL-4–producing cells generated was apparently not affected by the tolerance-inducing protocol. The results shown in Fig. 1 and those reported previously 26 suggest that the T cells involved in the breaking of tolerance have a lower avidity for antigen compared with normal immune T cells, as evidenced by the shift in the antigen dose–response profile. To study in greater detail the functional activity of the T cells involved in the breaking of tolerance to pMCC, and in order to analyze the structure of their TCRs, a series of T cell hybridomas was made from animals whose tolerance to pMCC was broken by immunization with pMCC-A. As controls, hybridomas were also derived from normal animals immunized with either pMCC or pMCC-A. The response of representative hybridomas derived from normal or tolerance-broken animals to stimulation with pMCC is shown in Fig. 2 . The two hybridomas derived from pMCC or pMCC-A normal immune animals behaved similarly to stimulation with pMCC. In contrast, and similar to the data obtained with the bulk T cell response, the hybridoma derived from the tolerance-broken group required ∼10-fold more pMCC to give an IL-2 response comparable to that of the hybridomas derived from the normal immunized animals. The decreased antigen responsiveness of the hybridomas derived from tolerance-broken animals was not due to a decrease in surface expression of either the TCR or the CD4 coreceptor . Fig. 3 summarizes the antigen response data from all hybridomas that reacted to both pMCC and pMCC-A, as a plot of the quantity of pMCC versus pMCC-A required to generate an IL-2 response in each hybridoma analyzed. Several conclusions can be derived from the data shown in Fig. 3 . (a) All but one of the hybridomas derived from the pMCC-immunized group responded to lower concentrations of pMCC-A than pMCC, as evidenced by a location on the graph below the diagonal. This indicates that the heteroclitic nature of pMCC-A, originally described with a single T cell clone, is generalizable to the majority of the pMCC-specific T cell repertoire. (b) The hybridomas in the pMCC tolerance-broken group, in general, required higher concentrations of pMCC to stimulate an IL-2 response than the hybridomas from either the pMCC or pMCC-A normal immune groups. Thus, the hybridomas from the tolerance-broken group required an average of 0.88 μM of pMCC to stimulate an IL-2 response, whereas the pMCC immune group required 0.01 μM, and the pMCC-A immune group required 0.07 μM pMCC to stimulate a response. (c) In contrast to the greater antigen requirement of the tolerance-broken group for pMCC, the response to pMCC-A was similar for all three groups. In addition to the cross-reactive hybridomas shown in Fig. 3 , a small number of hybridomas from pMCC or pMCC-A immune animals were found to be non–cross-reactive (1 of 10 in the pMCC immune group failed to respond to pMCC-A, and 3 of 14 of the pMCC-A group failed to respond to pMCC; data not shown). The antigen dose–response data for pMCC indicated that the hybridomas from the tolerance-broken group had a lower avidity for pMCC than hybridomas from normal immune animals. To determine if this functional difference between T cells derived from normal and tolerance-broken animals was correlated with structural features of the TCR, we undertook an analysis of their Vα and Vβ chains. Since previous studies of the Vα and Vβ utilization of MCC-specific T cells indicated that the response to this antigen was dominated by T cells that express Vα11 and Vβ3 32 33 34 35 36 37 38 , initially flow cytometric analysis of the T cell hybridomas for expression of Vα11 and Vβ3 was performed. Almost one half (5 of 12) of the T cell clones derived from pMCC-immunized animals were Vα11 + /Vβ3 + , with the rest being either Vα11 + /Vβ3 − (4 of 12) or negative for both Vα11 and Vβ3 (3 of 12). The repertoire of clones derived from pMCC-A immune animals differed somewhat from this distribution, in that only about one fourth of the clones (3 of 13) possessed the canonical Vα11 + /Vβ3 + phenotype, and about one half (7 of 13) were Vα11 + /Vβ3 − . The data obtained with the T cell clones derived from the tolerance-broken group were strikingly different, in that all eight clones analyzed were Vα11 + /Vβ3 − . These data suggest that the typical Vα11 + /Vβ3 + MCC-reactive clones were irreversibly inactivated by the tolerizing dose of pMCC and did not participate in the termination of tolerance induced by pMCC-A. Rather, a subset of the Vα11 + /Vβ3 − clones responsive to pMCC-A was involved in the breaking of tolerance. The difference in the repertoire elicited by pMCC and pMCC-A immunization with respect to the relative numbers of Vβ3 + /Vα11 + expressing T cells was substantiated by the analysis of short-term T cell lines . Whereas the T cell line generated from pMCC-immunized animals contained 33% Vα11 + /Vβ3 + cells and 25% Vα11 + /Vβ3 − cells, the pMCC-A immune line contained less than half the percentage of Vα11 + /Vβ3 + cells (14%) and more than twice the percentage of Vα11 + /Vβ3 − cells (59%). These data are consistent with the data obtained with the T cell hybridomas derived from pMCC-A–immunized animals that also demonstrated a skewing toward a Vα11 + /Vβ3 − phenotype. A second independently derived set of T cell lines gave similar results. To further analyze the TCR repertoire involved in breaking tolerance and to compare it with the repertoire normally involved in the response to MCC, cDNA sequence analysis of the junctional regions encompassing the CDR3 of the α and β chains was performed, together with the identification of the Vβ families other than Vβ3 that were used . Previous sequence analyses of the Vα11 genes associated with a cytochrome c–specific response indicated that the CDR3 regions were usually 8 residues long, with a conserved glutamic acid (E) at position 93 and S, A, or G at position 95 34 35 36 37 38 . Of the 8 Vα11 CDR3 regions analyzed from T cell hybridomas derived from pMCC-immunized animals, all had E at position 93, 5 had S at position 95, and 6 were 8 residues in length . Thus, this set of pMCC-specific clones was similar in the structure of their Vα CDR3 regions to previously studied TCRs. The Vα11 CDR3 region from the tolerance-broken group was, in general, very similar to that of the pMCC immune group, but with a slightly greater deviation from the canonical structure . Thus, all 8 clones had E at position 93, but only 4 had G or A at position 95, and only 4 were 8 residues in length. Of particular interest was the finding of a 4-asparagine (N) repeat from positions 95–98 that was present in 4 of the 8 sequences analyzed. This feature was observed in only one of the sequences from the pMCC immune group. The Vα11 CDR3 region from the pMCC-A immune group had characteristics intermediate between the pMCC immune group and the tolerance-broken group : all 10 sequences contained E at position 93, 6 of 10 contained G, A, or S at 95, 6 of 10 had CDR3 regions 8 residues in length, and 3 sequences contained the asparagine repeat from residues 95–98. With respect to the Vβ gene analysis, as described above, the most striking difference between the pMCC immune group and the tolerance-broken group was the complete absence of Vβ3 in the latter group of hybridomas. 4 of 8 Vα11 + TCR genes sequenced in the pMCC immune group were Vβ3, with the other 4 β chains being derived from Vβ8, 14, 15, and 16 gene families . In contrast, the tolerance-broken group expressed chains from only 2 gene families: Vβ16 (3 of 8) and Vβ8 (5 of 8). The motif and length of the Vβ CDR3 that has been previously described to be associated with anti-cytochrome c activity is a length of 9 residues, with N at position 100 and a G or A at position 102 34 35 36 37 38 . In the pMCC immune group, 6 of 8 β chains analyzed had CDR3 regions 9 residues in length, 6 had N at position 100, and 7 had G or A at 102. In striking contrast to this, none of the 8 Vβ CDR3 regions analyzed from the tolerance-broken group had a length of 9 residues, and none had N at position 100, although 4 of 8 had a G or A at 102. Again, the pMCC-A immune group had characteristics intermediate between those of the pMCC immune and tolerance-broken groups. 3 of the 10 β chains analyzed were Vβ3, with the remainder being either Vβ8 2 or Vβ16 5 . 4 of the 10 CDR3 regions were 9 residues long, 2 had N at position 100, and 6 contained G or A at 102. These data suggest the following conclusions: (a) MCC recognition, even the low-avidity response associated with the tolerance-broken group, is very strongly associated with the presence of an α chain from the Vα11 family, with a CDR3 region that has an E at position 93, a residue previously implicated as being involved in the interaction with one of the immunodominant TCR contact residues in pMCC, K99 39 ; (b) although the expression of Vβ3 is not necessary for MCC reactivity, it appears to be associated with the higher avidity interactions of the pMCC-immunized group; and (c) the lower avidity TCRs found in the tolerance-broken group expressed Vβ8 or Vβ16 and did not contain the canonical N at position 100. Vβ16 expression appeared to be particularly associated with decreased avidity for MCC. Two of the three clones from the pMCC-A immune group that failed to cross-react at all with pMCC were Vβ16, as was the clone with the least cross-reactivity with pMCC. Also, in the tolerance-broken group, the two lowest avidity clones for pMCC (requiring >1 μM MCC for stimulation) expressed Vβ16. The data shown in Fig. 3 and Fig. 5 were obtained with hybridomas derived, in the case of the tolerance-broken group, from a pool of lymphocytes from two animals, and in the case of the normal immune group, from two separate fusions of cells from individual mice. A second fusion of cells from another two tolerance-broken animals was consistent with the data shown in Fig. 3 and Fig. 5 , in that they had lower avidity for pMCC than normal immune animals, and none of them expressed Vβ3 (data not shown). To evaluate the possibility that breaking tolerance by pMCC-A might involve reversal of anergy, an adoptive transfer system was established in which the effect of pMCC-A on the tolerance induced in a single clone of cells could be evaluated 21 . For this purpose, T cells from cytochrome c–specific TCR transgenic mice (AND) were used. The AND TCR is Vβ3 + /Vα11 + and recognizes pMCC-A as a heteroclitic antigen. As recipient mice, H-2K + invariant chain–deficient animals were used. These animals had previously been shown to lack a T cell repertoire capable of responding to pMCC 40 41 . Thus, if tolerance could be broken in adoptively transferred invariant chain–deficient animals, the T cells involved would have to have been derived from the adoptively transferred TCR transgenic cells. In preliminary experiments, the failure of invariant chain–deficient mice to respond to either pMCC or pMCC-A was confirmed (data not shown). These mice were injected with 2.5 × 10 6 CD4 + Vβ3 + /Vα11 + T cells from AND transgenic mice. 3 d after transfer, one group of mice was tolerized by the intravenous injection of 300 μg of pMCC. 10 d after the induction of tolerance, these animals and groups of nontolerized, adoptively transferred animals were immunized with pMCC or pMCC-A and their immune responses were evaluated by analyzing the in vitro response to pMCC of regional lymph nodes harvested 10 d after immunization. Adoptively transferred and tolerized mice contained 1.61 ± 0.4% Vβ3 + /Vα11 + T cells, compared with 0.72 ± 0.23% Vβ3 + /Vα11 + cells in mice that did not receive an adoptive transfer. This difference was significant ( P < 0.01), and therefore it could be concluded that after adoptive transfer and tolerance induction, there was a residual transgenic T cell population derived from the adoptively transferred transgenic T cells (1.61 – 0.72 = 0.89%) that could be studied for reversal of anergy. The immune response of T cells derived from adoptively transferred, tolerized, and subsequently immunized animals is shown in Fig. 6 . The data, expressed as the amount of IFN-γ per Vβ3 + /Vα11 + cell put into culture, indicated: (a) tolerance could be established in this adoptive transfer system, since animals that received an intravenous injection of pMCC before immunization with pMCC in CFA were severely impaired in their capacity to produce IFN-γ compared with the nontolerized controls (0.15 vs. 0.95 pg/cell); and (b) immunization with the heteroclitic analogue pMCC-A failed to break this tolerant state. If the model suggested by our data is correct, namely that breaking tolerance involves the activation of T cells with low affinity for the tolerogen but high affinity for the heteroclitic antigen, then it must be further postulated that these T cells, once stimulated by the heteroclitic antigen, have a lowered threshold for stimulation and become responsive to stimulation by the lower affinity tolerogen. To evaluate this postulate, we studied the response of naive and recently primed TCR transgenic T cells to stimulation with either the cognate antigen used to prime the T cells or a nonantigenic TCR antagonist analogue of the antigen with presumably lower affinity for the TCR. T cells from the cytochrome c–specific TCR transgenic line AD10 were used for this purpose, since this TCR had been extensively analyzed previously for reactivity to a large panel of pMCC analogue peptides. Naive T cells or T cells that had been stimulated with pMCC 8–12 d previously were stimulated with varying doses of pMCC or the TCR antagonist peptide T102G, which has a glycine (G) substituted for one of the major TCR contact residues, T102. The data shown in Fig. 7 demonstrate that the T102G analogue, which was nonantigenic for naive T cells, was an agonist when assayed on recently primed T cells, albeit requiring a high concentration (1–100 μg/ml) to achieve this effect. This study was undertaken to evaluate two potential mechanisms by which antigen analogues might operate in the termination of T cell tolerance to an immunodominant epitope: stimulation of low-affinity nontolerized clones and reversal of anergy. Three sets of data presented in this study favor the conclusion that the first mechanism is operative. First, T cell hybridomas derived from tolerance-broken animals required, on average, 88-fold higher concentration of antigen than hybridomas derived from normal immune animals in order to be stimulated to produce IL-2. This difference in antigen dose requirement is most likely due to differences in affinity of the TCRs from these two groups of hybridomas, but other possibilities need to be considered, such as differences in the levels of expression of molecules that contribute to the stimulation of the T cells; these include the TCR itself, the CD4 coreceptor, and adhesion molecules such as LFA-1. Two lines of evidence suggest that differential expression of these molecules does not contribute to the antigen dose requirements observed. First, no correlation between the levels of expression of TCR and CD4 and the antigen dose requirement for stimulation was observed . Second, when I-E k –transfected fibroblasts that lacked intercellular adhesion molecule (ICAM), B-7, or other known ligands for T cell adhesion/costimulator receptors were used as APC, the large difference in antigen dose requirements for the tolerance-broken and normal immune animals was still observed (576 vs. 22 nM). The second set of data that supports the hypothesis of stimulation of low-affinity nontolerized clones as a mechanism for breaking tolerance is the finding of a different repertoire of TCRs on T cells from tolerance-broken animals and normal immune animals. Although there was no absolutely unique feature to the TCRs from the tolerance-broken group of hybridomas, there was pronounced skewing of the TCRs compared with those found in the normal immune group. Vβ3, the major Vβ family found in the normal immune group 33 34 35 36 37 38 , was not represented at all in the tolerance-broken group. Instead, Vβ8 and Vβ16 were expressed in the tolerance-broken group—Vβ families that were only occasionally expressed in the MCC-specific T cells from normal immune animals. Similarly, the canonical Vβ CDR3 motif and length associated with the response to pMCC were not observed in any of the Vβ CDR3 regions from the tolerance-broken group of hybridomas. If reversal of anergy were the mechanism for breaking tolerance, there is no apparent reason for it to be restricted to a minor subset of clones with the exclusion of the major Vβ3 + /Vα11 + subset. On the other hand, a distinctly different repertoire of TCRs with different Vβ/Vα composition would be consistent with a mechanism of breaking tolerance that involved the stimulation of low-affinity clones that were not capable of being stimulated after MCC immunization nor were tolerizable when MCC was administered in a tolerogenic form. The third observation in support of the concept that tolerance was broken by low-affinity nontolerized clones was our failure to terminate the tolerant state of adoptively transferred and tolerized transgenic T cells. When transgenic T cells bearing a typical Vβ3 + /Vα11 + TCR specific for MCC were transferred and subsequently tolerized to MCC, the same heteroclitic peptide that was capable of breaking tolerance in conventional animals failed to break tolerance in these animals with a clonal population of tolerized cells. Taken together, these three sets of data strongly support the hypothesis that breaking tolerance involves the stimulation of nontolerized, low-affinity clones by the MCC heteroclitic analogue. For reversal of anergy to be operative, it would be necessary to postulate that certain (Vβ3 + /Vα11 + ) cells are selectively incapable of undergoing reversal of anergy, whereas others (Vβ8 + /Vα11 + and Vβ16 + /Vα11 + ) are able to undergo anergy reversal. Although this is theoretically possible, we consider it unlikely. Our data are consistent with previous reports that found that tolerance to certain self-epitopes was incomplete and that immunization with the cognate antigen could generate an immune response 42 43 44 . However, the responding T cells that were elicited were of relatively low affinity. For instance, immunization of beef insulin (BI) transgenic mice with BI generated a BI-specific T cell response that required higher concentrations of antigen to be elicited than the response elicited in nontransgenic mice 42 . Similarly, low-avidity CD8 T cell responses were obtained after immunization with a p53 self-epitope when p53 + mice were used. In contrast, p53-deficient mice generated a higher avidity response 43 . These reports suggest that when self-tolerance is incomplete, active immunization with the unaltered self-protein or an epitope derived from it can elicit a response of low-avidity T cells that had escaped tolerance. Our data extend these observations to situations in which tolerance to the epitope in question is complete and cannot be reversed by immunization with the native epitope but can be overcome by immunization with a heteroclitic analogue. The hypothesis that termination of tolerance to pMCC involves the stimulation of low-affinity clones that are neither tolerized nor stimulated by pMCC but can be stimulated by the heteroclitic analogue, pMCC-A, requires the further postulate that after stimulation by the heteroclitic antigen, the affinity threshold for stimulation is lowered such that restimulation with the tolerogen results in a recall response. To test this postulate, we studied the response of naive and previously primed MCC-specific transgenic T cells to a ligand of presumably lower affinity, a TCR antagonist peptide. It was found that, whereas naive and primed T cells responded with similar vigor to the wild-type agonist peptide, only primed cells proliferated in response to stimulation with the antagonist peptide. This finding further supports the low-affinity T cell model of breaking tolerance. The mechanism by which priming results in a lowering of the affinity threshold for TCR-mediated stimulation is unknown. One possible factor that could influence the sensitivity of the T cell response is the level of expression of the CD4 coreceptor. Several studies have documented that T cell clones with the same TCR have heightened responses to suboptimal concentrations of agonist peptide when the T cell is CD4 + compared with CD4 − variants or when CD4 + cells are pretreated with anti-CD4 antibodies 45 46 47 48 . Although we have not been able to document any differences in the level of expression of CD4 on the naive and recently primed T cells that we analyzed (data not shown), it is possible that CD4 function differs in the two cell types; e.g., the activity of the CD4-associated Lck may be greater in primed compared with naive T cells. This and other possible differences between naive and recently primed T cells that may be important in the conversion of TCR antagonists into agonists are being investigated. | Study | biomedical | en | 0.999997 |
10510089 | Specific pathogen-free BALB/c and C.B-17 SCID mice were obtained from Simonsen Laboratories or Harlan Olac and maintained in the Animal Care Facility of the DNAX Research Institute or in the Biomedical Services Unit (BMSU) at the John Radcliffe Hospital. 129/SvEv WT, 129/SvEv recombination-activating gene (RAG)-2–deficient (RAG-2 −/− ) and 129/SvEv IL-10–deficient (IL-10 −/− ) mice were bred in isolators and maintained in microisolator cages with filtered air. Mice were used at 8–12 wk of age. The following mAbs were used for cell purification: 2-43, anti–mouse-CD8 (TIB210; American Type Culture Collection); M1/70, anti–mouse Mac-1 (TIB128; American Type Culture Collection); RA3-6B2, anti–mouse B220 12 ; FITC-conjugated anti–mouse CD45RB (clone 16A; PharMingen); and Cy-Chrome–conjugated anti–mouse CD4 (clone RM4-5; PharMingen). FITC-conjugated anti–mouse IFN-γ (clone AN-16; gift of Dr. A. O'Garra, DNAX Research Institute) and PE-conjugated anti–mouse TNF-α (clone XT-22; PharMingen) were used for intracellular staining. The following mAbs were used in vivo: 1B1.2 (rat IgG1), a blocking mAb reactive with mouse IL-10 receptor (IL-10R) 13 , JES5-2A5 (rat IgG1), an anti–mouse IL-10 mAb 14 , and GL113 (rat IgG1), an isotype control mAb reactive with β-galactosidase. Unless otherwise indicated, antibodies were purified from supernatants of hybridomas by ion exchange chromatography and shown to contain <3 EU endotoxin/mg of protein. CD4 + T cell subsets were isolated from spleens as described previously 4 . In brief, single cell suspensions were depleted of B220 + , MAC-1 + , and CD8 + cells by negative selection using sheep anti–rat coated Dynabeads (Dynal Ltd.). The resulting CD4 + -enriched cells were stained with Cy-Chrome–conjugated anti-CD4 and FITC-conjugated anti-CD45RB mAb and sorted into the CD45RB high CD4 + and CD45RB low CD4 + fractions by two-color sorting on a FACS Vantage™ (Becton Dickinson). All populations were >98% pure on reanalysis. RAG-2 −/− mice or C.B-17 SCID mice were injected intraperitoneally with sorted CD4 + T cell subpopulations in PBS. mAbs were injected intraperitoneally in PBS the day after T cell reconstitution and weekly for the duration of the experiments. Colons were removed from mice 8–12 wk after T cell reconstitution and fixed in buffered 10% formalin. 6 μm paraffin-embedded sections were cut and stained with hematoxylin and eosin. Tissues were graded semiquantitatively from 0 to 5 in a blinded fashion. A grade of 0 was given when there were no changes observed. Changes typically associated with other grades are as follows: grade 1, minimal scattered mucosal inflammatory cell infiltrates, with or without minimal epithelial hyperplasia; grade 2, mild scattered to diffuse inflammatory cell infiltrates, sometimes extending into the submucosa and associated with erosions, with minimal to mild epithelial hyperplasia and minimal to mild mucin depletion from goblet cells; grade 3, mild to moderate inflammatory cell infiltrates that were sometimes transmural, often associated with ulceration, with moderate epithelial hyperplasia and mucin depletion; grade 4, marked inflammatory cell infiltrates that were often transmural and associated with ulceration, with marked epithelial hyperplasia and mucin depletion; and grade 5, marked transmural inflammation with severe ulceration and loss of intestinal glands. Lamina propria (LP) lymphocytes were purified as described 5 . Colons were cut into 0.5–1-cm pieces and incubated in Ca- and Mg-free PBS containing 10% heat-inactivated FCS (GIBCO BRL) and 5 mM EDTA to release intraepithelial lymphocytes. The remaining tissue was further digested with collagenase/dispase (100 U/ml; Sigma Chemical Co.), and the LP cells were then layered on a Percoll gradient (Amersham Pharmacia Biotech). The lymphocyte-enriched population was recovered after centrifugation (600 g , 20 min) at the 40/75% interface. For cytokine detection, freshly isolated LP lymphocytes were cultured for 12 h in RPMI 1640 (PAA Laboratories GmbH) containing 10% FCS, 2 mM l -glutamine, 0.05 mM 2-ME, and 100 U/ml each of penicillin and streptomycin in 24-well flat-bottomed plates coated with anti-CD3∈ 145-2C11 . Brefeldin A (10 μg/ml; Sigma Chemical Co.) was added for the final 2 h of incubation, and surface and cytoplasmic stainings were performed as described previously 15 . Labeled cells were analyzed on a FACSort™ using CELLQuest™ software (Becton Dickinson). Colitis scores were compared using the Mann-Whitney test, and differences were considered statistically significant with P < 0.05. To determine whether the CD45RB low CD4 + population from IL-10 −/− mice contained regulatory T cells, this population was compared with CD45RB low CD4 + cells from WT mice for the ability to inhibit colitis induced in RAG-2 −/− mice by transfer of WT CD45RB high CD4 + cells. Expression of CD45RB was similar on CD4 + T cells from 10–12-wk-old IL-10 −/− and WT mice (data not shown). WT CD45RB high CD4 + cells (4 × 10 5 ) were transferred to syngeneic RAG-2 −/− mice either alone or in combination with 2 × 10 5 CD45RB low CD4 + cells from WT or IL-10 −/− mice. As controls, the CD45RB low CD4 + populations from IL-10 −/− or WT mice were transferred alone and some mice were not reconstituted. 10–12 wk after T cell reconstitution, mice were killed and the development of colonic inflammation was assessed. As described previously 3 4 , transfer of CD45RB high CD4 + cells into RAG-2 −/− mice resulted in the development of severe colitis in the majority of mice (63.4%; Table , top). This colitis was characterized by an extensive inflammatory cell infiltrate, marked epithelial cell hyperplasia, and depletion of mucin-secreting cells. Cotransfer of the CD45RB low CD4 + population significantly inhibited ( P < 0.005) the development of colitis, as the majority of mice in this group (83.8%; Table , top) had minimal changes in the intestine . In contrast, cotransfer of CD45RB low CD4 + cells from IL-10 −/− mice failed to protect mice from colitis, as the majority of mice in this group (64.2%; Table , top) developed intestinal inflammation identical to that seen in mice restored with CD45RB high CD4 + cells alone. Not only did the CD45RB low population from IL-10 −/− mice fail to protect from colitis, this population actually induced disease with similar characteristics and incidence (59.0%; Table , top) to that induced by WT CD45RB high CD4 + cells. As described previously 4 , immune-deficient mice either unreconstituted or restored with WT CD45RB low CD4 + cells exhibited no pathological changes in the intestine (data not shown). These data indicate that production of IL-10 by CD45RB low CD4 + cells is essential for cells within this population to mediate their immune-suppressive function. The finding that CD45RB low CD4 + cells from IL-10 −/− mice lacked T cells capable of regulating inflammatory responses in the intestine appears at odds with previous studies from this laboratory which showed that treatment with a neutralizing anti–IL-10 mAb failed to abrogate protection from colitis transferred by CD45RB low CD4 + cells 8 . The simplest explanation for these data is that the anti–IL-10 mAb used (JES5-2A5) in these studies failed to sufficiently neutralize IL-10. Recently, an mAb reactive with the murine IL-10R has been generated 13 and shown to efficiently neutralize the effects of IL-10. To test whether treatment with anti–IL-10R mAb was able to affect the function of the CD45RB low population, mice restored with a mixture of CD45RB high and CD45RB low CD4 + cells were treated weekly with anti–IL-10, anti–IL-10R, or isotype control mAb. As can be seen in Table (bottom), treatment with anti–IL-10R abrogated protection from colitis induced by the CD45RB low CD4 + population, as all of the mice in these groups, treated with anti–IL-10R alone or in combination with anti–IL-10, developed colitis. Antibody treatment alone did not induce immune pathology in the absence of T cells, as unreconstituted recipients treated with anti–IL-10R did not develop colitis (data not shown). As reported previously, anti–IL-10 treatment had no effect on the immune-suppressive activities of the CD45RB low population, as mice in this group, like mice treated with isotype control mAb, failed to develop colitis. Colitis induced by the CD45RB high CD4 + population was characterized by the accumulation of IFN-γ– and TNF-α–secreting Th1 CD4 + T cells in the lesions . A similar expansion of Th1 cells was also present in the colon of mice that developed colitis after transfer of WT CD45RB high CD4 + cells in the presence of WT CD45RB low CD4 + cells plus anti–IL-10R or after transfer of IL-10 −/− CD45RB low cells alone. Development of disease correlated with a significant increase in the total number of CD4 + T cells expressing IFN-γ and TNF-α after polyclonal stimulation compared with noncolitic mice restored with a mixture of CD45RB high and CD45RB low cells. The increase in total Th1 cells characteristic of colitis was due to the marked increase of CD4 + cells in the intestine ( Table ), as analysis of cytokine production at the single cell level revealed that the few CD4 + T cells present in nondiseased colons were capable of producing similar levels of IFN-γ and TNF-α as those isolated from colitic mice . The finding that in cotransfers of CD45RB high and CD45RB low CD4 + cells, neutralization of the function of IL-10 (by treatment with anti–IL-10R or the transfer of IL-10 −/− CD45RB low cells) led to the development of colitis with identical immunological and histological features to that induced by transfer of pathogenic CD45RB high cells indicates that IL-10 is a key mediator of the regulatory activity of the CD45RB low CD4 + T cell population. One way regulatory T cells could mediate their immune-suppressive properties is to induce the differentiation of naive T cells into cells with a similar function, rather than into pathogenic cells. This phenomenon has been termed infectious tolerance 16 17 . Given that production of IL-10 by CD45RB low CD4 + cells was essential for their regulatory function and that culture of T cells in the presence of IL-10 can lead to the generation of regulatory T cells 18 , it is possible that immune suppression by these cells requires IL-10 production by the normally pathogenic CD45RB high CD4 + population. To test this, CD45RB high CD4 + cells from IL-10 −/− mice were transferred alone or in combination with WT CD45RB low CD4 + cells to RAG-2 −/− mice. As described previously 19 , mice restored with IL-10 −/− CD45RB high CD4 + cells developed a colitis with similar incidence and severity (68.0%; Table ) to that transferred by this population isolated from WT mice. However, protection from colitis was equally effective when WT CD45RB low CD4 + cells were cotransferred with IL-10 −/− CD45RB high CD4 + cells, as the majority of mice (81.2%; Table ) had no colitis or minimal changes in the intestine. These results indicate that inhibition of inflammatory responses in the intestine mediated by CD45RB low CD4 + cells is not dependent on the differentiation of the progeny of CD45RB high CD4 + cells into IL-10–secreting cells. Data presented herein provide direct evidence that IL-10 plays an obligate role in the function of regulatory T cells that control inflammatory responses in the intestine. In contrast to CD45RB low CD4 + T cells from WT mice, which inhibit colitis induced in immune-deficient mice after transfer with CD45RB high CD4 + T cells, the CD45RB low CD4 + population from IL-10 −/− mice failed to mediate this function. In addition, they induced severe colitis when transferred alone to immune-deficient recipients. Previous studies from this laboratory showed that TGF-β was essential for inhibition of colitis by CD45RB low cells 8 . These results, together with the findings reported here, provide the first clear evidence that IL-10 and TGF-β play nonredundant roles in the functioning of regulatory T cells which control inflammatory responses towards intestinal antigens, as the neutralization or absence of one of these cytokines is sufficient to abrogate protection. Furthermore, IL-10 produced by regulatory T cells themselves is crucial for the normal functioning of these cells. Colitis in the SCID model involves the development of Th1 cells responding primarily to intestinal flora, as transfer of CD45RB high CD4 + T cells to germ-free SCID mice failed to induce disease 20 . The fact that the regulatory cells express the phenotype of antigen-experienced cells (CD45RB low ) would suggest that their generation in normal mice is antigen driven; however, whether these antigens are of bacterial or self origin is not known. Recent studies of the immune response elicited by Helicobacter hepaticus infection, a bacterium that colonizes the cecum, showed that normal mice mounted an IL-10–dependent response, whereas IL-10 −/− mice developed a pathogenic Th1 response towards the bacterium 21 . These studies support the hypothesis that in immunocompetent hosts, enteric antigens induce IL-10–secreting T cells that are immune suppressive and prevent inflammatory responses towards intestinal antigens. Mucosal T cell unresponsiveness to enteric antigens has similarly been shown in humans to be mediated by antigen-specific CD4 + T cells and production of IL-10 and TGF-β 22 . Injection of rIL-10 inhibited the development of colitis in CD45RB high CD4 + T cell–restored SCID mice 5 and in IL-10 −/− mice treated from weaning 10 . However, the inhibitory effects of IL-10 treatment were only transitory, as colitis developed after the treatment was stopped, whereas the transfer of CD45RB low CD4 + T cells provided long-lasting protection. This may reflect the capacity of regulatory T cells to provide a constant source of IL-10 upon stimulation by endogenous antigen (bacteria or self). Alternatively, they may produce or induce other regulatory molecules, such as TGF-β, which are not induced by exogenous administration of IL-10. The finding that CD45RB low CD4 + cells incapable of synthesizing IL-10 failed to inhibit colitis induced by transfer of CD45RB high cells to SCID mice illustrates that the regulatory T cell population itself is the critical source of IL-10 in this model, despite the fact that both the CD45RB high CD4 + T cells and host cells in the SCID mice were capable of making IL-10. It is not clear whether the lack of regulatory T cells in IL-10 −/− mice is a result of abnormalities in the development or effector function of this population. However, the finding that treatment with anti–IL-10R was able to neutralize the immune-suppressive function of differentiated regulatory cells in cotransfers of CD45RB high and CD45RB low cells from normal mice supports the idea that IL-10 is required for the effector function. The reasons for the striking difference in effectiveness between the anti–IL-10 mAb JES5-2A5 and the anti–IL-10R antibody 1B1.2 are not clear, as both are effective neutralizing antibodies in vitro. However, the relative ineffectiveness of JES5-2A5 has been observed in other experimental situations, such as Leishmania -infected mice (Mauze, S., and R.L. Coffman, unpublished observations). Precisely how IL-10 mediates its immune-regulatory function is not known. Inhibition of colitis mediated by IL-10–secreting CD45RB low CD4 + cells was characterized by substantial reductions in total number of Th1 cells recovered from the intestine. This difference was attributable to the reduced number of CD4 + cells present in the intestine (8–24-fold; Table ), as analysis of cytokine production at the single cell level revealed a similar percentage of CD4 + T cells capable of producing IFN-γ and TNF-α in diseased and nondiseased colons. This suggests that the major activity of IL-10–secreting regulatory T cells is to inhibit the accumulation of pathogenic Th1 cells in the intestine. Whether this is due to reduced expansion, or migration, of these cells is not known. IL-10 has been shown to mediate a range of antiinflammatory activities, including the inhibition of antigen-induced proliferation and cytokine secretion by T cells. Inhibition is thought to be mediated mainly by effects on APCs, particularly downregulation of molecules involved in T cell costimulation 23 . It seems likely that IL-10–secreting regulatory T cells act to inhibit Th1 cell activation, and that IL-10 produced locally in the intestine acts on macrophages to prevent their activation and elaboration of proinflammatory molecules and chemokines, thus inhibiting T cell recruitment into the intestine. Consistent with this, mice in which macrophages and neutrophils are unable to respond to IL-10 as a result of a cell type–specific deletion of Stat-3 developed enterocolitis 24 , suggesting that IL-10–mediated macrophage and neutrophil deactivation contributes to the immune-suppressive properties of IL-10 in the intestine. Recently, regulatory cells with activities similar to those within the CD45RB low CD4 + population were cloned in vitro by culturing with IL-10 18 . These cells, termed T regulatory 1 (Tr-1) cells, are characterized by their ability to produce IL-10 but not IL-4 and to inhibit T cell activation in vitro and in vivo. Although immune suppression in vitro was shown to be dependent on IL-10 and TGF-β, the mechanism of action of Tr-1 cells in vivo has not been established. It is likely that regulatory T cells contained within the CD45RB low CD4 + subset represent the in vivo counterpart of in vitro–derived Tr-1 cells. This is based on the fact that the function of the former population is dependent on IL-10 but not IL-4 synthesis, a defining feature of Tr-1 cells in vitro. In addition, a subset of CD45RB low CD4 + T cells, identified by expression of CD38, was shown to be immune suppressive in vitro and to produce IL-10 but not IL-4 after stimulation with anti-CD3 and IL-2 25 . However, further comparison between the in vivo–derived regulatory cells described here and in vitro–derived Tr-1 cells awaits identification of cell surface markers specific for Tr-1 cells and elucidation of their mechanism of action in vivo. The finding that IL-10 leads to the differentiation of Tr-1 cells in vitro raised the possibility that part of the mechanism by which IL-10–secreting regulatory T cells contained within the CD45RB low CD4 + population inhibit colitis is to induce the differentiation of the progeny of CD45RB high CD4 + cells into IL-10–secreting cells. In some systems, regulatory T cells have been shown to induce the differentiation of naive cells into cells with similar regulatory function, a phenomenon termed infectious tolerance 16 17 . However, differentiation of Tr-1 cells among the progeny of the CD45RB high population did not appear to be a crucial part of the immune-suppressive activities of the CD45RB low population, as these cells were able to inhibit colitis induced by IL-10 −/− CD45RB high CD4 + cells which could not differentiate into IL-10–secreting cells. However, this result does not rule out the possibility that IL-10 secretion by the CD45RB low population leads to the differentiation of the progeny of CD45RB high cells into regulatory T cells secreting other cytokines, for example TGF-β, and experiments are currently underway to test this hypothesis. In summary, these studies identify IL-10 as an essential mediator of the function of regulatory T cells contained within the CD45RB low CD4 + T cell population. Previous studies identified TGF-β as a critical component of the immune-regulatory function of these cells, and the finding that IL-10 has an identical effect is the first demonstration that both of these immune-suppressive cytokines play mandatory roles. How the functions of these two cytokines are linked is not known. The finding that IL-10 −/− mice have immune pathology restricted to the intestine 9 whereas TGF-β1 −/− develop multiple organ disease 26 suggests that IL-10 is not required for the production of TGF-β1. However, TGF-β has been shown to induce IL-10 secretion by APCs 27 , making it a possibility that TGF-β alters antigen presentation in favor of the generation of IL-10–secreting regulatory T cells. Alternatively, IL-10 and TGF-β may act entirely separately, and further experiments are required to distinguish between these possibilities. There is now good evidence that regulatory T cells can be induced after oral exposure to antigen and that their function is dependent on TGF-β 28 29 . In addition, regulatory T cells that inhibit the development of autoimmune disease have been shown to exist naturally in mice and rats (for a review, see reference 30 ). However, information regarding the role of IL-10 and TGF-β in the function of these cells is incomplete, as in most cases only one or the other has been examined. Thus, TGF-β–dependent mechanisms were shown to be involved in the regulation of autoimmune nephritis 31 and thyroiditis 32 , whereas inhibition of diabetes by NK1.1 T cells was dependent on IL-4 and IL-10 33 . It remains to be established whether regulatory T cells that control inflammatory responses in the gut are the same as those shown to regulate organ-specific autoimmune disease. However, more information on the relative roles of IL-10 and TGF-β in the function of these different populations of cells will facilitate their comparison. Differentiation of IL-10–secreting TGF-β–dependent regulatory T cells is one of the host's natural mechanisms for avoiding immune pathology. Further understanding of the antigen specificity, development, and mechanism of action of these cells is crucial for the design of effective immune interventions that seek to capitalize on this potent immune-regulatory mechanism. | Study | biomedical | en | 0.999997 |
10510090 | The following cells were used in these experiments: the RNK-16 49A.9 rat NK leukemia (RNK.Ly-49A) and the YB2/0 (YB) and YB2/0-D d (YB-D d ) rat B cell lines have been described previously 19 . The cells were cultured in RPMI 1640 glutamax (GIBCO BRL) supplemented with 50 μM 2-ME, 10 U/ml penicillin, 10 μg/ml streptomycin, and 10% FCS in 5% CO 2 at 37°C. The transfectants were maintained in 1 mg/ml G418 (GIBCO BRL) for selection purposes. All cells were mycoplasma free. To distinguish target cells from RNK.Ly-49A cells, 5 × 10 5 target cells were first labeled with biotin (300 μg/ml) in 100 μl PBS for 25 min at room temperature, extensively washed, and labeled with StreptaLite™ Cascade Blue ® , streptavidin-FITC, or streptavidin-TRITC (Molecular Probes) for 30 min at 4°C. Cells were then washed and used as target cells in subsequent cell–cell conjugation experiments. This labeling procedure has been demonstrated not to alter NK cell cytotoxicity 23 . Chromium-release assays were performed as described previously 24 . YB target cells were labeled with 100 μCi of Na 2 51 CrO 4 (Nycomed Amersham plc) for 1 h and washed before their use as targets in subsequent cytotoxicity assays. Varying amounts of unlabeled target cells were then added to the effector cells in triplicate wells of U-bottomed 96-well microtiter plates followed by the addition of “hot” YB targets in a final volume of 200 μl. E/T ratio was 40:1. After 4 h of incubation, 100 μl of the supernatant was harvested and its radioactive content was assayed in a gamma-irradiation counter. The mean percent specific lysis of triplicate wells was calculated using the following formula: % specific lysis = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. The main components of the optical tweezers system were an ion laser–pumped titanium-sapphire laser with a tuning range of 675–980 nm and an average peak power of 2.3 W, and an inverted microscope (model IX 70; Olympus). The microscope was rebuilt to work as an optical tweezers system by replacing the holder for the bottom mirror with a custom-built holder for a dichroic mirror (Plane plate, BK7, dichroitic coating FLP-5 Lam0 = 590 nm; Spindler & Hoyer). The dichroic mirror directed the infrared laser light into the microscope and onto the specimen plane while reflecting the visible observation light. An extra protective short-pass edge filter, 640 nm cut off , was mounted in the detection path to prevent scattered laser light from reaching the eyes or the camera. The trapping beam at 810 nm and 200 mW laser output power was focused to a diffraction-limited spot by a high numerical aperture objective (UPlanApo100×OI/1.35NA; Olympus). The cells were moved in the objective plane relative to the laser focus by a motor-driven scanning stage, Scan IM 100×100 (Märzhäuser). The microscope was combined with an Argus-20 image processor and a C2400-75i camera (Hamamatsu) for video microscopy. NK cells were first allowed to settle and adhere onto a small area at the bottom of a PetriPerm™ dish (Heraeus Instruments) placed on a heated stage. The temperature was continuously monitored with a temperature probe near the area where the cells were observed, and temperature was maintained at 35–37°C. Fluorescently labeled tumor target cells were then carefully added to these NK cells. The target cells were then captured in the optical trap, their identity confirmed by fluorescence microscopy, and placed at the leading edge of a migrating NK cell. Effector–target interactions were then analyzed using bright field microscopy and recorded onto videotapes for detailed analysis. For the dual target experiments, an H-2D d –expressing target (YB-D d ) was trapped by the optical tweezer, placed in front of a migrating NK cell, and allowed to interact for ∼2 min. Thereafter, a second target (YB) was placed next to the NK cell and allowed to bind. The identity of the cells was confirmed by fluorescence before selecting the target cells. Cellular events were then recorded as described in the single target experiments. We have included three videoclips from representative experiments for Fig. 2 , Fig. 3 , and Fig. 6 . Video 1, Fig. 2 : 7× speed, 0.5× original magnification; second part of clip is shown at 1× original magnification for a better view of cell–cell interactions. Video 2, Fig. 3 : 7× speed, 1× original magnification. Video 3, Fig. 6 : 7× and 21× speed, 1× original magnification. Still images at various time points are presented in the videos, also indicated by the timer. To determine how inhibitory MHC class I receptors modulate the cellular responses of individual NK cells, we used an optical tweezers system 25 combined with videomicroscopy to analyze RNK.Ly-49A cell interactions with susceptible (YB) or resistant (YB-D d ) target cells. In these assays, individual NK cells were allowed to settle and adhere to the surface of a dish filled with warm medium on a heated microscope stage. With the temperature confirmed at 35–37°C, a migrating NK cell was selected as an “active” effector cell. Tumor target cells were then carefully added above the NK cell, and a healthy target cell was trapped using laser optical tweezers before it could adhere to the surface of the dish. The identity of the target cell was confirmed by fluorescence, and the cell was then directly placed near the leading edge of the designated NK cell. Thereafter, the effector–target cell interaction was observed and recorded in real time. During trapping, positive identification, and positioning, the total time the target cell spent in the optical trap was between 20 and 60 s. The data in Fig. 1 illustrate typical morphological changes induced in a susceptible YB tumor target cell during an interaction with an NK cell. Early in the interaction, the NK cell began to round up, and small moving lamellipodia were still seen . Next, the NK cell completely rounded up , and the YB target cell began to swell . 8 min later, the target further increased its volume and lost intracellular integrity . In the final panel, the target cell had completely lost its original appearance and had acquired the typical morphology of a dead target cell . To rule out that laser light was harmful to the cells, target cells were held in the trap for >15 min, and no obvious morphological changes were induced (data not shown). We analyzed NK cell interactions with susceptible (YB) and resistant (YB-D d ) target cells (summarized in Table ). Upon first contact with a susceptible target cell (YB), the NK cell typically stopped migrating, rounded up within minutes, and acquired a dark rim along its membrane surface. We categorized the responses of NK cells after target cell encounters into two general groups: the NK cell became static and rounded after binding the target cell, or the NK cell maintained continuous motion when engaging the target cell. Effector cell rounding was observed in 81% of the NK–YB cell conjugates analyzed (31 interactions total), whereas only 21% of the NK–YB-D d cell interactions resulted in this type of effector response (28 interactions total). In the group where NK cells became static and rounded, cell death of susceptible YB cells was often noted (88%), whereas death of the YB target cells was rarely seen in the group where the NK cells maintained their motion (17%). There was no evidence for death of the YB-D d target cells in these experiments. These two major categories were further divided into two subcategories each, giving a total of four types of NK cell responses. In type I responses, the NK cell stopped migrating, rounded up, and stayed attached to the target cell for the remainder of the experiment (17–55 min). In type II, the NK cell responded to the target by cell rounding, stayed attached to the target for several minutes, and then moved away. In type III, the NK cell bound the target cell, but maintained its motion during the entire encounter, although the NK cell changed its direction of movement. The NK cell remained attached to the target for some minutes, but no NK cell rounding was seen. Eventually the NK cell left the target. Finally, in type IV responses, the NK cell continued to move after target cell contact and migrated under the target without significantly altering its direction of movement. In some cases, the NK cell attached to the target cell and carried it along for a period of time as it moved forward. A typical example of an interaction included in the first group, where the NK cell became static and rounded, is presented in Fig. 2 . In this example, an NK cell started to interact with a YB target cell . A second NK cell approached the first NK cell, and out of focus, a third NK cell was also seen attaching to the second NK cell . The first NK cell rounded up, whereas the second NK cell made contact with the first NK cell . Then, the second NK cell also started to interact with the YB target cell, and an extensive membrane interaction could be seen . 8.5 min after contact , the first NK cell formed lamellipodia on the opposite side from the YB cell, and pulled itself away from the YB target cell. 2 min later , the first NK cell separated from the YB cell, pulling two or three membrane filaments with it, while the YB cell began to swell, undergoing obvious morphological changes. By the last panel , the YB cell had lost its membrane integrity, and was eventually lysed. In the upper part of the panel, the second NK cell had started to separate from the YB target cell. The data in Fig. 3 show an example from the group where the NK cell maintained continuous motion . The NK cell made contact with a YB-D d target cell and failed to stop or round up . Instead, the NK cell continued to move under the YB-D d cell, carried it along , and then released the target cell unharmed into the surrounding medium . We next compared effector–target contact times between RNK.Ly-49A cells and YB tumor target cells that did (YB-D d , 21 interactions total) or did not (YB, 19 interactions total) express the protective MHC class I molecule for Ly49A . These experiments showed that the NK–target cell interaction times varied for each target cell type. However, there was a small difference in the duration of interaction between the two targets. NK–YB-D d cell conjugates preferentially lasted for shorter times, and a higher percentage (73%) of the NK–YB-D d cell conjugates separated earlier (<10 min) compared with NK–YB cell pairs (38%). Occasionally, longer interaction times (>25 min) were observed with both target cell types (YB, six interactions; YB-D d , six interactions). In 74% of the NK–YB cell interactions, the encounter resulted in target cell death, whereas none of the NK–YB-D d cell conjugates resulted in obvious morphologic changes in the target cell. Taken together, these data suggest that NK cells interact for somewhat shorter times with targets expressing an inhibitory MHC class I ligand, compared with target cells that do not. To investigate bystander effects of protected YB-D d cells on the killing of susceptible target cells, we performed cold target competition experiments using susceptible YB cells as “hot” targets. Increasing amounts of unlabeled YB-D d target cells did not compete for YB target killing by NK cells, yet unlabeled “cold” YB cells competed very efficiently with their hot labeled YB counterparts . These data indicated that killing of susceptible target cells (YB) by NK cells was not significantly affected by the presence of increasing numbers of resistant neighboring cells, regardless of the expression of H-2D d inhibitory ligands by bystander YB-D d targets. These data are similar to those reported with other target cell combinations, and they suggest that H-2D d positive target cells fail to deliver global inhibitory signals to Ly49A + NK cells 18 . There are several possible explanations for the lack of competition between YB-D d and YB cells, including differences in binding of YB and YB-D d cells, shorter release times for YB-D d cells (temporally restricted inhibition), or compartmentalization of Ly49A signaling (spatially restricted inhibition). As such, only YB-D d was protected from lysis, but not other targets. We wondered whether Ly49A + NK cells could bind to both YB-D d and YB cells simultaneously, and if so, were NK cells able to lyse YB, even though the NK effectors were bound to YB-D d targets? To test this idea directly, we first allowed the NK cell to establish contact with a YB-D d cell for ∼2 min before the addition of a susceptible target (YB). The videomicroscopic laser tweezers system allowed us to freely move the target cells and to monitor the resulting interactions. Fig. 6 shows a sequence of images from an interaction between an RNK.Ly-49A cell bound to a resistant YB-D d and a susceptible YB target. We found that in most cases (73%, 11 total interactions), the susceptible YB target cell underwent morphological changes associated with cell death, whereas the resistant YB-D d cell did not . The percentage of YB cells killed (73%) was similar to that observed when the NK cells encountered YB targets alone (74%). These data indicate that NK cells can bind to both susceptible and resistant target cells simultaneously and still retain the ability to selectively kill the susceptible target cell. Thus, inhibitory signals mediated via Ly49 receptors do not completely inhibit cellular effector functions. Rather, Ly49A directionally inhibits NK cell activation against protected, but not unprotected target cells. NK cells express inhibitory receptors which prevent the lysis of cells that express sufficient quantities of self MHC class I molecules. This system likely allows NK cells to distinguish between normal self cells and abnormal, or nonself, cells due to differences in MHC class I expression and polymorphism. There are significant variations in the expression of MHC class I molecules between different cell types and tissues, and it has been proposed that some cell types are unable to activate NK cells under normal circumstances. These cell types are likely to be ignored by NK cells. Data have accumulated from a variety of laboratories indicating that inhibitory receptors of the Ly49, KIR, and CD94 families are able to mediate inhibition of all NK cell effector functions 8 10 26 . Such “global” inhibition is believed to act as a fail-safe mechanism, protecting normal tissue from deleterious NK cell responses. In vivo, cells with reduced or absent expression of MHC class I molecules due to malignant transformation or viral infection are likely to be outnumbered by normal cells with high MHC class I expression. Thus, if NK cells continuously encounter normal cells able to engage their inhibitory receptors, how are NK cells able to mediate effector functions within the in vivo environment? There are many possible scenarios that could explain inhibitory NK cell functions in vivo. It is clear from in vitro and in vivo analyses that NK cells can readily distinguish target cells expressing the proper MHC class I from those that do not 27 . The final experimental readout from such bulk assays is the result of numerous individual cell–cell encounters, each making a small contribution to the overall response. To understand how NK cells function, we examined how inhibitory receptors affect individual NK cells when they encounter various target cell types. In these studies, we have directly tested the effects of inhibitory signals on individual NK cells using an optical tweezers system. This system has allowed us to temporally and spatially direct the entire NK cell encounter with divergent target cell types. We have used this system to understand how the response of individual NK cells accounts for the overall response of an NK cell population. Using videomicroscopic analysis of NK–target cell interactions, we found typical changes in NK cell movement upon contact with a susceptible target cell (NK cell rounding). These experiments are consistent with previously published studies using a T cell hybridoma, where a similar behavior was observed upon T helper cell interactions with B cells 28 . In addition, similar observations were seen in studies of a human T cell clone interacting with MHC class II–transfected L cells 29 . Our studies have uniquely enabled us to examine receptor-mediated NK cell inhibition during effector–target cell interactions in real time. Recognition of H-2D d MHC class I molecules on targets by Ly49A inhibitory receptors on NK cells considerably altered the behavior of the rat NK cell line (RNK.Ly-49A) towards potentially susceptible tumor target cells ( Table ). In the majority of cases, Ly49A ligation by target H-2D d prevented NK cell migratory arrest and the associated rounding seen when susceptible target cells were engaged. There was continuous NK cell movement even though NK–YB-D d contact had been made. Thus, inhibitory receptors prevent NK cell effector functions, and they prevent target-induced migratory arrest. These data also suggest that inhibitory receptors fail to globally inhibit the biochemical pathways required for cell movement. The NK–target interaction times varied for both protected and unprotected target cells (from 2 to 55 min). However, there was a trend toward shorter NK–YB-D d interactions compared with NK–YB interactions, although the observed differences were not statistically significant. It is possible that under physiological conditions in vivo, NK cells interacting with, or binding to, normal cells are likely to continue migrating in the presence of nearby cells and tissues, which may shorten the time spent with a resistant target cell compared with in vitro conditions in a petri dish. With respect to susceptible target cells, we believe that the absence of such mechanical forces imposed upon the isolated effector–target cell couple may increase the duration of the interaction we have observed in vitro, and account for some of the variation in Fig. 4 . Interestingly, the separation of an NK cell from a susceptible target during type II interactions was in many cases associated with the formation of one or more very flexible, thin membrane filaments between the cells . The significance of these filaments is unclear, but they may induce additional physical damage to susceptible targets during NK cell–mediated cytotoxicity. The experiments analyzing single NK–target cell interactions demonstrated that inhibitory receptors did not prevent all cell functions. In vivo, NK cells are surrounded by normal cells, and are thus likely to encounter normal and resistant cells simultaneously. Therefore, we have addressed the question of whether an NK cell surrounded and in contact with normal cells would recognize and kill an aberrant target. Using a cold target competition assay, our data demonstrated that expression of protective MHC class I molecules on surrounding targets does not significantly affect lysis of susceptible targets . The slight decrease in killing of YB cells in the presence of YB-D d cells may be due to the fact that NK cells bind to the YB-D d cells, and although they can continue to move, the NK cells will be slightly delayed due to membrane interactions with abundant YB-D d cells. Previous in vitro data with RNK.Ly-49A cells support these data 18 . Murine RMA cells did not influence YB cell lysis , and this could be correlated with the fact that these cells were not themselves killed and showed little or no binding to NK cells on videomicroscopic analysis (data not shown). These data indicate that RMA cells are “ignored” by the RNK.Ly-49A cells, perhaps due to lack of the proper adhesion and/or triggering molecules. Previous cold target competition studies have shown that NK cells are able to kill unprotected cells in the presence of MHC-protected targets 18 27 30 . One interpretation of these data is that NK cells might not kill susceptible targets when coengaged with MHC-protected targets, but susceptible cells might be killed after NK cell disengagement from protected cells (temporally restricted inhibition). Alternatively, the inhibitory effects of target MHC class I might be localized to the effector–target interface, allowing for the lysis of susceptible targets while simultaneously coupled to an MHC-protected target (spatially restricted inhibition). Our videomicroscopic demonstration that an NK cell coupled to an MHC-protected target can kill a susceptible target while leaving the protected cell undamaged proves that NK cell inhibitory receptors such as Ly49 do not deliver global inhibitory signals per se, but rather interrupt activation signals in a spatially restricted manner at the effector–target interface. The NK cell response to the YB target cell was similar to the behavior of an NK cell interacting with a susceptible target cell alone . Our data analyzing the individual cell response support the conclusions from molecular studies that demonstrated tyrosine phosphorylation in human NK cells upon conjugation with either sensitive or resistant target cells, which suggested that there was not complete effector cell inhibition 31 32 . However, the data presented here go much further since they demonstrate that an NK cell can mediate effector functions while bound to a resistant cell. In summary, our data show that Ly49 inhibition occurs in a spatially restricted manner and help explain how the inhibitory receptor system allows NK cells to distinguish normal cells from abnormal cells in an efficient manner. | Study | biomedical | en | 0.999995 |
10510091 | AND H-2 k/k TCR-Tg mice were bred from a C57BL/6 × SJL founder, provided by Dr. S. Hedrick (University of California, San Diego, CA), by successive backcrosses (12N) onto the B10.Br background. These mice express a Vβ3/Vα11 TCR transgene that recognizes PCCF in the context of I-E k . The mice were housed at either the animal facilities at the Trudeau Institute or the Scripps Research Institute (La Jolla, CA) until their use at 2–4 (young) and 13–16 mo old (aged). Mice with evidence of gross pathology were excluded from the study. Each experiment was performed at least two times with pooled spleens and peripheral lymph nodes from two young and two or three aged mice. The isolation of spleen cells enriched for CD4 cells has been described previously 23 . In brief, the cells were passed through a nylon wool column or a mouse CD4 enrichment column (R & D Systems, Inc.), and the nonadherent cells were stained with Cy-Chrome–anti-CD4, FITC–anti-Vα11, and PE–anti-Vβ3 or APC–anti-CD4, PE–anti-Vα11, biotin–anti-Vβ3, and streptavidin red613. The CD4 + Vβ3 + Vα11 + cells were sorted using either FACStar PLUS™ or FACSVantage™ (Becton Dickinson). Alternatively, small resting CD4 cells were prepared as described 25 by passage over nylon wool and antibody and complement depletion of CD8 + and class II + cells followed by Percoll gradient separation. Both methods of CD4 enrichment yielded similar results in all experiments. All staining was done at 4°C in PBS with 1% BSA and 0.1% NaN 3 . The following antibodies and fluorescent reagents were used: Cy-Chrome– and APC–anti-CD4 (clone RM4-5; PharMingen), FITC–anti-CD44 (clone IM7), FITC–anti-CD62L (clone Mel 14), FITC– and PE–anti-Vα11 (clone RR8-1; PharMingen), PE– and biotin–anti-Vβ3 (clone KJ25; PharMingen), FITC–anti-CD25 (IL-2R α chain; clone PC61), FITC–anti-CD122 (IL-2R β chain; clone 5H4), biotin–anti-CD132 (common γ [γc] chain; clone 4G3) streptavidin red613 (GIBCO BRL), streptavidin Per-CP (Becton Dickinson), streptavidin–PE, and streptavidin–FITC (both from Southern Biotechnology). All isotype control antibodies were purchased from PharMingen: FITC–hamster Ig, FITC–rat IgG1, FITC–rat IgG2a, and FITC–rat IgG2b. Flow cytometry was carried out using FACScan™, FACStar PLUS™ , or FACSCalibur™ flow cytometers, and the data were analyzed with CELLQuest™ software (all from Becton Dickinson). Naive cell populations were stained with the dye CFSE (carboxy-fluorescein succinimidyl ester; Molecular Probes, Inc.) as previously described 26 . In brief, cells were resuspended in PBS at 5 × 10 7 cells/ml. CFSE (1 mM stock) was added to the cell suspension at 1:250 and incubated in a 37°C water bath for 13 min. Cells were then washed two times, recounted, and cultured as described below. Cells were cultured in RPMI 1640 (GIBCO BRL) supplemented with penicillin (200 μg/ml), streptomycin (200 μg/ml), glutamine (4 mM), 2-ME (50 μM), Hepes (10 mM), and 8% fetal bovine serum (Intergen). DCEK-ICAM, a fibroblast cell line that expresses B7.1 constitutively and is stably transfected with intercellular adhesion molecule (ICAM)-1 and class II MHC (I-E k ) molecules, was used as APC at 2:1 T cell/APC. These cells do not express other costimulatory molecules such as lymphocyte-associated function antigen (LFA) type 1, CD48, or heat-stable Ag. Recombinant murine cytokines IL-2, IL-4, IFN-γ, and IL-5 were obtained from culture supernatant of X63.Ag8-653 cells transfected with cDNA for the respective cytokines 27 . Recombinant murine IL-12 was a gift of Dr. Stanley Wolf (Genetics Institute, Cambridge, MA). For experiments involving the addition of cytokines to T cell proliferation assays, IL-2 (35 U/ml), IL-4 (25 ng/ml), IL-7 (950 U/ml; a gift from Dr. Albert Zlotnik, DNAX, Palo Alto, CA), and human IL-15 (60 ng/ml; R & D Systems, Inc.) were added at the initiation of the cultures. CD4 effectors were generated by culturing Tg + CD4 cells (2 × 10 5 cells/ml) with 5 μM PCCF and mitomycin c–treated (100 μg/ml for 30 min at 37°C) DCEK-ICAM cells (2:1 T cell/APC) in the presence of polarizing cytokines. Th1 effectors were generated with IL-2 (80 U/ml), IL-12 (2 ng/ml), and anti-IL4 (11B11; 10 μg/ml). Th2 effectors were generated with IL-2, IL-4 (200 U/ml), and anti–IFN-γ (XMG1.2; 10 μg/ml). IL-2 effectors were generated with IL-2 (80 U/ml). “No cytokine” effectors were generated in the presence of PCCF/DCEK-ICAM alone. Day 4 effectors were used in all experiments and were restimulated in 1-ml cultures with PCCF/DCEK-ICAM; 24-h supernatants were collected and assayed for cytokine secretion. [ 3 H]thymidine incorporation assays to detect DNA synthesis were performed in 96-well plates. Varying numbers of Tg + CD4 cells (10 4 –5 × 10 5 cells/ml; 0.2-ml cultures) were incubated along with DCEK-ICAM cells and 5 μM PCCF for 3 d. The cultures were pulsed for the last 16 h with 0.4 μCi [ 3 H]TdR (6.7 Ci/mmol; NEN Research Products), harvested, and counted on a Wallac 1205 Betaplate counter. Culture supernatants collected after 24 h of culture or as indicated and were assayed for the presence of IL-2 in a bioassay with NK-3 cells and for IL-4, IL-5, and IFN-γ by ELISA as previously described 23 . IL-4 and IFN-γ concentrations are expressed in ng/ml; IL-2 and IL-5 concentrations are expressed in U/ml. 1 U of IL-2 is equal to 1.2 ng. Differences in IL-2 production between young and aged effector populations were analyzed by paired Student's t test. Values of P < 0.05 were considered significant. Cytokine production by effectors was detected by intracellular cytokine staining as previously described 28 29 . Effectors (5 × 10 5 per milliliter) were restimulated overnight with DCEK-ICAM cells (2:1 T cell/APC) with or without 5 μM PCCF. Brefeldin A (Epicentre Technologies; 10 μg/ml final concentration) was added 2 h after culture initiation. 16 h later, cells were collected and surface stained for CD4 and Tg + expression as described above. The cells were then divided into two tubes, washed, and fixed in 75 μl 4% paraformaldehyde plus 25 μl PBS containing 10 μg/ml Brefeldin A and incubated for 20 min at room temperature. The tubes were washed once with PBS, resuspended in 50 μl saponin buffer (PBS containing 1% fetal bovine serum, 0.1% NaN 3 , and 0.1% saponin, pH 7.4–7.6) containing anti–mouse IL-2–FITC (clone S4B6; PharMingen) or isotype control (FITC–IgG2a; PharMingen) and incubated for 30 min at room temperature. All samples were then washed with PBS and analyzed on a FACScan™ or FACSCalibur™ cytometer. To analyze the impact of the low IL-2 levels produced by naive CD4 cells from aged mice and to determine whether the defects could be reversed simply by providing IL-2, we used the AND TCR-Tg model to follow effector generation and the function and phenotype of the effectors generated in detail. As almost all Tg + cells are naive and because we used peptide Ag plus exogenous fibroblast APCs to stimulate effector generation, we can assess the intrinsic defect(s) due to age on naive T cell responses without the complications of age effects on other T cells or APCs. We have previously shown that when naive young and aged Tg + CD4 cells were stimulated with PCCF and DCEK-ICAM, the young cells synthesized DNA at a higher rate in a 3-d [ 3 H]TdR incorporation assay 23 . To pinpoint when the response of aged naive CD4 T cells becomes defective, similar numbers (2 × 10 5 ) of young and aged naive Tg + CD4 cells were stimulated with PCCF/DCEK-ICAM, and the number of cells on each day of a 5-d culture was determined . Both young and aged Tg + CD4 populations expanded at a similar rate up to day 3 (early phase proliferation). After day 3, the young cells showed a large burst of expansion that was not seen in the aged cultures (late phase proliferation). This burst of expansion resulted in a greatly increased number of cells by days 4 and 5 in the young cultures when compared with the aged cultures. We suspected that this lack of late phase proliferation of the aged cultures could potentially be attributed to decreased IL-2 production in these cultures. Fig. 1 B shows the IL-2 recovered in supernatants of young and aged cultures on each day. The young cultures contain large quantities (∼3,000 U/ml) of IL-2 on days 1 and 2 that is then consumed by day 3, just as the cells begin to undergo rapid expansion. In contrast, the supernatants of cultures of aged CD4 T cells contain only small quantities of IL-2 (<500 U/ml), and expansion does not continue beyond day 3, supporting the concept that availability of IL-2 may be the critical factor in driving later expansion. The decreased late phase proliferation of aged cells can also be visualized by labeling with the dye CFSE. CFSE is distributed equally between cells upon each cell division, resulting in the sequential halving of fluorescence intensity with each round of division. This sequential loss of fluorescence is visualized as distinct peaks when analyzed by flow cytometry 26 . Fig. 1 C shows a kinetic analysis of young and aged CFSE-labeled Tg + CD4 cells stimulated with Ag plus APC. On days 1–3, responding CD4 cells from both young and aged populations have undergone similar numbers of cell divisions, as shown by similar CFSE fluorescence profiles. On day 1, no divisions have yet occurred; on day 2, both young and aged populations show three peaks of division; and on day 3, both show four peaks of division. Importantly, these profiles also show that all, not just a subset, of both the young and aged Tg + CD4 cells undergo several rounds of division in response to stimulation. If no Ag is present, neither young nor aged Tg + CD4 cells divide (i.e., profile remains a single peak with high levels of CFSE staining; data not shown). This data suggests that all of the aged naive Tg + CD4 cells are capable of being activated to respond and divide. On days 4 and 5, the recovered cells from cultures of young naive CD4 cells continue to divide (up to six peaks of division), whereas the aged cells seem to stop dividing after day 3 (day 4 and 5 profiles look very similar to those of day 3). These two additional cell divisions in the young population translate theoretically into a fourfold greater expansion, as is seen in Fig. 1 A. These results further confirm the observation that both young and aged Tg + CD4 cells can initially proliferate (days 1–3) in response to Ag stimulation, but the aged cells are unable to sustain late phase proliferation. As IL-2 levels were significantly decreased in the cultures of aged Tg + CD4 cells, we examined the ability of aged Tg + CD4 cells to expand in the presence of exogenous IL-2. As shown in our previous experiments, young CD4 cells synthesized DNA at a twofold higher rate on day 3 than aged cells (as measured by [ 3 H]TdR incorporation) when no exogenous IL-2 was added . When IL-2 was added on day 0 to the Ag-stimulated cultures of naive young and aged CD4 cells, both effector populations incorporated radiolabel to the same extent . Fig. 2 B shows a kinetic analysis of cell counts from young and aged Tg + CD4 cell cultures with and without exogenous IL-2 through day 5. As in the experiment shown in Fig. 1 A, all cultures expanded similarly through day 3. On days 4 and 5, both young and aged cells cultured with IL-2 expanded to the same extent, as did young cells cultured with Ag/APC alone. Only the aged Tg + CD4 cells cultured without added IL-2 showed greatly reduced expansion. These results indicate that the aged Tg + CD4 cells are capable of proliferating in response to Ag in a manner comparable to young cells if they are provided with adequate amounts of exogenous IL-2. This suggests that there is no defect in signaling through the IL-2R in these aged naive T cells. As IL-2 can induce naive aged Tg + cells to proliferate and differentiate like young cells, we examined the effect of other common receptor γ chain signaling cytokines on proliferation of young and aged cells to determine if the important components of IL-2 signaling were related only to γc or unique to the IL-2R chains. IL-2, IL-4, IL-7, and IL-15 were titrated over a broad range to determine the concentration for optimal proliferation in our assay (data not shown). The optimal doses were then added to cultures of young and aged naive CD4 T cells, and [ 3 H]TdR incorporation was determined at day 4. Fig. 3 A, like the experiment shown in Fig. 2 , shows that aged cells are dividing less but that addition of exogenous IL-2 can induce aged Tg + cells to synthesize DNA to the same extent as young cells. IL-15 and IL-4 can induce similarly high levels (greater than threefold) of DNA synthesis of aged cells as are observed with IL-2. IL-7 can also somewhat enhance proliferation (about twofold) of aged cells but has little effect on young cells. The expansion and IL-2 production of young and aged effectors generated in the presence of these cytokines was also examined. Fig. 3 B shows the fold expansion of young and aged cells from day 0 to 4 of culture. Young cells expanded two to three times as much as aged cells when no cytokines were added, whereas expansion of both young and aged cells was equivalent when IL-2 was present. IL-4, IL-7, and IL-15 also augment the expansion of aged cells to some extent. IL-4, however, also enhances young CD4 T cell expansion so that young cell expansion remained greater than that of aged CD4 T cells. The effect of IL-15 on expansion was equivalent to that of IL-2. Because the most important outcome of the primary CD4 T cell response is the generation of effectors, we also examined the function of recovered effectors by assaying for IL-2 production upon restimulation with Ag/APC. Fig. 3 C shows mean ± SE of IL-2 secretion from all experiments performed, and Fig. 4 shows a representative experiment with both intracellular staining and cytokine secretion. IL-2 is the only cytokine that can cause an increase in IL-2 production by aged effectors to the levels seen for young cells. IL-4, IL-7, and IL-15 have little or no enhancing effect on IL-2 production by aged effectors. As IL-4 causes differentiation to a Th2 cytokine pattern, the effect of IL-4 on IL-2 production is negative in young as well as aged CD4 T cells. Despite the ability of IL-7 and IL-15 to restore proliferation, neither cytokine restores levels of IL-2 production by the aged cells to those achieved by the young CD4 T cells. These results suggest that IL-2 causes generation of effectors both by inducing proliferation and, via a separate mechanism, upregulation of IL-2R expression, whereas IL-4, IL-7, and IL-15 can support proliferation but not all aspects of further differentiation. We also examined the cell surface phenotype of effectors generated from Tg + CD4 cells of young and aged mice in the absence of exogenous cytokines (no cytokine effectors) and presence of exogenous IL-2, IL-4, IL-7, or IL-15 in addition to Ag/APC. Previously, we reported 23 that the Tg + CD4 cells obtained from both aged and young mice express a naive phenotype , i.e., they express low levels of CD44 and high levels CD62L (L-selectin) and do not express CD25 (IL-2Rα). As shown in Fig. 5 A, the aged no cytokine effectors express a partially activated phenotype (CD44 hi CD62L hi CD25 lo ) by day 4 after stimulation, whereas the young no cytokine effectors express a more differentiated phenotype (CD44 hi CD62L lower CD25 hi ). The addition of IL-2 at the initiation of the day 4 cultures produced IL-2 effectors from aged and young mice that expressed a similar and fully differentiated activated phenotype (CD44 hi CD62L lower CD25 hi ). This again supports the role of IL-2 in the full differentiation to an effector stage 9 . Both young and aged IL-4 effectors showed a more pronounced downregulation of CD62L compared with the other effectors, also consistent with a more differentiated state. But IL-4, IL-7, and IL-15 cannot induce upregulation of CD25 on the aged effectors. Therefore, effectors that were generated under conditions where the level of IL-2 was limiting (aged no cytokine effectors and IL-4, IL-7, and IL-15 effectors) expressed lower levels of CD25, whereas effectors that were generated under conditions where IL-2 was not limiting, either due to the adequate production of IL-2 by the effectors (all groups of young effectors) or the addition of exogenous IL-2 to the cultures (aged IL-2 effectors), expressed high levels of CD25 with no apparent age-related differences. To further investigate IL-2R expression, we examined the expression of all three chains of the receptor. Fig. 5 B shows IL-2Rα, -β, and -γc chain expression on young and aged no cytokine and IL-2 effectors. As shown in Fig. 5 A, α chain (CD25) expression on aged no cytokine effectors is lower than on young no cytokine effectors and is upregulated on the aged effectors when they are provided with exogenous IL-2. There are no age-related differences in β (CD122) or γ chain (CD132) expression on either no cytokine or IL-2 effectors. To further evaluate the functional impact of aging, we examined whether naive CD4 T cells from aged mice could become polarized effectors in the presence of polarizing cytokines and blocking antibodies. Polarized Th1 and Th2 effector populations can be generated from naive Tg + CD4 cells in vitro by the addition of cytokines and anticytokine blocking antibodies in addition to Ag and APC. For optimum polarization, Th1 effectors require IL-2, IL-12, and anti-IL-4, whereas Th2 effectors require IL-2, IL-4, and anti–IFN-γ. Although not shown here, the expansion by young versus aged naive Tg + CD4 cells cultured with Th1 or Th2 polarizing conditions were comparable. When Tg + Th1 and Th2 effectors are restimulated, they secrete high levels of specific cytokines. Th1 effectors secrete IL-2 and IFN-γ, whereas Th2 effectors secrete IL-4, IL-5, and a small amount of IL-2. To determine whether clear polarization could occur with aged CD4 T cells, we generated effectors from both young and aged CD4 T cells in the presence of cytokines (including IL-2) and blocking antibodies. When we restimulated Tg + Th1 and Th2 effectors after 4 d of culture, effectors generated from both young and aged populations secreted similar levels of the appropriate cytokines. Fig. 6 shows that Tg + Th1 effectors produced IL-2 and IFN-γ and Tg + Th2 effectors produced IL-4 and IL-5 with no age-related deficiencies. These results demonstrate that aged naive Tg + CD4 cells are just as capable of differentiating into highly polarized Th1 and Th2 effectors as young Tg + cells, given the appropriate polarizing conditions and adequate amounts of IL-2. IL-15 acts by binding to a three-chain receptor composed of the IL-2Rβ and IL-2Rγ chains and a unique α chain. To see if there was any difference in the ability of IL-2 and IL-15 to support effector generation of aged CD4 T cells, we tested the ability of IL-15 to substitute for IL-2 in the differentiation of young and aged Th1 and Th2 effectors. Polarized effectors were generated in the presence of polarizing cytokines and antibodies (IL-12 and anti–IL-4 for Th1 and IL-4 and anti–IFN-γ for Th2) alone, with exogenous IL-2, or with exogenous IL-15. Fig. 7 A shows cytokine production data from a representative experiment, whereas Fig. 7 B shows the mean percent increase ± SE in cytokine production in cultures generated with added IL-2 or IL-15 over that seen with polarizing cytokines alone for three separate experiments. IL-2 and IFN-γ production is from Th1 effectors, and IL-4 and IL-5 production is from Th2 effectors after restimulation with Ag and APC. Th1 effectors from CD4 T cells of young mice make high levels of IL-2 and IFN-γ regardless of whether IL-2 or IL-15 is added. Aged Th1 effectors make enhanced levels of IL-2 (a 150% increase) when exogenous IL-2 is added, compared with a 70% increase when IL-15 is added; IFN-γ production by Th1 cells from aged mice is for the most part unaffected by exogenous IL-2 or IL-15. Th2 effectors from CD4 T cells of young mice produce IL-4 and IL-5, which can be enhanced up to 100% when IL-2 is added. Importantly, the production of high levels of IL-4 and IL-5 by aged Th2 effectors is uniquely IL-2 dependent (a 110% increase for IL-4 and a 235% increase for IL-5) and cannot be induced by the presence of IL-15 (a 0.7% increase for IL-4 and a 20% increase for IL-5). The cell recoveries of the effectors generated in Fig. 7 A are shown in Fig. 7 C. Both IL-2 and IL-15 can enhance the expansion of young and aged Th1 effectors but have little effect on Th2 cells, because the Th2 effectors expand due to the presence of IL-4. These results indicate that even though both IL-4 and IL-15 can induce high levels of proliferation of aged Tg + CD4 cells, other additional factors are necessary to induce differentiation into highly polarized Th1 or Th2 effectors in vitro. Fig. 7 D shows CD25 expression by the young and aged effectors from Fig. 7 A. As expected, only the addition of IL-2 can increase CD25 expression by aged effectors (both Th1 and Th2) to the levels seen on young effectors. Aged Th1 and Th2 effectors generated with polarizing cytokines alone or in the presence of IL-15 express lower levels of CD25 compared with young effectors. During a primary response, generation of large numbers of CD4 effectors able to secrete high titers of cytokines is critical in order to quickly clear the immunogen. Using a TCR-Tg mouse model, we have found several profound alterations in CD4 effector cell generation that are associated with aging, and our results suggest that the low production of IL-2 by aged naive cells is largely (or totally) responsible for these deficiencies. Aged CD4 T cell defects included lower production of IL-2, loss of late phase expansion, and failure to differentiate into effectors that express phenotypic markers associated with that stage and that secrete high levels of cytokines. Importantly, provision of exogenous IL-2 both supports the expansion and differentiation of aged naive CD4 cells and leads to generation of effectors that themselves are capable of optimum IL-2 production. Furthermore, we show here that whereas both IL-2 and IL-15 as well as IL-4 and IL-7 can enhance the expansion of aged CD4 T cells, only IL-2 can support the efficient generation of polarized Th2 effectors, indicating a unique role for IL-2 signaling through the high affinity IL-2R in effector development. Aged Tg + CD4 cells expand less than young cells during the 5-d period of in vitro effector generation . The early stage of expansion (days 1–3) is quite similar in both the young and aged cultures, whereas later expansion (days 4 and 5) is three to five times greater in the cultures of young cells. This is most dramatically observed by flow cytometric analysis of CFSE-labeled young and aged Tg + CD4 cells , in which we can actually visualize that the aged cells do not divide after day 3, whereas the young cells continue dividing. This effect is mirrored in the expansion of cells, which continues after day 3 in young but not aged populations. Moreover, the aged CD4 effectors expressed decreased levels of CD25 (IL-2Rα). We suggest that the early stage of proliferation is more dependent upon Ag-induced TCR stimulation, and the latter stage is more dependent upon IL-2, which is present in greater amounts in the young cultures. The phenotype of the day 4 effectors generated from aged naive cells was between that of a naive cell and a day 4 effector from young mice, i.e., the aged effector cells had a less differentiated phenotype that had high levels of CD44 but did not substantially downregulate CD62L and did not upregulate IL-2R (CD25) expression. This suggests that cytokines (IL-2 and perhaps others) acting midway (days 2–3) in the primary response (when IL-2 disappears from cultures of aged cells) are required for full differentiation into effectors. Effectors generated from these Tg + CD4 cells from aged mice produced less IL-2, and the frequency of effector cells secreting IL-2, as detected by intracellular staining, was also significantly reduced in the aged, supporting dependence on the continued presence of IL-2 for efficient effector generation. These results thus support a large impact of aging on CD4 effector development but also suggest that the spectrum of deficiencies found in effector generation of aged CD4 T cells may be attributable largely or totally to the decreased secretion of IL-2 upon initial TCR stimulation. In vitro, IL-2 supports the progression of activated T cells through the cell cycle, the regulation of the differentiation of T cell effectors, and the differentiation of CD4 T cells so they become susceptible to Fas/FasL-induced activation-induced cell death 30 31 32 . Although other cytokines that signal through the γc chain may substitute for IL-2, IL-2 is the only γc chain–binding cytokine produced when naive CD4 T cells are stimulated in vitro and is thus solely responsible under these circumstances where no other T cells are present and other cell populations are limited. Therefore, the finding that IL-2 secretion by CD4 Tg + cells upon TCR stimulation is markedly reduced in the aged is predicted to have profound effects on the generation of effector cells and the ensuing effector response. Moreover, as IL-2 regulates its own receptor expression 33 34 35 , the effects of any reduction in IL-2 production in the early stages of an immune response may be amplified enormously. It then becomes important to know whether these deficiencies may be overcome simply by the addition of IL-2 or other cytokines or if other intrinsic defects in the effector cells' ability to respond to IL-2 are present. When aged CD4 + Tg + effectors are generated in the presence of IL-2 (IL-2 effectors, Th1 and Th2 effectors), they become phenotypically and functionally similar to effectors derived from younger mice. In addition to upregulating CD44, they upregulate CD25 expression and downregulate CD62L. They also expand at the same rate and to the same extent as seen in cultures of young Tg + cells . When aged effectors generated in the presence of IL-2 are restimulated with Ag/APC, they secrete levels of cytokines that are the same as young effectors . Additionally, similar proportions of IL-2 effectors in both the young and aged populations are actively secreting IL-2 , and similar amounts of cytokines are secreted on a per-cell basis. These findings suggest that the major defect in effector cell generation in the aged is the inability to secrete sufficient IL-2 levels to sustain cell expansion and induce further differentiation to fully functional effector cells. However, the addition of sufficient levels of IL-2 during effector cell generation abrogates these age-related differences. Once adequately differentiated effectors are generated, it is not surprising that the effector progeny are capable of high levels of cytokine production, based on recent publications reporting the heritability of differential gene methylation patterns (epigenetic remodeling) that occurs during effector generation 36 37 38 . This model proposes that activation and effector generation of T cells involves demethylation and increased accessibility of the cytokine gene locus (IL-2 in this case) to transcription factors as a consequence of signaling from the appropriate cytokine receptor. This remodeling is permanent and heritable, thus explaining our finding that aged IL-2 effectors generated in the presence of high levels of IL-2 can produce high levels of IL-2 upon restimulation. Experiments to test this hypothesis on young and aged effectors are planned. When IL-2 remains low during effector generation in cultures of aged CD4 T cells, only a fraction of the potential effector generation is realized. It is not clear if this partially activated population can later be recruited into the responding population, as a lack of IL-2 during effector cell generation has been reported to lead to anergy in some models 39 40 . The presence of a significant population of nonresponding or poorly responding T cells in aged individuals is well documented 12 19 41 42 43 44 , and these cells have been shown to display a phenotype similar to the aged no cytokine effectors generated in our model. Furthermore, in IL-2 −/− , IL-2Rα −/− , and IL-2β −/− mice 45 46 47 , many CD4 T cells express an Ag-experienced phenotype but appear to be nonresponsive to TCR stimulation. There is also impaired in vivo peripheral deletion of these Ag-experienced cells in both models. This has lead to the hypothesis that in the absence of IL-2 induced signals, stimulation of T cells fails to activate the death pathway, which leads to the accumulation of Ag-experienced anergic cells in the periphery, and we suggest that this is what may be occurring in aged individuals. During an in vivo response, other cytokines that signal through the γc chain may be present and could possibly replace IL-2 and restore function of aged naive cells. The experiments shown in Fig. 3 demonstrate that other cytokines that signal through the γc chain (IL-15, IL-4, and IL-7) can indeed increase the proliferative capacity of naive aged Tg + CD4 cells. It seems likely that these effects are via an IL-2–independent mechanism that does not involve increased IL-2 production or upregulation of IL-2Rα expression. Most interesting, from a therapeutic viewpoint, is the effect of IL-15. IL-15 was able to support not only enhanced expansion of aged CD4 T cells without IL-2Rα expression but also development of Th1 polarized effectors. However, although IL-2 addition allows aged CD4 T cells to develop into polarized effectors under the influence of polarizing cytokines, IL-15 is unable to support optimum development of Th2 effectors. These data support the hypothesis that IL-2, acting through the high affinity IL-2R, plays a unique role in Th2 effector generation and that lower IL-2 production by aged CD4 T cells results in a defect in both expansion of responding naive cells and efficient generation of Th2-polarized effectors. Recent reports have shown that adjuvants such as LPS, poly I:C, and nonvertebrate DNA can induce type 1 IFN, which then induces APCs to secrete IL-15 48 49 . If these adjuvants can induce adequate IL-15 levels in vivo, they may be able to stimulate enhanced proliferation of activated T cells in aged individuals, which could lead to the development of more efficacious vaccines. Even though aged effectors generated in the presence of IL-15 in vitro are not as differentiated as those generated in the presence of IL-2, the increased expansion of these aged “IL-15 effectors” may boost the in vivo aged immune response sufficiently to gain an advantage over an invading pathogen. However, based on our results, one might expect defects in generation of Th2 polarized effectors and in response to pathogens cleared by Th2-dependent mechanisms. Experiments are currently underway in our laboratory to examine the in vivo potential of this approach. In conclusion, it is clear that the decline in IL-2 production by naive CD4 T cells that occurs with aging has a dramatic negative impact in the generation of T cell effectors in vitro. Assuming that such a decrease also occurs in vivo, this would lead to decreased and ineffective responses, thus helping to explain the increased incidence of mortality and infection with aging. In fact, preliminary studies suggest that aged naive CD4 T cells also respond poorly in vivo (Haynes, L., and S.L. Swain, unpublished data). Moreover, it is conceivable that any cell that partially undergoes a limited response and receives subthreshold signals from IL-2 may become anergic, and these anergic cells (which do not undergo apoptosis) could potentially accumulate with aging and fill the peripheral immune system with nonresponsive lymphocytes. However, the ability of exogenous IL-2 and other γc-binding cytokines to reverse the proliferative defect of aged CD4 cells in vitro raises the possibility that adjuvants that work to induce inflammatory cytokines (IL-15 or other γc reacting cytokines) might boost the primary response and restore effector generation in the elderly. | Study | biomedical | en | 0.999998 |
10510092 | Mouse B and T lymphoma cell lines A20 and L5178Y (mock and FasL transfectant) and the human retroviral packaging cell line phoenix-ampho (available at http://www.uib. no/mbi/nolan/NL-phoenix.html) were grown as described 11 12 . A20 is derived from a spontaneous reticulum cell neoplasm found in an old BALB/c mouse 11 . Sex- and age-matched (4–6 wk old) inbred BALB/c, (BALB/c × C57BL/6) F 1 , C57BL/6, and C.B-17SCID mice were obtained from Charles River Labs. Mice were maintained in the animal facility at the University of Stockholm. KSHV-FLIP was amplified by PCR from the BCBL-1 cell line, established from a BCBL 13 using the oligonucleotides K13EcoU 5′-ACTGGAATTCATGGCCACTTACGAGGTTCTCTG-3′ and K13BamL 5′-CATGGGATCCCTATGGTGTATGGCGATAGTGTTG-3′. The fragment was inserted into the EcoRI and BamHI sites of the retroviral expression vector pLXSN 14 and then used to transiently transfect the phoenix-ampho packaging cell line. Supernatants containing recombinant viral particles were used for retroviral transduction of A20 cells. Stable G418-resistant clones were obtained by limiting dilution. Mock and KSHV-FLIP–expressing clones were identified by RT-PCR, and the presence of helper virus was excluded by PCR amplification of viral env using the primers 5′-ACCTGGAGAGTCACCAACC-3′ and 5′-TACTTTGGAGAGGTCGTAGC-3′. Sensitivity of mock and KSHV-FLIP clones to Fas-mediated apoptosis was assessed by treating 10 6 cells with 40 ng/ml of the anti–mouse Fas mAb Jo2 (PharMingen) for 24 h at 37°C. Alternatively, the human retroviral packaging cell line phoenix-ampho was transfected with the FasL-hCD8α/pSG5 vector encoding the soluble mouse FasL, and 5 × 10 5 A20 cells were cultured with 1:16 diluted soluble FasL supernatant or 1:2 diluted supernatant from nontransfected φA cells in a total volume of 1 ml for 24 h at 37°C. Apoptosis was also induced with membrane-bound FasL by mixing mock- or mouse FasL-transfected L5178Y cells with 2 × 10 5 A20 cells at an E/T ratio of 1:4 for 24 h at 37°C. Cells were then stained with propidium iodide and annexin-V-fluos (Boehringer Mannheim) according to the manufacturer's instructions, and apoptosis was monitored by flow cytometry analysis. The mock- and KSHV-FLIP–transduced A20 cell lines and clones were treated with 200 ng/ml of the Fas antibody in a limiting dilution assay for 12 d at 37°C, and the frequency of clonal growth was determined by visual inspection on day 12 and calculated as described 15 . The expression of Fas on the mock- and KSHV-FLIP–transduced A20 clones was evaluated by incubating 10 6 cells with 1:100 PE-conjugated hamster anti–mouse Fas (Jo2) or 1:100 of an isotype-matched control in the presence of 1:100 anti–mouse CD16/CD32 (Fc-block). Dead cells and debris were excluded from the analysis by gating in forward and side scatter. 6 × 10 6 cells of mock and KSHV-FLIP clones (c)11 and 17 were subjected to 40 ng/ml of the anti–mouse Fas mAb Jo2 for 20, 40, 60, or 120 min at 37°C. Cells were then washed twice in PBS and immediately frozen in liquid nitrogen and stored at −80°C. DEVD- (caspase-3), IETD- (caspase-8), and LEHD-AMC (amino-methyl-coumarin; caspase-9) cleavage was measured using a protocol adopted from Nicholson et al. 16 . DEVD- and IETD-AMC were obtained from the Peptide Institute, Inc., and LEHD-AMC was purchased from Enzyme Systems Products. Cell lysates (10 6 ) and 50 μM substrate were mixed in a reaction buffer (100 mM Hepes for DEVD-AMC and IETD-AMC or 100 mM 2-( N -morpholino)-ethanesulfonic acid for LEHD-AMC, 10% sucrose, 0.1% 3-[(3-cholamidopropyl) dimethylammonio] propane-1-sulphonic acid [CHAPS], 5 mM dithiothreitol, and 10 −6 % NP-40, pH 7.2 for DEVD- and IETD-AMC and pH 6.8 for LEHD-AMC) and dispensed in duplicate in a microtiter plate. The cleavage of the fluorogenic peptide substrates was monitored by AMC liberation in a Fluoroscan II plate reader (Labsystems) at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Fluorescence was measured every 70 s for 30 min. The fluorescence units were converted to picomoles of AMC using a standard curve obtained with free AMC, and the data was analyzed by linear regression. Groups of four to eight BALB/c or (BALB/c × C57BL/6) F 1 mice were injected subcutaneously in the interscapular region with 2.5 × 10 5 , 10 6 , or 4 × 10 6 mock- or KSHV-FLIP–transduced A20 cells. Three groups of four C57BL/6 mice were injected subcutaneously in the interscapular region with 4 × 10 6 mock or KSHV-FLIP clones 11 and 17. Two groups of six C.B-17SCID mice were injected subcutaneously in the interscapular region with 10 6 mock- or KSHV-FLIP–transduced A20 cells (KSHV-FLIPc11). Tumor growth was monitored every second day for 110 d for BALB/c, (BALB/c × C57BL/6) F 1 , and C57BL/6 and daily for 25 d for C.B-17SCID mice. Tumors were measured using a caliper, and the square root of each tumor area was calculated. Mice were killed with CO 2 when the tumors had reached the maximal allowed size of ∼1 cm, as decided by the Stockholm Ethical Committee for Animal Experiments, or when the experiment was terminated. Tumor samples were obtained by surgical excision and frozen at −70°C or fixed in a 4% solution of paraformaldehyde and then processed to paraffin blocks. All material was cut into 6-μm-thick sections. The frozen material was mounted on Super Frost microscope slides (Menzel-Glaser), fixed in cold acetone for 10 min, and stored at −20°C until use for immunostaining. Paraffin sections were mounted on glass slides and used for morphological evaluation after hematoxylin and eosin staining or after immunohistochemistry. Frozen sections were immunostained for mouse antigens using rat primary antibodies specific for B cell (ID3, B220), cytotoxic T cell (Lyt 2), and macrophage markers (Mac 3), and an avidin–biotin complexes–peroxidase detection system was used according to the manufacturer's specifications (Vector Labs., Inc.). Bound antibodies were developed with 1 mg/ml diaminobenzidine (Sigma Chemical Co.) followed by counterstaining the slides with Meyer hematoxylin, and they were evaluated by light microscope. The percentage of apoptosis in the tumor samples was determined by TdT-mediated dUTP–biotin nick-end labeling (TUNEL). The TUNEL reaction was carried out by using an in situ cell death detection kit (Boehringer Mannheim) according to the manufacturer's indications, followed by Meyer hematoxylin counterstaining. To assess the antiapoptotic property of KSHV-FLIP in vitro , KSHV-FLIP was cloned into the retroviral expression vector pLXSN, followed by transduction of a Fas-sensitive subclone of the B lymphoma cell line A20. Two clones (KSHV-FLIPc11 and -c17) and a mock clone were chosen for further studies and tested for sensitivity to apoptosis induced by the agonistic anti-Fas mAb Jo2 or by soluble or membrane-bound Fas ligand. KSHV-FLIP conferred almost complete protection against Fas-mediated apoptosis, whereas the mock clone was as sensitive as the wild-type A20 cell line . The expression level of the Fas receptor and the proliferation rate (data not shown) were similar between sensitive and resistant clones, indicating that the difference in Fas sensitivity is not due to differential expression of Fas and that KSHV-FLIP confers no growth advantage to the cells in vitro. Limiting dilution analysis showed that KSHV-FLIP–positive cells were able to form clones in the presence of anti-Fas mAb at a frequency between 1/11 and 1/37, whereas the corresponding frequencies for the mock-transduced cells were between 1/5,000 and 1/27,000 ( Table ). Previous reports have established that the FLIP proteins prevent death receptor–mediated apoptosis by impeding the activity of the upstream caspases 8 and 10 6 7 , resulting in a disruption of the signal through the caspase cascade and, consequently, a suppression of the activity of the downstream caspases. The mechanism of action of KSHV-FLIP in the KSHV-FLIP clones 11 and 17 was therefore investigated by monitoring IETD-like activity (caspase-8), LEHD-like activity (caspase-9), and DEVD-like activity (caspase-3) in vitro in a continuous fluorometric assay. The mock and KSHV-FLIP clones 11 and 17 were challenged with 40 ng/ml of the mouse anti–Fas mAb Jo2 and collected at different time points for measurement of AMC release . The activity of caspase-8 was almost completely blocked in the presence of KSHV-FLIP . Similar results were obtained measuring caspase-9– and caspase-3–like activities . Some caspase-3–like activity was, however, observed in the KSHV-FLIP clones but was significantly lower and delayed in time . We have thus shown that KSHV-FLIP can act as an antiapoptotic protein in vitro by interfering with caspase activity, as anticipated from previous reports on other members of the FLIP family 6 7 . Furthermore, KSHV-FLIP allowed clonal cell growth in the continuous presence of death stimuli. As KSHV-FLIP presumably inhibits apoptosis through interaction with death effector domain–containing proteins, it is likely that it can interfere with death signals from other death receptors as well. We wanted to test the role of KSHV-FLIP in tumor progression in vivo. Therefore, the mock and KSHV-FLIP A20 clones 11 and 17 were injected subcutaneously into two recipient mouse strains, BALB/c (syngeneic) and (BALB/c × C57BL/6) F 1 (semiallogeneic), and tumor growth was monitored for 110 d. The semiallogeneic system was chosen to assess whether expression of KSHV-FLIP would be involved in the so-called hybrid resistance to parental tumors, possibly comprising NK cells 17 . The results are shown in Table and summarized in Fig. 4 . The frequency of tumor appearance in BALB/c recipient mice was dramatically higher for KSHV-FLIP– than for mock-transduced cells . A similar pattern was seen in the semiallogeneic mice . In addition, tumor appearance was delayed in mice injected with mock-transduced cells compared with mice injected with KSHV-FLIP–transduced clones. This holds true both in the syngeneic and semiallogeneic systems . Compared with the mock tumors, the KSHV-FLIP tumors reached the maximally allowed size of 1 cm with a higher frequency in both recipient strains (67 and 50% for c11 and c17 versus 11% for mock in BALB/c; 81 and 60% for c17 and c11 versus 11% for mock in [BALB/c × C57BL/6] F 1 ). Furthermore, the KSHV-FLIP tumors grew faster: ∼45 d after injection, most KSHV-FLIP tumors had progressed to a size of 1 cm and mice had to be killed. In contrast, the few progressive mock tumors attained a similar size first after 70–90 d after injection, as shown in Fig. 4C and Fig. D . These data strongly suggest that KSHV-FLIP promotes tumor establishment and progression in vivo and significantly increases uncontrolled growth of the tumor. The tumor-promoting property of KSHV-FLIP was, however, not sufficient to allow tumor establishment upon injection of 4 × 10 6 cells in a completely allogeneic tumor–host system. In contrast to the tumor cells transduced with KSHV-FLIP, most mock cells were rejected shortly after appearance in both mice strains. This prompted us to determine the nature of the immune response that is triggered and accounts for the elimination of the A20 tumor cells, against which KSHV-FLIP confers protection. Tumor samples were therefore stained for cell markers of B and T cells and macrophages. Histologically, the tumors appeared to be homogeneous growing tumors of large, undifferentiated lymphoid cells staining positively for B cell markers, confirming their origin as A20 cells. 5–30% of the cells within the tumors showed typical markers for macrophages. In tumors resulting from injection of KSHV-FLIP–transduced cells, <5% CTLs were observed, whereas the CTL infiltration in the mock tumors was more evident (5–30%). The percentage of apoptotic cells by TUNEL was higher in mock-transduced (2%) than in KSHV-FLIP tumors (0.5%) (data not shown). To investigate the role of conventional T cells and NK-like cells in tumor clearance, KSHV-FLIPc11 and mock were injected into BALB/c congenic C.B-17SCID mice lacking both the B and T cell compartments due to a recombination deficiency. KSHV-FLIP– as well as mock-transduced cells developed tumors with similar kinetics and size in all mice injected . As B cells are normally not involved in the rejection of solid tumors 18 , we conclude that conventional T cells, rather than NK-like cells, are necessary for the rejection of A20 tumor cells. The results suggest that T cells may have a role in immunosurveillance of death receptor–positive tumors. However, the observation that the frequency of tumor growth was somewhat lower in the semiallogeneic situation suggests that NK-like cells may participate in the rejection of tumor cells lacking KSHV-FLIP. Our work formally demonstrates that KSHV-FLIP can function as a tumor progression factor by blocking signaling through death receptors and thereby protecting the tumors against rejection mediated by conventional T cells. Mechanistically, this inhibition of apoptosis is shown by our data to be mediated through direct prevention of caspase-8 activation and subsequent inhibition of caspases 3 and 9. The kinetics of caspase-8 induction is very rapid, and caspase-3 activation is delayed as expected. Interestingly, caspase-9 activity is appearing as quickly as caspase-8 activity. This finding may suggest an involvement of the mitochondrial pathway in Fas-mediated apoptosis induced in A20 cells. In fact, it has previously been described that Fas-induced apoptosis through caspase-8 can be enhanced by release of cytochrome c and the induction of the caspase-9 pathway 19 . Therefore, the classification of cells into types I and II based on the apoptotic pathway (via the death-inducing signaling complex or mitochondria) preferentially used by cells undergoing Fas-induced cell death 20 cannot be applied to the A20 B lymphoma cells used in this study. The tumor-progressive activity of KSHV-FLIP not only provides insight into the role of this protein in pathogenesis of KSHV infection but also defines the family of inhibitors of death receptor signaling as a new class of tumor progression factors. To date, this family includes viral and cellular FLIPs and is likely to grow. It is expected from our data that dysregulated expression of inhibitors of death receptor signaling, such as the cellular FLIPs, will also be involved in tumor formation and progression in humans. Interestingly, the long form of cellular FLIP (cFLIP L ) is reported to be upregulated in human metastatic melanoma tumors 7 , compatible with a role for FLIP proteins in nonviral tumorigenesis. In addition, the apoptosis inhibitor survivin, which is structurally similar to the baculovirus inhibitor of apoptosis protein, has been shown to be highly expressed in cells of several common human cancers such as lung, colon, pancreas, prostate, and breast cancer and in ∼50% of high-grade non-Hodgkin's lymphomas 21 . The newly discovered TRAIL (TNFR-related apoptosis-inducing ligand) receptors 1 and 2 (TRAILR-1/DR4 and TRAILR-2/DR5) have been reported to play an important role in the clearance of tumors 22 . As the FLIPs have been reported to inhibit TRAIL signaling 6 7 , this sensitivity to TRAIL-induced apoptosis can be due to the fact that tumor cells express lower levels of FLIP, as observed in human melanoma cells 23 . This would predict that tumors expressing high levels of FLIP would be particularly aggressive. In fact, the inhibition of death receptor signaling by loss of Fas receptor expression on certain tumors can enable and enhance metastatic progression 24 . Further knowledge of the role of inhibitors of death receptor signaling in tumorigenesis is needed to comprehend the complex process that leads to malignancy so as to develop efficient diagnostic and therapeutic strategies. | Study | biomedical | en | 0.999996 |
10510093 | C57BL/6 perforin −/− (PKO, H-2 b ; reference 20; a gift from Dr. M. van den Broek) and C57BL/6 Kh (B6, H-2 b ) mice were bred at TNO-PG (Leiden, The Netherlands). C57BL/6 nu/nu (B6 nude, H-2 b ) mice were obtained from Bomholtgard (Ry, Denmark). Adenovirus type 5 E1A plus mutant EJ- ras –transfected (AR6; reference 19) and MBL2-Fas (MF; reference 20) (a gift from Dr. M. van den Broek, Zürich, Switzerland) were transfected by electroporation (240 V, 960 μF) with a plasmid encoding murine FLAG-tagged cFLIP under the control of a CMV promoter (cDNA provided by Dr. J. Tschopp, Lausanne, Switzerland) or a control plasmid. Sam-D b is a mouse embryo fibroblast transfected with a plasmid encoding the E1A epitope coupled to a signal sequence and murine H-2D b and therefore presents high levels of the Ad5E1A epitope in the context of H-2D b . CD95L-expressing C57BL/6 lpr/lpr mouse embryo cells (MECs) were obtained by Ca 3 (PO 4 ) 2 transfection with a plasmid encoding murine CD95L. Clone 1 was obtained by immunizing mice with RMA and is specific for an epitope encoded by the FMR-MuLV gagLeader sequence (CCLCLTVFL). The E1A (line 5)– and gagLeader (clone 1)–specific CTLs were cultured as previously reported 16 19 . Detection of anti-CD95–triggered apoptosis in MF was performed using the Nicoletti assay as described 21 . Cell-mediated cytotoxicity was determined either by incubating Na 51 CrO 4 - or [ 3 H]thymidine-labeled target cells (1 h, 100 μCi and 4 h, 5 μCi, respectively) with CTL clones or CD95L-expressing MECs at different E/T ratios. Release of 51 Cr was determined by collecting the medium after 6 h. Maximum release was determined by incubation of the labeled cells with 1 M HCl. [ 3 H]thymidine retention was determined by harvesting the cells and counting in the presence of scintillation fluid. To degrade perforin, CTLs were preincubated for 2 h with 75 nM concanamycin A (CMA; Sigma Chemical Co.), which was present throughout the assay 22 . E1A-specific CTL response in vivo was determined by isolation of splenocytes and subsequent restimulation with Sam-D b . After 6 d, CTL activity toward Sam-D b was analyzed with 51 Cr release. Cell lysates and immunoprecipitates were generated as described 6 21 . For immunoprecipitations, the polyclonal rabbit anti-FLAG antibody (Zymed Labs., Inc.) was used. SDS-PAGE and Western blot analysis were performed using a standard protocol 6 16 21 with the anti–FLAG-M2 antibody (Eastman Kodak Co.). Flow cytometry was performed as previously described 23 using Jo2–FITC (PharMingen) and anti–mouse mAb against H-2D b and H-2K b . AR6 tumors were injected subcutaneously into 6-wk-old male mice at 2 × 10 7 cells per mouse. Mice were killed when tumors reached a size >1,000 mm 3 . MF tumors were injected intraperitoneally into 6-wk-old female mice and were followed by weighing the animals. Mice were killed at >15% weight gain and/or at clear signs of intraperitoneal tumor growth. The inability of cFLIP to prevent CTL killing in vitro makes it questionable whether increased cFLIP expression, as observed in human melanomas (references 10 and 11; Medema, J.P., and J. de Jong, unpublished observations), can protect tumor cells from CTL-mediated cytotoxicity in vivo and thereby provide a mechanism of tumor escape. To analyze this directly, we used the murine tumor line MF. MF is a CD95 transfectant of MBL2 20 , a Moloney murine leukemia virus–induced lymphoma that can be controlled in vivo by virus-specific CTLs 18 . Due to the high sensitivity of MF to CD95-induced apoptosis, it is well suited to testing the potential of cFLIP to modulate tumorigenicity. Transfectants of MF were generated either expressing high levels (MF-FLIP high ) or, as a control, very low levels (MF-FLIP low ) of FLAG-tagged cFLIP . Both lines express identical amounts of MHC class I and CD95 (data not shown), but only MF-FLIP low is sensitive to apoptosis induced by the murine CD95–specific antibody Jo2 in vitro . Overexpression of cFLIP does not affect tumor-specific CTL–induced cytotoxicity . However, when the perforin pathway of this CTL is blocked by preincubation with CMA, a substance that results in specific degradation of perforin 22 , the sensitivity of MF-FLIP high is completely lost, whereas MF-FLIP low killing is only slightly reduced . These data also indicate that for the MF tumor, cFLIP expression only affects CD95- and not perforin-dependent killing and that both pathways need to be inhibited for MF to escape from CTL-induced apoptosis in vitro. We subsequently set out to analyze the role of perforin- and CD95-dependent cytotoxicity in vivo. Injection of 10 2 MF-FLIP low cells into syngeneic PKO mice results in clearance of the tumor cells in the majority of these mice, whereas injection of the same dose of MF-FLIP high cells results in progressive tumor growth . To our knowledge, this outcome represents the first direct proof that overexpression of cFLIP enables tumor cells to escape CD95-dependent cytotoxicity not only in vitro but also in vivo. Next we tested whether cFLIP expression also allowed these tumor cells to escape destruction by the immune response in wild-type mice, in which not only the CD95- but also the perforin-dependent pathway is functional. Wild-type mice are far more resistant to MF-FLIP low . Injection of up to 10 6 of these cells does not efficiently induce tumors , whereas such doses give rise to progressively growing tumors in PKO mice (data not shown). This indicates that perforin-dependent killing is an essential aspect of the immune defense against this tumor, an observation that is in line with previous observations 20 and our in vitro data . Nevertheless, we found that injection of 10 6 MF-FLIP high cells leads to much higher tumor-take in wild-type mice than injection of the same number of MF-FLIP low cells . Importantly, MF-FLIP low and MF-FLIP high are equally efficient in inducing tumor growth in T cell–deficient nude mice . This indicates that overexpression of cFLIP enables MF to escape from T cell–dependent immunity in immunocompetent mice. Even in the presence of perforin-dependent cytotoxicity, which increases the resistance of mice to these tumors and is sufficient to obtain complete lysis in vitro, the T cell–dependent immune response in vivo is not adequately equipped to eradicate MF-FLIP high tumors. These data underscore the crucial role of CD95-induced apoptosis in tumor clearance by a physiological T cell response and indicate that blockade of the CD95 pathway can tip the balance in favor of the tumor cells. Due to the high levels of CD95 on MF, CTL activity against this line may be skewed toward this pathway. Therefore, we analyzed the effect of cFLIP on a tumor that expresses only modest levels of endogenous CD95. Tumor line AR6 has been generated by transfection of B6 MECs with the adenovirus type 5 E1A and mutant EJ- ras oncogenes. Subcutaneous injection of AR6 results in initial tumor growth, after which the tumor regresses due to an efficient CTL response directed against an epitope encoded by E1A 19 . AR6 expresses low but detectable levels of CD95 . We transfected AR6 with FLAG-tagged cFLIP (AR6–FLIP) or as control with vector alone (AR6–vector) . Two subclones were selected on the basis of comparable MHC class I expression, growth kinetics, and morphological features in vitro. AR6–vector is resistant to Jo2 (anti–murine CD95)–induced apoptosis, probably due to the low cytotoxic potential of this antibody (Medema, J.P., and J. de Jong, unpublished observation). However, in cocultures with CD95L-expressing MECs, apoptosis of AR6–vector is induced but AR6–FLIP is fully resistant . Even though CD95-induced apoptosis is completely blocked, we find that cFLIP does not affect lysis of AR6–FLIP by E1A-specific CTLs . Inhibition of the CD95 pathway during CTL-induced apoptosis by cFLIP is, however, suggested by the partial cleavage of FLAG-tagged cFLIP in AR6–FLIP , which has been shown to correlate with resistance 10 24 . When injected into nude mice, the AR6 lines display equal and uncontrolled tumor growth (data not shown). In contrast, both PKO and wild-type mice can control the AR6–vector tumor . However, comparable to our findings with MF-FLIP high , neither of these mouse strains is capable of rejecting the cFLIP-overexpressing tumor AR6–FLIP . Thus, escape from T cell–mediated immunity in vivo by cFLIP overexpression is not limited to tumors with very high CD95 surface expression. It is important to note that the AR6–FLIP tumor grows almost as efficiently in PKO and wild-type mice , which suggests that eradication of this tumor depends almost exclusively on CD95. This observation is not without precedent, as other murine tumor lines, such as the T cell lymphoma RMA and the melanoma B16, were shown to be equally tumorigenic in PKO and wild-type mice 20 . Apparently, clearance of certain tumors does not critically depend on perforin-based cytotoxicity but can be achieved by other mechanisms, which obviously include CD95-induced apoptosis. Importantly, this dependance is not determined by a high sensitivity to CD95-induced apoptosis, as is clear from the two tumor lines tested here. Further evaluation is required to determine the relative importance of perforin-dependent cytotoxicity among different tumor systems, but these data clearly indicate that CD95-mediated apoptosis constitutes a highly important mechanism for tumor clearance even in situations where the perforin pathway is operational. Recent findings have suggested that apoptosis of tumor cells may be required for efficient uptake and cross-presentation of tumor antigens by dendritic cells and therefore may be essential for priming of effective T cell immunity 25 . To test whether immune escape by AR6–FLIP may involve prevention of T cell priming rather than escape from the T cell response, we injected B6 mice simultaneously with AR6–FLIP and AR6–vector in either flank. As expected, most mice were again capable of rejecting the AR6–vector cells, pointing to the induction of an efficient immune response. Despite this response, AR6–FLIP tumors in the other flanks of these mice developed progressively, indicating that these cFLIP-expressing tumors are capable of growing out in the face of an effective antitumor response . To directly examine whether anti-tumor CTL immunity is induced in mice challenged with AR6–vector, AR6–FLIP, or both, we analyzed the CTL response against the E1A epitope in splenocytes from these mice. Importantly, comparable E1A-specific CTL immunity was detected in all mice . Also in the MF model, we obtained evidence that cFLIP overexpression enables escape from antitumor immunity rather than preventing the induction of this response. As shown in Fig. 1 C, a minority of the PKO and wild-type mice challenged with MF-FLIP low developed progressively growing tumors. Intriguingly, isolation of these tumors and analysis in vitro showed that the cells had acquired resistance to CD95-mediated apoptosis, which correlates well with the increased expression of cFLIP in these cells . Moreover, reinjection of such tumor cells revealed that they are as tumorigenic as MF-FLIP high (data not shown). As challenge of nude mice with MF-FLIP low does not result in tumors with elevated cFLIP expression (data not shown), this process requires the selective pressure of the T cell immune system. In conclusion, our data show that cFLIP-overexpressing tumors escape from T cell immunity in vivo despite the fact that they are efficiently killed in vitro. This apparent discrepancy is most likely due to limitations of in vitro assays, which do not accurately reflect the microenvironment in the tumor. For instance, in vitro CTL assays are generally performed at aphysiological E/T ratios and under conditions that allow prolonged CTL–target interactions that may give rise to a different killing potential of the CTL. Alternatively, the experimental generation of CTL clones often favors clones with high affinity, which does not necessarily reflect the response against a tumor in vivo. In addition, in in vitro assays target cells are often pretreated with IFN-γ to increase their MHC class I expression and thereby the avidity of the CTL–target interaction. Together, this may change the specificity of the response. Indeed, it has been suggested that decreasing the affinity/avidity of the CTL–target interaction shifts the balance of CTL-mediated cytotoxicity toward the CD95-dependent pathway 26 27 28 . Although the exact reason for this disparity remains to be determined, our data do provide direct evidence that tumor clearance in vivo critically depends on the CD95 pathway. In view of this finding, it is interesting to note that a plethora of mechanisms has been reported by which tumors seem to block CD95-induced apoptosis. For instance, mutation 29 or simply downmodulation of CD95 30 31 32 33 is found in several tumors. Alternatively, secretion of CD95 decoy receptors 34 could provide a separate route to escape from CD95-dependent cytotoxicity. Our findings demonstrate that blockade of the CD95 pathway, for instance through overexpression of cFLIP as was found in human melanomas 10 11 , can indeed serve as an efficient mechanism of immune escape by tumors. | Study | biomedical | en | 0.999996 |
10523602 | Ikaros DN +/− and null +/− mice of a mixed background (C57BL/6 × SV129) were bred against RAG-1 −/− (gift of E. Spanopoulou, Mount Sinai School of Medicine, New York, NY), TCR-α 2/− and TCR-β 2/− (The Jackson Laboratory), and F5 (gift of D. Kioussis, Medical Research Council, The National Institute for Medical Research, London, UK) mice of the C57BL/6 background. DN +/− and null +/− daughters who were heterozygous for the RAG, TCR-α, and TCR-β mutations were then backcrossed with their RAG −/− , TCR-α 2/− , and TCR-β 2/− fathers. Colonies were expanded by intercrossing of littermates. Cells from the thymus, spleen, lymph nodes, and bone marrow were prepared and analyzed for expression of surface differentiation antigens as described previously 8 9 . All antibodies using for stainings were from PharMingen. Flow cytometric analyses and cell sorting were performed using a FACscan™ (Becton Dickinson) and high speed MoFlo sorter (Cytomation, Inc.), respectively. Thymocytes were prepared as described previously 8 . Mice were 4–8 wk of age. Thymocytes from two to six mice were pooled before depletion. Each pool was stained with the following antibodies provided as hybridoma supernatants for complement-mediated depletion: anti-CD4 (GK1.5) and anti-CD8 (3.168.8). In some experiments, purification was performed as described elsewhere 10 . In brief, the lightest 30% of thymocytes was selected by a density cut procedure, and all adherent cells were removed by culture for 1 h at 37°C. Cells bearing CD3, CD4, CD8, CD2, CD25, B220, Mac-1, Gr-1, Ter119, and class II MHC were removed by depletion using Dynabeads (Dynal). In some experiments, remaining double negative cells were further purified by removal of CD4 + and CD8 + cells using the VarioMacs™ (Miltenyi Biotec) or sorted using the MoFlo™ sorter (Cytomation, Inc.). RNA and cDNA were prepared as described previously 11 12 . The relative concentration of cDNAs for each sample was determined with primers that would amplify cDNA of the housekeeping gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT). Adjusted amounts of cDNAs were amplified with primers derived from sequences corresponding to the first exon of the constant region of the TCR α chain gene 13 . [α- 32 P]ATP and [α- 32 P]CTP (0.5 μCi each) were included in each PCR reaction. Cycling conditions were: 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min for 35 cycles. The two sets of primers used were: HPRT 5′, TGG CCC TCT GTG TGC TCA AG; HPRT forward, CAC AGG ACT AGA ACA CCT GC; Cα1, CCC AGA ACC TGC TGT GTA C; and Cα2, TGA ACT GGG GTA GGT GGC. PCR products were electrophoresed through an 8% polyacrylamide gel, which was then dried and exposed to autoradiography film. Exposures shown here are for <12 h for HPRT, lanes 1–5, and TCR-α, lanes 3–5, and for 30 h for TCR-α, lanes 1 and 2. Cells were prestained with FITC-conjugated mAbs against CD25 (double negative cells) or TCR-γ/δ ( Ikaros DN +/− × TCR-β 2/− cells) (both from PharMingen), and then fixed in 95% ethanol. Fixed cells were resuspended in a staining solution consisting of 10 μg/ml propidium iodide and 250 μg/ml RNase A, and incubated at 37°C for 30 min. Flow cytometric analyses were performed on a FACscan™ (Becton Dickinson). Analysis was carried out as reported previously 14 . To delineate the role of Ikaros in T cell differentiation, we analyzed T cell precursor populations within the thymi of young Ikaros null mice, which have a polyclonal thymocyte repertoire. The earliest identifiable T cell precursors express low levels of the CD4 coreceptor and can give rise to T, B, and thymic dendritic cells 15 . The number of CD4 low precursors in the Ikaros null thymus was 4.7-fold less than that present in the wild-type thymus . This decrease in numbers was also observed through the next four stages of T cell development, identified in part by cell surface expression of CD44 and CD25 (together designated as the double negative stage). The maturational sequence of these precursors is (A) CD44 + CD25 − to (B) CD44 + CD25 + to (C) CD44 − CD25 + to (D) CD44 − CD25 − . Ikaros null thymi contain an average 3.9-fold fewer double negative thymocytes, 88% of which do not express CD25 , whereas in the wild-type thymus, only 30–40% of these cells are CD25 − . As expected, the decrease in absolute numbers of double negative precursors is most dramatic within the CD25 + population (stages B and C), which is 6% of wild-type. In contrast, the numbers of CD25 − stage D precursors are similar to those seen in the wild-type . A similar trend was observed when thymocyte precursors were analyzed in mice heterozygous for the Ikaros DN mutation in which, due to the expression of a dominant negative interfering Ikaros isoform, there is a severe reduction in Ikaros activity. Thymi from young Ikaros DN +/− mice (1–2 mo of age) with polyclonal thymocyte repertoires contain, on average, twofold fewer double negative thymocytes, the majority of which, as seen in the Ikaros null thymi, do not express CD25 . A decrease to 48% of wild-type numbers is observed in the number of CD25 + cells. Taken together, these studies show that reduction in levels of Ikaros activity leads to a decrease in numbers of the earliest thymic T cell precursors (stages A–C). However, this decrease is not observed in the most mature of these immature precursors (stage D). The developmental stage at which thymocytes receive a pre-TCR signal necessary for their proliferative expansion and further differentiation is the stage directly preceding stage D, which is composed of CD25 + thymocytes . In the Ikaros DN +/− thymus, there is an increase in the percentage of CD25 + cells in the S/G2/M phases of the cell cycle . Therefore, the less dramatic reduction in precursor numbers observed in stage D is possibly due to their accelerated maturation through stage C as a result of pre-TCR–mediated hyperresponsiveness to differentiation signals provided by the microenvironment, or to a greater proliferative expansion that occurs as cells transit from stage C to D. To investigate the role of Ikaros in the transition from a double negative to a double positive thymocyte, mice homozygous for an Ikaros null mutation were bred onto a genetic background with a mutation in RAG-1 ( RAG-1 −/− ). Thymocyte differentiation in RAG-1 −/− mice is unable to proceed beyond the double negative C stage (CD44 − CD25 + ) due to lack of TCR β chain rearrangement . Unexpectedly, in Ikaros null −/− × RAG-1 −/− thymi, thymocytes expressing CD4/CD8 and CD4 were present . By reverse transcriptase (RT)-PCR analysis, it can be shown that at least some of these thymocytes express TCR-α germline transcripts, providing further evidence that they have developmentally progressed . As wild-type thymocytes transit from double negative to the double positive stage, there is a dramatic proliferative expansion that results in a double positive to double negative ratio of ∼80:1. In the absence of Ikaros, this ratio is <3:1. Therefore, in the absence of Ikaros activity, T cell differentiation, as evidenced by expression of the CD4 and CD8 coreceptor(s), proceeds in the absence of pre-TCR signaling, but without the accompanying dramatic proliferative expansion. The observed decrease in cellularity of the Ikaros null −/− × RAG −/− thymi is likely due to the reduction in numbers of thymic progenitors in these mice and to the lack of proliferative expansion as double negative cells proceed to the double positive stage, as discussed above. Ikaros DN +/− × RAG-1 −/− thymi have, on average, 50% fewer thymocyte precursors than their RAG-1 −/− counterparts . The decrease in cellularity is most dramatic at the stage where pre-TCR is initially expressed (stage C, CD44 − CD25 + ; data not shown), as observed with the DN +/− double negative populations. In contrast to the phenotype observed in Ikaros null −/− × RAG-1 −/− thymocytes, in the majority of the cases, Ikaros DN +/− × RAG-1 −/− thymocytes do not progress past the double negative stage of differentiation . Therefore, the reduced levels of Ikaros present in these thymocyte precursors suffice to provide a barrier to differentiation. Ikaros DN +/− mice develop leukemias and lymphomas with 100% penetrance within 3 mo of life. Interestingly, when a cohort of DN +/− × RAG −/− mice was followed for 11 mo, no lymphomagenesis was observed . When a transgene expressing a class I–restricted TCR (F5; reference 18) was bred onto the DN +/− × RAG −/− background, thereby restoring T cell differentiation from the double through to the CD8 single positive stage , the transformation phenotype could be restored . Therefore, expression of a functional pre-TCR or TCR complex at subsequent stages of differentiation is necessary for Ikaros deficiency to have a destabilizing effect on T cell homeostasis. These studies implicate Ikaros as a critical regulator of the pre-TCR–mediated checkpoint in T cell differentiation known as β-selection. In the absence of Ikaros, signals received through engagement of a pre-TCR are no longer necessary for differentiation from a double negative to a double positive and CD4 single positive thymocyte. This progression occurs in the absence of proliferative expansion, thereby uncoupling two critical events in T cell differentiation. To delineate the role of Ikaros during the transition from the double positive to the single positive stage of thymocyte differentiation, the Ikaros null −/− and DN +/− mutations were bred onto a TCR-α 2/− genetic background. Thymocytes that lack expression of TCR-α arrest at the double positive stage due to their inability to transit through positive selection . However, in Ikaros null −/− × TCR-α 2/− thymi, cells are detected that have downregulated expression of CD4 or CD8 coreceptor . Therefore, these thymocytes have the phenotype of transitional-stage intermediates, a population that arises upon the combinatorial engagement of a TCR of the appropriate specificity and its coreceptor. However, these intermediate populations never fully mature into CD4 and CD8 single positive T cells and are not observed in the periphery. Thymi from young (up to 1 mo of age) Ikaros DN +/− × TCR-α 2/− mice are indistinguishable phenotypically from their TCR-α 2/− counterparts, indicating no obvious differentiation defects . However, shortly thereafter, abnormal thymic profiles are observed. In many animals, CD8 + cells are seen in the thymus, indicating inappropriate downregulation of the CD4 coreceptor . These CD8 + cells express the TCR β chain on their cell surface, suggesting that they belong to the TCR-α/β cell lineage (data not shown). Cells of this phenotype are also observed in the periphery where they undergo dramatic expansions resulting in splenomegaly and lymphadenopathy (data not shown). In fact, between 2 and 4 mo of age, Ikaros DN +/− × TCR-α 2/− mice succumb to leukemias and lymphomas with 100% penetrance. Disease development takes about half the time in these mice compared with their Ikaros DN +/− × TCR-α 1/− littermates, which do not have a block in T cell differentiation . Therefore, in the absence of all Ikaros activity, thymocytes can progress in differentiation to the transitional intermediate stage (CD4 lo CD8 hi and CD4 hi CD8 lo ) in the absence of TCR-mediated signals. Nevertheless, their further differentiation to the mature single positive stage is prohibited. On the other hand, when levels of Ikaros are severely reduced ( Ikaros DN +/− × TCR-α 2/− ), a block in T cell differentiation at the double positive, pre-TCR + stage promotes an even more rapid development of leukemias and lymphomas. We next examined the effects of the Ikaros mutation on differentiation of the γ/δ T cell lineage. Block in α/β T cell differentiation caused by lack of expression of the TCR β chain is observed at the late double negative precursor stage . However, this differentiation block does not affect differentiation of thymocyte precursors along the γ/δ T cell pathway 4 . Analysis of the role of Ikaros in TCR-γ/δ lineage differentiation and homeostasis was studied using the Ikaros DN +/− mutation, since only a very small subset of TCR-γ/δ lineages, those found in the mucosal epithelia, develops on the Ikaros null −/− genetic background. The thymi of Ikaros wild-type × TCR-β 2/− mice are hypocellular, and the majority of thymocytes are double negative, with the percentage of double positive thymocytes varying between animals. Mature TCR-γ/δ + thymocytes are CD4 − CD8 − , and therefore fall within the double negative population . Thymi from young (up to 1 mo of age) Ikaros DN +/− × TCR-β 2/− mice contain two- to threefold more cells due to an increase in the numbers of double positive, TCR-γ/δ 2 thymocytes . In the majority, these double positive cells do not express TCR-γ/δ in either Ikaros wild-type or Ikaros DN +/− × TCR-β 2/− thymi . Evidence exists that these double positive thymocytes may represent TCR-α/β lineage precursors that can develop due to the presence of functional TCR-γ and TCR-δ gene rearrangements 20 . They normally cannot expand and are arrested in development due to the inability to express a pre-TCR. Upon reduction in Ikaros activity, larger numbers of these cells may indicate increased commitment into the α/β lineage or an increased ability of these TCR-α/β lineage precursors to survive in the absence of normally required survival signals mediated by a pre-TCR. Alternatively, this expanded population may represent TCR-γ/δ lineage precursors not normally observed due to the vast overrepresentation of TCR-α/β lineage precursors in a wild-type thymus. However, this does not seem likely, since we do not observe an increase in the numbers of mature TCR-γ/δ + cells in these young animals. Thymi from older animals contain up to 200-fold more cells than their Ikaros wild-type × TCR-β 2/− counterparts, again due to a dramatic increase in the number of double positive cells . This increase in thymic cellularity in older Ikaros DN +/− × TCR-β 2/− mice led us to investigate whether these cells have become transformed. Therefore, the cell cycle profiles of thymocytes from TCR-β 2/− animals with normal and reduced (Ik DN +/− ) levels of Ikaros were examined. An average 400-fold increase in the number of cycling cells was observed in thymi of Ikaros DN +/− × TCR-β 2/− mice . The majority of cycling cells express TCR-γ/δ (data not shown), suggesting a deregulation of growth among the normally quiescent thymic γ/δ T cells. In addition, in many cases these TCR-γ/δ + cells also express CD8 , a cellular phenotype not observed in normally differentiating thymic TCR-γ/δ lineage cells. An increase in numbers of TCR-γ/δ cells, with the same phenotype as the cycling cells observed in the thymus, is also observed in the periphery of Ikaros DN +/− × TCR-β 2/− animals, as determined by analysis of spleen and lymph node cell populations (data not shown). These cell populations are mono- or oligoclonal as shown by a PCR analysis of V to J rearrangements at the TCR γ chain locus , suggesting the outgrowth of a malignant clone(s). This evidence clearly shows that γ/δ T cells also depend on Ikaros activity for homeostasis. Differentiation of a double positive thymocyte to a CD4 or CD8 single positive T cell depends on TCR specificity. CD4 and CD8 T cells arise from double positive cells with a TCR specificity for an MHC class II/self-antigen complex and an MHC class I/self-antigen complex, respectively. This differentiation process is known as positive selection, and thymocytes that are positively selected upregulate the CD69 activation marker as a consequence of productive ligation of the TCR complex 21 . Ikaros null −/− thymocyte profiles are heavily skewed towards CD4 single positive T cells and intermediates in transition to this phenotype 7 . Although all Ikaros null −/− single positive thymocytes express high levels of TCR-α/β, the majority do not express CD69, suggesting that they may undergo this differentiation step without being positively selected . Therefore, in the absence of Ikaros, differentiation through the checkpoint imposed by positive selection may be occurring in the absence of signals delivered upon engagement of an appropriate TCR–coreceptor complex. Alternatively, positive selection may be occurring at an accelerated rate due to lowered differentiation thresholds, as we have shown occurs during the process of β-selection in Ikaros-deficient thymocytes. To clarify the effects of Ikaros on the double to single positive transition as regulated by the process of positive selection, we introduced a transgene expressing a class I MHC–restricted TCR (F5) onto the Ikaros null −/− × RAG-1 −/− background. The F5 TCR is expressed on all thymocytes and, in Ikaros wild-type mice, results in the exclusive positive selection of CD8 T cells . However, Ikaros null −/− , F5 × RAG-1 −/− thymocytes differentiate into both CD8 and CD4 T cells, showing approximately equal percentages of each population . However, these CD4 T cells do not appear in the periphery, indicating that they have not undergone final selection steps required for their exportation. These studies delineate the role of Ikaros in the development of the α/β and γ/δ T cell lineages. Ikaros proteins set thresholds that must be overcome by signaling through the pre-TCR and TCR complexes for T cell differentiation to occur. In the absence of Ikaros activity, these thresholds are reset to a lower level, allowing progression from the double negative through the double positive to the single positive stage to occur without the appropriate pre-TCR and TCR signaling. Thus, it appears that the existence of distinct TCR signaling thresholds that control the outcome of T cell differentiation pathways relies on the Ikaros family of nuclear factors. Thymocytes that do not express a pre-TCR complex (i.e., RAG-1 −/− ) arrest their development at an early precursor stage (double negative, CD44 − CD25 + ). Pre-TCR signaling is required for progression to the next stage of differentiation where both CD4 and CD8 coreceptors are expressed (double positive stage; reference 23). It is also required for the proliferative expansion of thymocytes during this transition. However, in the absence of Ikaros activity, thymocyte precursors can transit from the double negative to the double and CD4 single positive stages without the signaling events provided by pre-TCR and TCR engagement. However, this differentiation event occurs without the normally occurring proliferative expansion, resulting in a highly hypocellular thymus. Thus, lack of Ikaros activity in thymocyte precursors uncouples the process of differentiation from that of proliferation, allowing the one to occur in the absence of the other. Interestingly, the preferential “development” of CD4 lineage cells observed in Ikaros null −/− × RAG −/− thymi correlates with the phenotype previously reported for Ikaros null −/− thymi without the RAG mutation 7 . Therefore, we hypothesize that Ikaros plays a central role in establishing thresholds for differentiation that give rise first to double positive, and subsequently to CD4 single positive thymocytes. In support of this hypothesis, we have shown that within the double negative thymocyte population, reduction of Ikaros activity also causes an apparent increase in the rate of differentiation to the double positive stage, possibly by lowering thresholds of pre-TCR signaling. In Ikaros-deficient mice, there is a decrease in both the absolute and relative numbers of late double negative precursors (CD25 + , stage C), a greater percentage of which are in cell cycle. However, normal numbers of stage D precursors are observed. Therefore, precursors may be transitioning through the CD25 + stage more rapidly, leading to the decrease in the size of this precursor compartment and, in addition, undergoing a more dramatic proliferative expansion such that normal numbers of stage D precursors are generated. In mature T cells, Ikaros is a negative regulator of TCR-mediated proliferative responses 24 . Progressive reduction of Ikaros activity in peripheral T cells results in a progressive increase in the TCR-mediated proliferative response. In addition, reduction of Ikaros activity allows T cells to proliferate in response to lower levels of signaling that normally do not support proliferation. In a similar fashion, Ikaros may provide negative regulation for pre-TCR–mediated differentiation. In the absence of Ikaros, differentiation can proceed in the absence of pre-TCR, and in the presence of pre-TCR may proceed at a faster rate. Double positive thymocytes expressing TCR undergo a process known as positive selection as they differentiate to the CD4 and CD8 single positive stages. Using a TCR transgenic model system, we have shown that, in the absence of Ikaros, CD4 + TCR + thymocytes are produced without the signals provided by a class II MHC–restricted TCR. Ikaros null −/− mice with a polyclonal thymocyte repertoire also show a significant increase in the relative percentage of CD4 single positive thymocytes. The majority of single positive cells in the Ikaros null −/− thymus do not express the CD69 activation marker, an event indicative of positive selection. Therefore, transition from the double to the CD4 single positive stage is occurring in the absence of selection, possibly as a result of lowered signaling thresholds. Alternatively, lower thresholds of signaling at the double positive TCR + stage may allow for faster transition through the CD69 + stage. The CD4 + T cells that develop in the Ikaros null −/− , F5 × RAG-1 −/− thymus are not found in the periphery, indicating that the final step in the selection process is not taking place and, therefore, that Ikaros activity is not required for regulating this final step of T cell maturation which occurs in the thymus. We have shown that thymocytes express the highest levels of Ikaros 11 , and it is within this population that transformation occurs with 100% penetrance when these levels are reduced 7 9 . However, Ikaros-deficient thymocytes that lack expression of a receptor linked to TCR signaling pathways are refractory to transformation. Therefore, when levels of Ikaros activity are reduced, expression of a pre-TCR, TCR-α/β, or TCR-γ/δ is necessary to destabilize homeostasis and trigger proliferative events that eventually lead to malignancy. We have recently shown that when primary Ikaros-deficient T cells proliferate in response to engagement of the TCR complex, they consistently show chromosome instability 24 . Perhaps this same instability arises after receptor engagement within the thymocyte population. Supporting this hypothesis, we have observed that when Ikaros-deficient thymocytes are arrested in differentiation at a stage that is targeted for dramatic proliferative expansion (pre-TCR + stage), malignancies arise with increased kinetics. Expression of an active recombinase machinery in immature thymocytes may greatly contribute to the chromosomal aberrations mediated by decreased levels of Ikaros activity during their proliferation. In conclusion, we have provided evidence that Ikaros activity is required at several distinct steps of thymocyte differentiation. One function of Ikaros is to regulate the transit through pre-TCR and TCR-mediated differentiation checkpoints by providing signaling thresholds, while the second function is to control proliferation in response to pre-TCR and TCR engagement on immature thymocytes. We have strong evidence that Ikaros is a critical nuclear effector of multiple signaling pathways downstream of TCR complex engagement in mature peripheral T cells (i.e., Src protein tyrosine kinases, mitogen-activated protein kinase, calcineurin ). Many of these same pathways are used within the thymocyte population to regulate proliferation and differentiation 23 25 26 . How Ikaros activity is modulated by these signaling pathways to effect its function will be the focus of future research. | Study | biomedical | en | 0.999997 |
10523603 | In this study, we used the recombinant form of IgA1 protease expressed in Escherichia coli strain H2053, which harbors plasmid pIP11 containing the iga gene of N. gonorrhoeae MS11 with the temperature-inducible bacteriophage λ promoter P L , as described previously 18 . In brief, the supernatant of a 4-liter stationary phase culture was recovered by centrifugation, supplemented with 0.1 M EDTA, and diluted 1:1 (vol/vol) with potassium phosphate buffer (20 mM, pH 7.0). The bacterial supernatant was applied to a cation exchange column. After washing, bound protein was eluted with 500 mM potassium phosphate, 8.6% glycerol. Fractions of 10 ml were collected, precipitated with ammonium sulphate, and then redissolved in gel filtration buffer. Gel filtration was performed with a preequilibrated Superdex column (Amersham Pharmacia Biotech). Protein-containing eluates were recognized by UV absorption, pooled, and precipitated with ammonium sulphate. Finally, the pellet was suspended in PBS and dialyzed against PBS, pH 7.4, and stored in aliquots at −70°C. Purity of the protein was assessed by SDS-PAGE with subsequent Coomassie staining showing the characteristic protein profile with the typical 109- and 106-kD protein bands as described previously 18 . The protein concentration was determined using a commercial protein determination assay (Bio-Rad). Specificity for IgA1 protease was confirmed by immunoblotting using an IgA1 protease–specific rabbit antiserum 13 . The cleavage activity of the recombinant IgA1 protease was determined with human IgA1 (20 ng/μl; Dakopatts) in Tris buffer (0.05 M Tris-HCl, 10 mM CaCl 2 , and 10 mM MgCl 2 , pH 7.5) at 37°C according to the method described previously 36 . Heat denaturation (boiling for 10 min) completely destroyed the enzymatic activity. The enzymatic activity could also be blocked by addition of the specific peptide prolyl boronic acid inhibitor 37 . 100 μM of the inhibitor (provided by A.G. Plaut, New England Medical Center, Boston, MA) abrogated cleavage of IgA at concentrations up to 10 ng per μl of IgA1 protease (data not shown). The endotoxin contents of our IgA1 protease preparations were determined by Limulus amebocyte lysate assay (LAL; Haemochrom Diagnostica). All preparations contained <2 pg of endotoxin per μg protein. It is described that some proteins could interfere with the Limulus assay 38 . To ensure that IgA1 protease is free of LPS, we have tested in addition the priming potency of IgA1 protease for neutrophils, which are as sensitive as the Limulus assay in detecting LPS. Different types of LPS can prime neutrophils to increase their oxidative response after stimulation with the agonist fMLP (Sigma Chemical Co.) 38 . PMNs preincubated with 10 μg/ml IgA1 protease for 30 min stimulated with 1 μM fMLP showed no alteration of the oxidative response compared with the control when measured as luminol-dependent chemiluminescence in a six-channel Biolumat LB 9505 (Berthold; data not shown). PBMCs were isolated from buffy coats or freshly citrated blood of adult, healthy volunteers by density gradient centrifugation over Ficoll/Isopaque (Amersham Pharmacia Biotech) and washed twice in Ca 2 , Mg 2+ –free PBS. For some experiments, lymphocytes and monocytes were separated (6 × 10 6 cells/ml in PBS plus 0.5% BSA) by counterflow centrifugation (elutriation) using the Beckman J6ML/JE-5.0 centrifuge system. Elutriation was performed at constant rotor speed with increasing flow rates, and lymphocytes were collected. To obtain purified T cells, the lymphocyte suspensions were positively sorted by anti-CD3 magnetic beads (MACS; Miltenyi Biotec). Purity of CD3 + T cells was always >95% as determined by flow cytometry. Monocytes were prepared from PBMCs either by adherence to plastic dishes (1 h, 37°C) or by negative selection using the MACS monocyte isolation kit (Miltenyi Biotec). The purity of CD14 + cells was always >85% as measured by flow cytometry using a PE-conjugated anti-CD14 antibody (Leu M3; Becton Dickinson). The remaining 15% consisted of CD3 − , CD19 − , and CD56 − flow cytometric counting events. The monocytic cell line MonoMac 6 (no. 124; American Type Culture Collection) was obtained from the German Collection of Microorganism and Cell Cultures (Braunschweig, Germany) and maintained in 24-well plates under low endotoxin conditions (<10 pg/ml) in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, and 9 μg/ml bovine insulin as described 39 . The cell line normally expresses CD14 as controlled by flow cytometry. Cytotoxic effects of IgA1 protease preparations were excluded by staining of PBMCs with propidium iodide (10 μg/ml) and subsequent evaluation by flow cytometry. Cell viability of freshly isolated or cultured cells (24 h with 10 μg/ml IgA1 protease) was >95% in all experiments. All cell cultures were performed in RPMI 1640 supplemented with 20 nM l -glutamine, 25 mM Hepes (GIBCO BRL), 500 U/ml penicillin, 500 μg/ml streptomycin, and 5% heat-inactivated human AB serum (pooled male serum, <<1 ng/ml endotoxin; Sigma Chemical Co.). This medium is further designated as culture medium. A total of 10 5 cells was cultured in 200 μl culture medium in round-bottomed 96-well plates at 37°C, 5% CO 2 . MonoMac 6 cells (10 5 cells/well) were incubated for 24 h in 96-well flat-bottomed plates with culture medium. Cell-free supernatants were harvested after 18 h (150 μl/well, three wells per independent experiment), pooled, and frozen in 50 μl aliquots at −20°C until assayed. To investigate the effect of CD3 + T lymphocytes on the IgA1 protease–induced cytokine production of CD14 + monocytes, 2 × 10 4 CD14 + cells were cultured either with or without CD3 + cells keeping the monocyte/lymphocyte ratio constantly at 1:4. A total of 10 5 PBMCs were cultured as a control with a monocyte content ranging from 10 to 20%. The levels of the released cytokines TNF-α, IL-1β, IL-6, IL-8 (R&D Systems), and IFN-γ, GM-CSF, and IL-10 (PharMingen) in the culture supernatants were quantified with commercially available sandwich ELISA kits. The assays were performed as recommended by the manufacturer. In brief, polystyrene microtiter plates were coated with the catching mAb overnight, washed intensively (washing buffer: PBS, 0.02% Tween 20), and blocked with PBS, 5% AB serum. Different dilutions of all samples and the standard cytokine dilutions were assayed in duplicate or triplicate, respectively, with a total volume of 100 μl for 2 h at room temperature. After removing the unbound detecting mAb by washing (10 times), streptavidin-peroxidase solution was added to each well and incubated for 20 min. After 10 washes, the chromogenic substrate (TMB substrate with H 2 O 2 ; Kirkegaard & Perry) was added to each well and color development was stopped after 20 min by adding 50 μl 1 M H 3 PO 4 . The absorbance of the samples was measured at 450 nm. The cytokine concentrations were determined by extrapolating from the standard curve. The results are expressed as means of triplicates in ng/ml. The detection limits of the ELISA kits were as follows: TNF-α, IL-1β, IL-10, GM-CSF, and IFN-γ ≤ 0.01 ng/ml; IL-6 and IL-8, ≤ 0.05 ng/ml. For immunofluorescence staining, we used the following: anti-CD14–PE as a monocytic marker (LeuM3, IgG2a; Becton Dickinson), and FITC-conjugated anti–HLA-DR (B.812.2, IgG2a; Coulter Immunotech) and anti-CD25 for detection of the IL-2 receptor (ACT-1, IgG1; Dako). Control mouse Ig (IgG1-FITC/IgG2a-PE) were obtained from Becton Dickinson. An FITC-conjugated goat anti–mouse F(ab) fragment (Coulter Immunotech) was used as a secondary antibody. The cells were washed, adjusted to 2 × 10 5 /200 μl in PBS with 0.2% BSA, and incubated with the antibodies for 30 min on ice. After washing, the samples were analyzed on a FACScalibur™ (Becton Dickinson) using the CELLQuest™ program. The cells were gated due to their forward scatter/side scatter profile. Background levels of immunofluorescence were determined using isotype control Ig. For the detection of IL-2 receptor, cells were first labeled with unconjugated anti-CD25, washed, and then incubated with FITC-conjugated goat anti–mouse F(ab) 2 fragment (5 μg/ml). Proliferation assays with PBMCs (10 5 cells/well in culture medium) were performed at 37°C in 96-well round-bottomed plates at a total of 150 μl/well. The cells were cultured for various time periods (1–4 d) and pulsed with 18.5 kBq of [ 3 H]thymidine per well. Cells were then harvested on filters, and incorporation of [ 3 H]thymidine was measured using a Micro-beta Scintillation Counter (Wallac Instruments). Unstimulated cells and PHA-stimulated (5 μg/ml) cells were used as controls. The nonparametric Wilcoxon rank test was performed for comparison of cytokine data of PHA-, LPS-, and IgA1 protease–stimulated PBMCs. Statistical differences were considered significant at P < 0.01. Data from separate experiments were expressed as means ± SEM and were evaluated for statistical significance by Student's t test. Data from a single representative experiment are presented as means ± SD obtained by triplicate determination. To assess the production of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8, PBMCs from healthy human donors were incubated with purified IgA1 protease. LPS, PHA, and medium alone were used as control. Initial experiments showed that release of proinflammatory cytokines from PBMCs induced by IgA1 protease reached maximal levels between 12 and 24 h (data not shown). Furthermore, a rapid decrease in IgA1 protease–induced cytokine production could be observed after 24 h, and only small amounts of TNF-α were detectable after 72 h. Therefore, in subsequent experiments a single end-point of 18 h culture was chosen that allowed detection of TNF-α and other proinflammatory cytokines. IgA1 protease stimulated the release of proinflammatory cytokines by PBMCs in a dose-dependent manner . Significant amounts of TNF-α in the supernatants were already detectable in the presence of 1 μg/ml IgA1 protease, and highest levels were reached using 10 μg/ml. Similar dose–response patterns were obtained for other proinflammatory cytokines such as IL-1β, IL-6, and the chemokine IL-8. Nonstimulated PBMCs produced negligible amounts of TNF-α, IL-1β, and IL-6, but some IL-8, which was probably induced by adherence as previously postulated by Kasahara et al. 40 . To determine whether the cytokine induction was due to LPS present in the preparation of the recombinant IgA1 protease from E . coli , we tested the potential of E . coli LPS to induce TNF-α. The endotoxin content present in the protease preparation of 10 μg/ml did not exceed 20 pg/ml. As shown in Fig. 1 , no significant cytokine production compared with the control was detected at 10–30 pg/ml LPS, indicating that endotoxin contamination was not the cause of cytokine induction by purified IgA1 protease. TNF-α synthesis in response to 10 μg/ml IgA1 protease reached the level synthesized by cells stimulated with a much higher dose of E . coli LPS (30 ng/ml). In subsequent experiments, low dose (30 pg/ml) and high dose (30 ng/ml) controls of LPS were always included. The cytokine release of PBMCs showed similar variations between different donors upon stimulation with either IgA1 protease, LPS, or PHA. As shown in Fig. 2 (A–D), the median levels of the proinflammatory cytokines detected in the presence of 10 μg/ml IgA1 protease were approximately similar to those found after LPS stimulation and sometimes even higher than those incubated with the lectin PHA. Heat denaturation abolished the cytokine-inducing capacity of IgA1 protease (data not shown). To test whether the enzymatic activity is involved in that process, PBMCs were incubated in the presence of 100 μM of a specific IgA1 protease inhibitor (peptide prolyl boronic acid ). Although at this concentration the inhibitor completely blocked the cleavage of IgA1, it altered neither the LPS- nor the IgA1 protease–induced production of proinflammatory cytokines (data not shown). This demonstrates that the native form but not the enzymatic activity of IgA1 protease is required for the induction of proinflammatory cytokines. IgA1 protease triggers a strong release of proinflammatory cytokines from PBMCs after only a short incubation period, indicating that monocytes might be the main producer of these mediators. To determine the source of cytokine release, we cultivated lymphocyte-depleted peripheral blood monocytes (purity varied between 85 and 95%). As demonstrated in Fig. 3 , IgA1 protease induced significantly lower amounts of TNF-α, IL-1β, IL-6, and IL-8 compared with LPS, which readily stimulated TNF-α production in purified monocytes. Direct comparison with PBMCs revealed that IgA1 protease–stimulated PBMCs can elicit up to 20-fold higher amounts of these cytokines compared with the respective lymphocyte-depleted monocyte cultures, which was particularly true for IL-6 and IL-8 . We also tested the monocytic cell line, MonoMac 6. This cell line shows phenotypic and functional characteristics of mature blood monocytes and produces IL-1, IL-6, and TNF-α upon stimulation with LPS or PMA 39 41 . Compared with LPS, we found a significantly lower production of these cytokines and IL-8 after stimulation with IgA1 protease ( Table ). Direct contact with T cells can augment monocyte cytokine production 42 . To examine the necessity of cell contact for TNF-α release in the different types of stimulation, monocytes stimulated with IgA1 protease, LPS, or PHA were incubated with autologous active and fixed (15 min, paraformaldehyde at 0°C) lymphocytes at a monocyte/lymphocyte ratio of 1:2. In parallel, the lymphocytes and monocytes were incubated alone. Such lymphocytes and purified monocytes produced only low levels of TNF-α upon stimulation with 10 μg/ml IgA1 protease . The same results were obtained after stimulation with PHA. The addition of viable lymphocytes increased the TNF-α secretion in nonstimulated, IgA1 protease–, and PHA-treated monocytes, whereas the addition of prefixed lymphocytes did not. Monocytes stimulated with IgA1 protease or PHA also showed a significantly stronger TNF-α induction in the presence of lymphocytes compared with the control. Only LPS was able to induce TNF-α in purified monocytes alone, and this was not further enhanced by lymphocytes. To examine the particular role of T cells for IgA1 protease–induced TNF-α release, monocytes were incubated with autologous CD3 + T lymphocytes at a monocyte/lymphocyte ratio of 1:4. While T lymphocytes produced no or very low amounts of TNF-α, the monocytes released reduced levels of TNF-α in response to IgA1 protease compared with crude PBMCs. Such reduced TNF-α secretion of monocytes was enhanced in the presence of autologous purified T cells . However, when the two cell populations were separated by a permeable membrane during the cell culture, the TNF-α release by monocytes was not increased in response to IgA1 protease (data not shown). These results suggest that direct contact between T cells and monocytes is necessary for a maximum of cytokine production by monocytes in response to IgA1 protease. It is well known that lymphokines such as GM-CSF or IFN-γ can augment cytokine release in monocytes 43 44 . We found increased levels of GM-CSF in PBMC cultures after incubation with IgA1 protease for 18 h, whereas IgA1 protease failed to induce release of IFN-γ after that time . Very low levels of IFN-γ were finally detectable after 4 d (data not shown). However, the IFN-γ levels in these cultures were always more than 100-fold lower than peak levels of IFN-γ in PHA-stimulated PBMC cultures. Furthermore, in the first 4 d of culture, neither cell proliferation nor increase of IL-2 receptor–positive lymphocytes could be measured after incubation with IgA1 protease (data not shown). These results suggest that viable T cells are required for the cytokine release by monocytes stimulated with IgA1 protease, but unlike PHA, IgA1 protease does not seem to provide a strong activation signal for T lymphocytes. IL-10 is a cytokine with immunosuppressive and antiinflammatory effects on monocytes. Reportedly, IL-10 synthesis is delayed relative to the synthesis of inflammatory cytokines TNF-α, IL-1β, and IL-6 45 . In contrast to the induction of proinflammatory cytokines, IgA1 protease failed to induce IL-10, whereas LPS as well as PHA could stimulate IL-10 production in PBMCs . Kinetic experiments revealed that IgA1 protease does not induce IL-10 production in PBMCs within a period up to 96 h. In contrast, culture supernatants of PHA- or LPS-stimulated PBMCs contained high IL-10 levels at different time points (data not shown). IgA1 protease, a putative virulence factor of human pathogenic Neisseriae , was initially discovered as an enzyme that cleaves human IgA1 molecules, thus interfering with the biological functions of IgA1 10 . Here we report on a novel property of neisserial IgA1 protease, the induction of the proinflammatory cytokines TNF-α, IL-1β, and IL-6, and the chemokine IL-8 in PBMCs. The stimulation of cytokine synthesis by IgA1 protease occurred in the nanomolar range and increased in a dose-dependent manner. The level of cytokine release was substantial but varied to some extent depending on the donor. The rapid cytokine induction within 24 h indicates a role of monocytes/macrophages in this process. However, compared with PBMCs, significantly lower amounts of TNF-α and IL-8 and practically no IL-6 and IL-1β were elicited by purified monocytes in response to IgA1 protease, whereas LPS induced a strong cytokine secretion that included IL-6 and IL-1β. The monocytic cell line MonoMac 6 also showed a low production of TNF-α, IL-1β, IL-6, and IL-8 in response to IgA1 protease. These results suggest that the presence of lymphocytes is required for full cytokine induction by IgA1 protease. It is known that the activation of monocytes/macrophages and the release of monocyte-derived cytokines can be provoked by either direct cell–cell contact with activated antigen-specific T cells or secreted lymphokines 42 43 44 46 . This is consistent with our finding that IgA1 protease–induced TNF-α secretion by monocytes was enhanced by the addition of lymphocytes or CD3 + T cells to the cultures. To investigate the role of activated T cells in this process, we measured IFN-γ secretion, CD69, and IL-2 receptor expression, representing typical markers of activated T cells. Although CD69 was slightly upregulated, the low number of IL-2 receptor–positive cells suggests that no large T cell population was activated or that only partial activation occurred ( 46 ; and data not shown). The T cell cytokine IFN-γ can enhance the cytokine expression in monocytes synergistically in combination with GM-CSF 43 44 . Although GM-CSF was detected after IgA1 stimulation, the release of IFN-γ was low and the addition of anti–IFN-γ antibodies had no effect on the monokine production (data not shown). However, the fact that cocultivation with T cells separated from the monocytes by a permeable membrane could not enhance the production of proinflammatory cytokines indicates that T cells may also participate by a yet unknown contact-dependent mechanism or by nonclassical presentation of antigen 47 48 49 . We also did not find binding of IgA1 protease to T cells, whereas CD14 + monocytes were clearly stained with FITC-labeled IgA1 protease (data not shown). The profile of the cytokine response of PBMCs to IgA1 protease shows some similarities to LPS stimulation. Although the IgA1 protease was produced as a recombinant protein in E . coli , our preparations contained usually <2 pg LPS/μg protein. This LPS content could not sufficiently induce the observed cytokine production. Furthermore, effects which are typically triggered by LPS, such as priming of phagocytes for an enhancement of the oxidative burst, could not be confirmed in cells incubated with IgA1 protease ( 38 ; and data not shown). On the other hand, LPS does not require T cell engagement, suggesting that IgA1 protease functions by a different mechanism. Both molecules can have synergistic effects in vitro (data not shown) and may also act concertedly in vivo. Activated monocytes/macrophages also produce the antiinflammatory cytokine IL-10. This cytokine mediates important autoregulatory effects by downregulating inflammation, and its maximal production is delayed compared with the initial burst of proinflammatory cytokines 45 . IL-10 production was augmented by stimulation with LPS or PHA, but not with IgA1 protease 50 . Thus, IgA1 protease seems unique in stimulating an asymmetric synthesis of proinflammatory cytokines from monocytes and fails to induce strong regulatory feedback loops. Besides the neisserial IgA1 protease reported here, other bacterial products have been found to induce proinflammatory cytokines. These components have been classified as a separate class of bacterial virulence factors termed modulins 51 34 . Examples include the listeriolysin from Listeria monocytogenes 52 , the E . coli hemolysin 53 , and the streptococcal pneumolysin 54 . Other proteins with enzymatic activity, such as the urease of Helicobacter pylori 55 and the sphingomyelinase from Staphylococcus aureus 56 , also modulate cytokine production. Only a few modulins, like the anthrax lethal toxin from Bacillus anthracis 34 and the clostridium toxin from Clostridium difficile 57 , act in the attomolar or femtomolar range, while most other modulins including IgA1 protease exhibit their activity in the nanomolar range. Some modulins, such the staphylococcal toxic shock syndrome toxin 1 (TSST-1) from S. aureus or the erythrogenic exotoxin (ET) from Streptococcus pyogenes possess superantigenic activity, thereby inducing T cells to proliferate and to produce cytokines 58 . IgA1 protease is different from those superantigenic factors because it is not a T cell mitogen. The property of neisserial IgA1 protease to induce proinflammatory cytokines may be highly relevant for the disease-causing properties of gonococci and meningococci. A hallmark of the pathophysiology in these diseases is the intense inflammatory response associated with the substantial release of proinflammatory cytokines. TNF-α, IL-1, IL-6, and IL-8 have been detected in local secretions and sera during mucosal infections with gram-negative species, including N . meningitidis and N . gonorrhoeae 29 35 . Increased levels of IL-8 and TNF-α in particular in plasma and in urine, and smaller amounts of IL-1β and IL-6 were found in experimental gonorrhea after intraurethral application of gonococci 32 . High levels of these cytokines were also found in the CSF and in the serum during meningeal inflammation. These mediators promote the inflammatory response with subsequent tissue damage and in some cases septic shock 28 29 30 . The level of TNF-α in the CSF directly correlates with the severity of the damage to the blood–brain barrier 29 . The most important bacterial component of gram-negative bacteria known to stimulate the release of inflammatory cytokines is LPS. Indeed, meningococcal LPS was found to be a major cytokine-inducing component in bacterial meningitis, since the cytokine profile and levels found in serum or CSF correlated with the LPS concentrations. However, Prins et al. 59 could not confirm the relationship between levels of cytokines and bacterial endotoxin produced by various strains of young meningitis patients, suggesting that factors other than endotoxin may be involved in cytokine induction during acute meningococcal septic shock. In vivo, LPS and IgA1 protease might be released together and act synergistically. Strikingly, though, the three major causative agents of bacterial meningitis, N . meningitidis , H . influenzae , and Streptococcus pneumoniae , all produce an IgA1 protease 11 13 14 60 . Therefore, IgA1 protease may constitute an underestimated virulence factor in sepsis and meningeal infection. Besides the cleavage of IgA1, several additional functions have been attributed to neisserial IgA1 protease. IgA1 protease was recently shown to cleave LAMP-1, a lysosomal protein associated with the endosomal maturation 36 61 , but only one report suggested that, as a consequence, IgA1 protease facilitates the intracellular survival of pathogenic N . gonorrhoeae 61 . Furthermore, other human proteins have been found to be proteolytic substrates in vitro. However, the biological relevance of their cleavage remains unclear. In a parallel study, we recently observed that IgA1 protease efficiently protects phagocytes from TNF-α–mediated apoptosis (our unpublished results). Considering the strong potential of IgA1 protease to induce TNF-α, this observation is intriguing since Neisseria is capable of entering phagocytic cells by an active mechanism 62 . Hence, the pathogens may have an interest in preventing lethal effects of TNF-α on their intracellular niches. The induction of proinflammatory cytokines may contribute to the characteristic inflammatory reaction caused by the pathogenic Neisseriae . Understanding the mechanism of IgA1 protease in the regulation of these cytokines has potential therapeutic significance and is currently in progress. Future research should reveal strategies for blocking this cytokine-inducing pathway in response to pathogenic Neisseriae and probably to other IgA1 protease–secreting human pathogenic bacteria. | Study | biomedical | en | 0.999997 |
10523604 | Phage clones representing the γ c locus were isolated from a 129/SV library (Stratagene Inc.) using a γ c cDNA probe. The SalI inserts of the phage were inserted into pGEM11Zf and further characterized. A 4-kbp BamHI fragment carrying all coding exons of the γ c gene was replaced by the pgk-hygromycin selection cassette to generate the targeting construct. Homologous recombination results in the deletion of the complete coding region of the γ c gene. The resulting SalI targeting fragment was excised from the pGEM11 vector and electroporated into 129/Ola (E14) ES cells as described 30 . Hygromycin-resistant colonies were analyzed for homologous recombination by Southern blot. Targeted ES cell clones were used for injection into B6 blastocysts as described 31 . Chimeric males were mated to B6 or FVB/N females to obtain γ c heterozygous female offspring. Mice deficient for γ c were obtained by subsequent intercrosses. The generation and typing of Rag2-deficient mice 14 , CD3γ-deficient mice 32 , and Eμ- Pim1– 33 and Bcl2 -Ig–transgenic 34 mice have been described elsewhere. Transgenic mice were crossed with Rag2-deficient mice. Transgenic offspring was further crossed with Rag2 knockout mice. The resulting transgenic and nontransgenic heterozygous and homozygous Rag2-mutant mice were used for analysis. Mice were kept in isolators under specific pathogen–free conditions. 200 one- to three-day-old Rag2-deficient mice were injected with 2.5 × 10 5 MoMuLV 35 , and 185 mice were monitored two to three times weekly over a period of 200 d for the development of tumors. Mice were killed when moribund and single-cell suspensions of lymphomas were analyzed by flow cytometry. Upon euthanasia, thymi were dissected and single-cell suspensions were prepared by mincing the lymphoid tissues through a nylon mesh (Cell Strainer; Becton Dickinson). Lymphocyte counts were performed in a Sysmex Toa F800 microcell counter or in a Coulter Counter (Coulter Electronics Ltd.). Single cells were kept at 4°C in PBA (1× PBS, 0.5% BSA, and 0.05% sodium azide). The mAbs anti-CD3∈ (clone 145-2C11), anti-CD4 (clone RM-4-5), anti-CD5 (clone 53-7.3), anti-CD8 (clone 53-6.7), anti-CD24 (heat-stable antigen, clone M1/69), anti-CD25 (clone 7D4), anti-CD44 (Pgp1, Ly-24, clone IM7), anti-CD90.2 (Thy1.2, clone 53-2.1), anti-Nk1.1, and anti–TCR-β (H57-597) were purchased from PharMingen. FITC, PE, and/or biotin conjugates thereof were used for immunofluorescence. Cells (10 6 ) were incubated in 96-well U-bottom plates for 20 min at 4°C in 20 μl PBA and saturating amounts of mAbs. Cells were washed twice with PBA. Cells incubated with biotin-conjugated mAb were subsequently either stained by incubation with PE-conjugated streptavidin (double stainings) or by incubation with Cy-Chrome–conjugated streptavidin (triple stainings). After washing twice, flow cytometry was performed on a FACSCalibur™ (Becton Dickinson) and analyzed using CellQuest™ software. CD4 − 8 − 25 + 44 − thymocytes were identified and sorted directly from Rag2-deficient thymi by four-color staining using anti-CD4 (APC-conjugated), anti-CD8 (PE-conjugated), anti-CD25 (biotinylated), and anti-CD44 (FITC-conjugated) mAbs. The biotinylated CD25-specific mAb was indirectly stained with streptavidin–Red613. To induce pre-T cell development in Rag2-deficient mice, Rag2-deficient mice were inoculated intraperitoneally with 100 μg of HPLC-purified, CD3∈-specific mAb 145-2C11 in 200 μl PBS. CD4 − 8 − 25 − 44 − (quadruple negative), CD4 − 8 + 25 − 44 − (ISP), and CD4 + 8 + 25 − 44 − (DP) thymocytes were sorted 2–4 d after treatment with anti-CD3. The sorted thymocyte fractions were pelleted and kept at −70°C. Total RNA was isolated using RNA-easy anion exchange columns (QIAGEN Inc.). 10 ng of total RNA from each thymocyte fraction was used to analyze the expression of Pim1, Pim2, c-myc, N-myc , and β-actin by reverse transcriptase (RT)-PCR. For this purpose, we used the SuperScript™ One-Step™ RT-PCR System (GIBCO Life Technologies) in combination with one of the following gene-specific primer sets: β-actin , GCACCACAACCTTCTACAATGAGCTG (sense) and CACGCTCGGTCAGGATCTTCATGAG (antisense); Pim1 , GACTTCTGGACTGGTTCGAGAGG (sense) and CCCTTGATGATCTCTTCATCGTGC (antisense); Pim2 , TTGCGCTGCTGTGG-AAGGTGGG (sense) and GGGAGACATGAGCAGGGAAGTG (antisense); N-myc , AGAGTCGGCGTCGGTGCCCGC (sense) and GGGCGTGGAGAAGCCTCGCTC (antisense); and c-myc , CCGCTCAACGACAGCAGCTCG (sense) and CCAATTCAGGGATCTGGTCACGC (antisense). The PCR program was as follows: 50°C for 30 min and 94°C for 2 min, followed by 30 or 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. One-fifth of each reaction was analyzed on a 1.5% agarose gel. Very similar results were obtained with total RNA isolated from independent sorts. The analysis of proviral integrations was performed as described 36 . Total RNA was isolated from frozen tissue samples using anion exchange columns (QIAGEN Inc.). TCR-α germline transcripts were identified by Northern blot analysis as described in 37 . Previously, we have shown that provirus tagging in Rag-deficient mice might be a suitable technique to identify genes involved in the control of early T cell development 38 . Therefore, 185 Rag2-deficient newborn mice were inoculated intraperitoneally with MoMuLV. Thymic lymphomas developed at very high incidence. The average latency period was 150 d . Within 200 d, 81% (150/185) of the mice had developed lymphomas. The tumor incidence of MoMuLV-infected Rag-mutant mice was comparable to that observed previously in wild-type mice 35 . These data further indicate that (a) MoMuLV infects and transforms pro-T cells of Rag-mutant mice and (b) MoMuLV-induced lymphomagenesis does not depend on a functional V(D)J recombinase nor on the presence of mature lymphocytes. The differentiation stage of each tumor was determined by flow cytometry using mAbs specific for CD4, CD8, CD25, CD24 (heat-stable antigen), CD44, CD45R (B220), CD90 (Thy1.2), and NK1.1. Interestingly, in addition to the expected phenotype of differentiation-arrested DN Rag-mutant thymocytes, the phenotype of most tumors (87%) resembled that of further matured T cell precursors. Representative examples showing the differentiation spectrum of these tumors from DN, ISP/DP, DN/DP, and DP and differentiating DN/ISP/DP tumors are depicted in Fig. 2 . These results indicate that proviral activation of host gene(s), rather than the MoMuLV infection itself, can compensate for the lack of a pre-TCR signal in Rag-mutant mice. However, the identification of DN tumors indicates that transformation of the CD25 + DN thymocyte subset can also occur independent of differentiation. To identify the genes responsible for pre-TCR–independent differentiation in MoMuLV-induced Rag-deficient tumors, the presence of proviral insertions in the loci of known protooncogenes was determined. This was performed for 76 selected tumors that were classified according to the expression of CD4 and CD8 markers. Fig. 3 A summarizes the analysis of proviral insertions into the Pim1 and Pim2 loci of these tumors. 27% (15/56) of the tumors that expressed CD4 and/or CD8 (i.e., differentiated tumors) harbored a proviral insertion near Pim1 , as compared with only 10% (2/20) in CD4 − 8 − (undifferentiated) tumors. The differentiation markers of these tumors (79 and 116) displayed a very similar marker spectrum: CD4 − 8 − 25 lo 44 hi 90 lo and CD24 − in the case of tumor 79, and CD24 + in the case of tumor 116. Possibly, these tumors are derived from very early thymocytes, where PIM1 is placed in a signaling context able to amplify growth factor receptor signals, which control differentiation and proliferation of DN thymocytes 39 . Of the 18 DP tumors, 50% (9/18) carry an insertion near Pim1 . As β-selection is associated with an induction of TCR-α germline transcription, the identification of these transcripts in the majority of differentiated tumors supports the differentiation to immature DP T cells (data not shown). No proviral integrations in the Pim2 locus were found in the 76 tumors analyzed from Rag-deficient mice . As proviral integrations in the Pim1 locus were found in T cell lymphomas at all developmental stages, albeit at different frequencies, we wanted to address the function of PIM in early T cell development in a more controlled setting. Previous studies on the function of PIM1 implied that this kinase acts downstream of several cytokine receptors expressed on different hematopoietic cell lineages 24 25 26 40 41 . As the IL-7–IL-7R complex is critical in controlling the cellularity of the pro-T cell compartment, we crossed Eμ- Pim1 –transgenic mice 33 to γ c - and IL-7–deficient mice. For comparison, we also introduced the Bcl-2-Ig 34 and LckF transgenes 17 into the γ c -mutant background. Although these transgenes are expressed in DN thymocytes (data not shown), only Pim1 was capable of restoring the thymus cellularity to an appreciable extent ( Table ), whereas the relative distribution of CD4/CD8 subsets in these thymi was unaltered . These data indicate that Pim1 can compensate to a significant extent for the lack of cytokine signaling, allowing Pim1 -transgenic, γ c - or IL-7–deficient thymocytes to expand. In line with a recent report 42 but in contrast to data reported by others 43 , Bcl2 was only very marginally if at all capable of compensating for the lack of cytokine signaling in the γ c -deficient background ( Table ), resulting in an unaltered number of thymocytes in these mice . The failure of transgenic TCR and LckF (reference 6 and data not shown) to restore thymic cellularity in γ c -deficient thymocytes further supports an important role of cytokine signaling in controlling thymic cellularity independent of pre-TCR signaling. Taken together, these data suggest a role for PIM kinases in T cell cytokine signaling. The high incidence of proviral insertions into the Pim1 locus, 50% (9/18) in DP tumors as compared with 10% (2/20) in DN tumors, suggests a potential of PIM1 to facilitate the generation of DP thymocytes. The question of whether the frequent proviral insertions into the Pim1 locus of DP tumors were causally involved in the differentiation into pre-T cell–like tumors was addressed by introducing the Eμ- Pim1 transgene into the Rag-deficient background. Indeed, in Eμ- Pim1 –transgenic Rag-deficient mice, we observed differentiation and slow expansion of large DN, CD25 + thymocytes into small resting DP CD25 − pre-T cells . The PIM1-mediated differentiation is age dependent. Only 1–2 million DP thymocytes are found at an age of 4–5 wk, but the numbers increase to normal levels (100–200 million) at an age of 8–9 wk . This is in contrast to the transgenic expression of LckF in the Rag-deficient background, which results in a normally sized thymus at birth. Both the reduction in cell size, as measured by forward scatter analysis, and the reproducible number of DP thymocytes at a given age argue against transformed DP cells, which is also in line with the slow transforming activity seen in Pim1 -transgenic mice 23 . These data suggest that PIM kinases synergize with TCR signaling in generating DP thymocytes. As differentiation-arrested Rag-deficient thymocytes still have residual CD3 signaling capacity 44 on the basis of γ∈ modules, we questioned whether differentiation of DN pre-T cells in Eμ- Pim1 –transgenic Rag-deficient mice still requires particular CD3 components on the surfaces of pro-T cells of Rag-deficient thymocytes 44 45 46 . As a first approach to addressing this question, the Eμ- Pim1 transgene was introduced into the CD3γ-deficient background, in which most thymocytes are blocked at the CD25 + DN stage 32 . Strikingly, independent of the age of the mouse, no further differentiation and expansion of Pim1-transgenic CD3γ-deficient pro-T cells was found, which is in strong contrast to Pim1 -transgenic Rag-deficient mice . These data suggest that PIM1 cannot act on its own but requires CD3-mediated signals to overcome the T cell differentiation arrest seen in Rag- and CD3γ-deficient mice. In contrast, introduction of the LckF transgene into the CD3γ-deficient background results in restoring the number of thymocytes in these mice ( Table ). The constitutively active LckF kinase is capable of bypassing CD3γ deficiency. Differentiation and proliferation of DN to DP thymocytes is regulated by the expression of a pre-TCR–CD3 complex 2 . The exponential expansion of precursor thymocytes during β-selection accounts for a 100-fold increase in the number of precursor T cells. As only a minority of thymocytes will finally fulfill the thymic selection criteria and mature into functional immunocompetent T cells, the expansion of precursor thymocytes during β-selection is a prerequisite for the efficient formation of a diverse peripheral α/β T cell repertoire. Specific growth and/or differentiation signals are required for this expansion and could also play a role in malignant transformation. However, the observation that ∼10% of the T cell lymphomas isolated from MoMuLV-infected Rag-deficient mice had retained a DN phenotype indicates that transformation of the DN thymocyte subset can occur, i.e., the induction of proliferation per se does not necessarily imply differentiation of DN thymocytes. Almost 90% of the T cell lymphomas isolated from MoMuLV-infected Rag2-deficient mice had bypassed the block in T cell differentiation, as indicated by the expression of either CD4 and/or CD8. Given this fact, we reasoned that the analysis of common proviral insertions in a large panel of these tumors, and specifically in DP tumors, might allow us to identify genes with a function in early T cell development. Subsequently, this function can be tested by expressing these gene(s) in thymocytes of compound recombination-deficient mice. 150 lymphomas derived from MoMuLV-infected Rag2-deficient mice were characterized for the presence of CD4, CD8, CD24, CD25, CD44, CD45R, CD90, or NK1.1 surface markers and were classified according to the expression of CD4 and/or CD8 markers. The Pim1 locus was identified as a proviral integration site in T cell lymphomas at all developmental stages from MoMuLV-infected Rag-deficient mice. Two tumors with a provirus in the Pim1 locus had retained the DN marker profile. Interestingly, both tumors displayed a very similar marker profile specific for very early thymocytes 47 : CD4 − 8 − 25 lo 44 hi 90 lo and CD24 − in the case of tumor 79, and CD24 + in the case of tumor 116. Possibly, PIM1 is placed in a signaling context able to amplify other growth factor receptor signals, which control proliferation of early DN pro-T cells 39 . These data suggested that PIM1 can support growth of pro-T cells. As previous functional studies on PIM1 indicated a role in cytokine signaling and proliferation of B cell progenitors 24 and mast cells 25 , we further investigated whether PIM1 fulfills a similar role in the T cell lineage. Indeed, elevated PIM1 levels can rescue to a significant extent the thymic proliferation defect of γ c - and IL-7–mutant strains. This effect is specific for PIM, as a range of other oncogenes, such as Bcl2 and LckF, failed to do so. The high frequency of proviral insertions into the Pim1 locus of DP tumors suggested that PIM1 can also be involved in compensation of defective β-selection in Rag-deficient thymocytes. A subsequent independent gain of function analysis making use of Pim1 -transgenic Rag-deficient mice directly confirmed this involvement. We observed further development of DN, differentiation-arrested, Rag-deficient thymocytes into small DP CD25 − thymocytes in a time-dependent fashion. The DP thymocytes show a reduced cell size, arguing against the possibility that these cells are transformed by PIM1 or have become a target of a secondary transforming event. These and recently described data 27 indicate that PIM1 kinase can compensate for defective pre-TCR–CD3 signaling. A potential PIM1 target that might mediate this effect has been reported recently 48 . PIM1 binds and phosphorylates the ubiquitously expressed transcriptional coactivator p100 and thereby stimulates the transcriptional activity of c-Myb in a p100-dependent manner 48 . In this respect, it would be of interest to investigate whether Myb plays a role in thymic expansion. To elaborate further on the role of PIM kinases in T cell signaling pathways, the same Eμ- Pim1 transgene was crossed into the CD3γ-mutant background. Similar to the situation in Rag-deficient mice, T cell development is arrested at the CD4 − 8 − 25 + 44 − in CD3γ-mutant mice 32 . Rag-deficient thymocytes express CD3∈-containing complexes at the cell surface that permit β-selection upon cross-linking with CD3∈-specific mAbs in fetal thymic organ cultures as well as in vivo 44 45 46 . These data are in line with other observations indicating that the cytoplasmic domain of CD3∈ suffices in signaling β-selection 49 . However, in PIM1-transgenic CD3γ-deficient mice, we did not observe any expansion of the DP compartment. Two models can explain the apparent requirement for CD3γ in PIM1-mediated T cell differentiation of Rag-deficient thymocytes. First, PIM1 might directly function in the CD3γ pathway in addition to its role in cytokine signaling. Second, and in our view more likely, PIM1 might act in the cross-talk between cytokine and TCR signaling in which the effect of PIM1 depends on CD3 signaling. It is possible that in Rag-deficient mutant mice, occasional dimerization of CD3γ∈ modules provides a weak signal in DN pro-T cells. The frequency of dimerization might be too low and, consequently, this signal might normally fall below a critical threshold required to signal β-selection in DN thymocytes. However, in the presence of higher PIM1 levels, as is the case in Eμ- Pim1 –transgenic Rag-deficient mice, the proliferating DN population would be larger and occasional dimerization could occur in sufficient frequency to permit β-selection in some of the cells in Pim1 -transgenic Rag-deficient mice. The rare occurrence of this CD3 signal might explain the correlation between the numbers of DP thymocytes and the age of the Pim1 -transgenic Rag-deficient animals. In this model, PIM functions as an integrator of cytokine and TCR signaling. The concept of integrated signaling pathways is also supported by data obtained from crosses between mice deficient for the γ c and pre-T cell α genes 50 and between mice deficient for γ c and c-kit genes 51 . The phenotype of the compound mutant mice is far more severe than would be expected on the basis of the additive effects of the single knockouts. Future experiments will address the biochemical basis of this synergistic interaction. | Study | biomedical | en | 0.999995 |
10523605 | The syntheses of biotinylated α- and β-GalCer (biotin–α-GalCer and biotin–β-GalCer) have been described elsewhere 29 . After synthesis, the biotinylated compounds were purified by silica gel column, which completely removed the biotinylating reagent 29 . The purity of biotinylated α- and β-GalCer compounds was verified by mass spectrometry. No unbiotinylated galactosyl ceramide was detectable in the final preparation of the compound 29 . N -(biotinoyl)dipalmitoyl- l -α-phosphatidylethanolamine was purchased from Pierce Chemical Co. Gangliosides GM1 and GD1a were purchased from Calbiochem. Hexasaccharide disialyllacto- N -tetraose (α-Neu5Ac-[2→3]-β-Gal-[1→3]-(α-Neu5Ac-[2→6])-β-GlcNac-[1→3]-β-Gal-[1→4]-Glc) was purchased from Sigma Chemical Co. Polyethylene glycol (PEG) 2000 ceramide was purchased from Northern Lipids, Inc. P99 peptide (YEHDFHHIREWGNHWKNCLAVM) and NH 2 terminus biotinylated P99 peptide were synthesized at Research Genetics, Inc. For expression of soluble hCD1d molecules in insect cells, we generated the plasmid hCD1d-pRmHa-3, which encodes a truncated form of the hCD1d cDNA containing 32 bp of 5′ untranslated region, the signal peptide for insertion into the endoplasmic reticulum, and the cell surface α1–α3 domains. The hCD1d coding sequence was amplified using PCR from a plasmid encoding hCD1d (a gift of Dr. Steven Balk, Beth Israel Hospital, Boston, MA). Oligonucleotides corresponding to the 5′ untranslated region upstream of the hCD1d AUG start codon (TTGCAGCCATGGAGGTCCCCACG) and the distal end of the cytoplasmic domain (CGGGATCCCCAGTAGAGGACGATGTCCTG) introduced 5′ NcoI and 3′ BamH1 restriction enzyme sites, which allowed for cloning of the truncated cDNA into a modified version of the insect cell expression vector, pRmHa-3 ( 30 ; a gift of Dr. A. Raúl Castaño, Instituto de Salud Carlos III, Madrid, Spain). Following the BglII cloning site in this vector are sequences encoding 2 protein tags; the 10 amino acid sequence, YPYDVPDYAS, derived from influenza hemagglutinin (HA), which is recognized by the 7F11 mAb; and a 6-histidine tag for protein purification on Ni-NTA agarose (Qiagen). Drosophila melanogaster cells in tissue culture were cotransfected with 15 μg of hCD1d-pRmHa-3, 15 μg of hβ2m-pRmHa-3, encoding hβ2m, and 1 μg of pUChsneo by the calcium phosphate method and selected with 1–1.5 mg/ml of G418 for 2 mo. The hβ2m-pRmHa construct was a gift from Dr. Jeffrey E. Miller (IVS Technologies LLC, Carlsbad, CA). Cultures of parental and mCD1- and hCD1d-transfected D . melanogaster embryonic SC2 cells were carried out as described previously 31 . For the culture of transfected insect tissue culture cells, the medium was supplemented with 1 mg/ml G418 (Sigma Chemical Co.). The mCD1-transfected D . melanogaster cells and untransfected parental insect cell lines were a gift of Dr. A. Raúl Castaño. The mCD1-transfected A20 cell lines have been described previously 32 . Antibodies used for ELISA were 7F11 (mouse IgG 2b ) for detection of HA epitope–tagged polypeptides; BBM.1 for hβ2m 33 , biotinylated 1B1 (rat IgG 2b ) for mCD1 32 , and NOR3.2 (mouse IgG 1 ) for hCD1d 34 . Horseradish peroxidase (HRP)-conjugated goat anti–mouse Ig (Caltag Labs) and HRP-conjugated streptavidin (Jackson ImmunoResearch Labs) were used as secondary reagents in ELISA. 3–10 liters of insect cells, stably cotransfected with the CD1d- and β2m-encoding plasmids, were induced with 0.7 mM CuSO 4 for 3 d to transcribe the metallothionein promoter. After induction, cell supernatant was concentrated and dialyzed against 0.15 M sodium phosphate buffer (pH 7.4) using an Amicon Y1S10 Spiral Wound Membrane cartridge with a 10-kD mol wt cut-off (Millipore). Soluble CD1d–β2m complexes were first affinity-purified on Ni-NTA agarose column (1 ml bed volume; Qiagen) and then FPLC purified with Mono Q anion-exchange column (1 ml bed volume; Amersham Pharmacia Biotech). Gel filtration was performed in PBS on a Superdex 200 column (Amersham Pharmacia Biotech) to verify that purified protein did not form aggregates. Protein concentration was determined from the absorbency at 280 nm and by using the bicinchoninic acid protein assay reagent (Pierce Chemical Co.). Soluble mouse class I molecule H2-M3 was a gift of Drs. Santai Shen and Johann Deisenhofer (Howard Hughes Medical Institute, University of Texas, Southwestern Medical Center, Dallas, TX). The Vα14Vβ8 NK T cell hybridomas N38-3C3 and N38-2C12 (a gift of Dr. Kyoko Hayakawa, Fox Chase Cancer Center, Philadelphia, PA) and DN3A4-1.2 (a gift of Dr. M. Bix, University of California San Francisco, San Francisco, CA) have been described previously 19 . For the assays, T hybridoma cells were cultured for 16 h at 5 × 10 4 cells/well in the presence of mCD1-transfected A20 cells that had been prepulsed for 2 h with the indicated concentrations of glycolipids or with the vehicle DMSO. IL-2 release was evaluated in a sandwich ELISA using rat anti–mouse IL-2 mAbs (PharMingen), and the levels of cytokine release were evaluated using a recombinant IL-2 standard (PharMingen). For experiments in which α-GalCer was presented by immobilized CD1d proteins, 96-well tissue culture plates were coated for 2 h at 37°C with 1 μg/well of soluble protein that had been premixed with the indicated concentrations of lipid antigen in 100 μl PBS. Control wells were coated with protein only in the presence of DMSO (0.25–1% final concentration) or with lipids alone. After coating, the plate was washed five times in PBS and 5 × 10 4 hybridoma cells/well were added immediately. IL-2 release was measured after 16 h of culture. For competition experiments, gangliosides were dissolved in PBS and added to CD1d proteins before the addition of α-GalCer. A BIAcore X biosensor system was used for real time binding experiments, which were all done at room temperature. 1 mg/ml stock solutions in DMSO of biotin–α-GalCer or biotin–β-GalCer were diluted to 50 μg/ml into BIAcore flow buffer, Hepes-buffered saline (HBSt: 0.01 M Hepes, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, 0.005% [vol/vol] surfactant P20), and captured onto a streptavidin sensor chip (BIAcore) at a flow rate of 5 μl/min, which yielded 200–400 response units (RU) of immobilized ligand. Presence of immobilized biotin–α- and biotin–β-GalCer was additionally confirmed by binding of the galactose-specific lectins Ricinus communis agglutinin I (RCA I) and Isolectin B4 of Griffonia simplicifolia lectin I (GSL I isoform B4). 0.1 mg/ml stock solution of biotin-DPPE in DMSO was diluted to 2 μg/ml into BIAcore flow buffer for immobilization. 2 mg/ml stock solution of biotin-P99 in water was diluted to 20 μg/ml into BIAcore flow buffer for immobilization. For biotin-GalCer, kinetic studies were performed by injecting various concentrations of mCD1 or hCD1d in PBS or HBSt at 10–50 μl/min flow rate to measure the association phase, and washed with the same buffers and at the same flow rate to measure the dissociation phase. For biotin-DPPE, kinetic studies were done at 2 μl/min flow rate. Lipid-coupled surfaces were regenerated using 0.5–2-min pulses of PBS containing 0.05–1% Triton X-100 (Sigma Chemical Co.). For competition experiments, mixtures of protein with competitor lipids were preincubated at room temperature for 15–20 min before injection. Evaluation of the data was performed using the BIAevaluation software version 2.1 (BIAcore). For determination of the association rate constant, the association phase of the sensogram was fitted to a single exponential model and the k on rate was determined from the plot of the observed association rate k obs against protein concentration, according to the formula k obs = k on C + k off . The dissociation phase of the sensogram was directly fitted to a single or double exponential model, and the contribution of each component to the overall dissociation was calculated. To biochemically define the interaction of group II CD1 molecules with glycolipids, soluble hCD1d–hβ2m and mCD1–mβ2m heterodimers were produced in the D. melanogaster –derived SC2 cell line. This insect tissue culture system has previously been shown to produce functional human and mouse MHC class I, class II, and mCD1–mβ2m heterodimers 28 35 36 . CD1d heterodimers were purified to 90–99% purity by nickel affinity chromatography and anion-exchange chromatography. The identity of the CD1d molecules produced was verified in ELISA in which specific mAbs were used to detect wells coated with the purified proteins . The mAbs used include the hCD1d heavy chain–specific antibody NOR3.2, the hβ2m-specific antibody BBM.1, and the mCD1-specific antibody 1B1 . The 7F11 antibody specific for the COOH-terminal influenza HA epitope tag on the soluble heavy chains recognized both CD1d proteins. Purified CD1d proteins were free of aggregates, as assessed by size exclusion FPLC chromatography (data not shown). Because of the tendency of lipids to interact nonspecifically with macromolecules, we wished to determine if physiologically relevant or antigenic lipid plus CD1d complexes could be formed in vitro. Soluble mCD1 and hCD1d molecules were preincubated with glycosphingolipids, and were used to coat the wells of microtiter plates that were subsequently cultured with N38-3C3 (hereafter 3C3), a Vα14/Vβ8 + mouse NK T cell hybridoma 19 . Previously, we demonstrated that some Vα14 + mouse NK T cell hybridomas could recognize α-GalCer presented by either mCD1- or hCD1d-transfected APCs 19 . In agreement with those results, we found that plate-bound mCD1 and plate-bound hCD1d could both present α-GalCer to the 3C3 T cell hybridoma, whereas plates coated with CD1d molecules without antigen, or with CD1d preincubated with β-GalCer, were not recognized . Plates coated with the glycosphingolipid antigen alone also did not stimulate the hybridoma. Anti-mCD1 mAb 1B1 inhibited NK T cell recognition of α-GalCer presented by plate-bound mCD1, but had no effect on presentation by plate-bound hCD1d . Thus, the T cell stimulation we observe is not due to the reuptake of α-GalCer and mCD1-mediated autopresentation by the hybridoma cells, which are weakly mCD1 positive. The 3C3 hybridoma has a low level of autoreactivity to mCD1-transfected A20 cells in the absence of α-GalCer antigen 19 20 . Yet we did not observe any autoreactivity of this hybridoma to plates coated with soluble mCD1 that had not been preloaded with α-GalCer. The inability to react to plate-bound mCD1 suggests that the endogenous ligand present in soluble mCD1 molecules purified from Drosophila cells (Degano, M., and I. Wilson, personal communication) may be different from the endogenous ligand(s) bound to mCD1 in A20 cells. Most interestingly, the pattern of autoreactivity of two other Vα14 + NK T hybridomas, DN3A4-1.2 (hereafter 1.2) and N38-2C12 (hereafter 2C12), to mCD1 and hCD1d molecules was recapitulated in their autoreactivity to soluble CD1d proteins. The 1.2 hybridoma is strongly reactive to A20 hCD1d and weakly reactive to mCD1 transfectants, even without α-GalCer 19 20 . It also reacts to plates coated with soluble hCD1d , producing significant amounts of IL-2 . Similarly, 2C12 has reactivity to both A20hCD1d and A20mCD1 transfectants without α-GalCer, and it produces IL-2 in response to plates coated with either CD1d protein . While the reactivity of the 1.2 and 2C12 hybridomas to the insect cell–derived CD1d molecules is significant, this response can be enhanced by exogenously added α-GalCer, similar to the results with CD1d + APCs 19 20 . Finally, Vα14 + mouse NK T cell hybridomas that express either Vβ7 (N38-2H4) or Vβ10 (DN3A4-1.4) TCR β chains also could recognize α-GalCer presented by immobilized mCD1 molecules, although they did not show reactivity to CD1d in the absence of this antigen (data not shown). We synthesized biotinylated versions of α-GalCer and the nonantigenic β-GalCer in order to develop a CD1d binding assay in which the lipid could be immobilized onto a solid support with hydrophobic moieties exposed, thereby minimizing the problem of micelle or bilayer formation. The antigenicity of the biotinylated compounds was tested using parental and mCD1-transfected A20 cells . mCD1 + A20 cells can present both α-GalCer and the modified biotin–α-GalCer to the 3C3 hybridoma, whereas untransfected A20 cells could not. Other Vα14 + NK T cell hybridomas could also recognize this compound (data not shown). Biotin-modified β-linked GalCer (biotin–β-GalCer) did not stimulate the T cell hybridoma . These data indicate that the addition of a biotin group did not eliminate the ability of biotin–α-GalCer to be presented by mCD1 to NK T cells, nor did it cause β-GalCer to be recognized. However, it should be noted that the modification of the acyl chain of this compound, including the presence of the biotin group and two amide bonds, makes it a weaker antigen. In dose–response experiments with mCD1 + APCs, biotin–α-GalCer gave a 1.5–2-fold lower stimulation than the unmodified α-GalCer (data not shown). Similarly, in antigen presentation experiments using CD1d-coated plates, it stimulated approximately three- to fivefold lower IL-2 release when incubated with either mCD1 or hCD1d molecules (data not shown). The ability of mCD1 and hCD1d molecules to bind to biotin–α-GalCer was examined using SPR. In our experimental set-up, biotin-GalCer compounds were immobilized on flow cell 2 of the streptavidin biosensor chip. No ligand was immobilized on flow cell 1, which therefore provided a control surface that allowed monitoring refractive index changes and nonspecific protein sticking. After the coupling procedure and every protein injection, the active surface is regenerated by washing with a solution containing Triton X-100, which removes any noncovalently bound lipid or protein. Therefore, binding to the exposed acyl chains of tethered α-GalCer molecules, which should be relatively free of aggregates, can be directly measured. At neutral pH, soluble mCD1 and hCD1d proteins, but not H2-M3, could bind immobilized biotin–α-GalCer . The nonclassical class Ib molecule H2-M3 was chosen as a negative control, because, similar to mCD1, it has a hydrophobic antigen-binding groove 37 . Additionally, soluble hCD1b protein was also able to bind to immobilized biotin-GalCer (data not shown). mCD1 binding to biotin–α-GalCer is dose dependent , and similar dose–response data were obtained using hCD1d (data not shown). Binding of the plant lectins specific for the galactose portion of the immobilized GalCers (GSL I-B4 and RCA I) gave a signal similar in magnitude to the CD1d signal 29 , suggesting that CD1d molecules bound to almost all of the glycosphingolipids on the chip that are accessible to macromolecules. Fig. 4 C shows the dissociation curve of mCD1 from biotin–α-GalCer. The data can be best fitted to a two-component dissociation model with k off fast 0.1 s −1 and k off slow 0.004 s −1 , which correspond to half-lives of 7 s and 3 min, respectively. In different binding experiments, the faster component contribution varied between 20 and 30%. We estimated the association rate for mCD1 binding to α-GalCer from a single site model, plotting apparent association rate constant k obs versus the concentration . In different experiments, k on values between 5 and 13 × 10 4 M −1 s −1 were observed. By taking the weighed average of the dissociation rate (20% × 0.1 + 80% × 0.004 = 0.023 s −1 ), we obtain a K D of 0.34 μM (with the range of 5.9 × 10 −8 M for the slow component of dissociation to 1.5 × 10 −6 M for the fast component of dissociation). The calculated rate constants for hCD1d binding to α-GalCer are essentially equal to those obtained for mCD1 ( Table ). It has been speculated that the α-anomeric form of the sugar is essential for contacting the invariant NK T cell TCR, rather than for CD1d binding 18 19 20 21 22 23 . Consistent with this hypothesis, both mCD1 and hCD1d could bind strongly to biotin–β-GalCer . The binding of β-GalCer to either mCD1 or hCD1d was surprisingly similar to the binding of α-GalCer, with respect to the two-component dissociation and the rate constants for association and dissociation ( Table ). However, in several experiments the absolute number of RU for binding of either CD1d molecule to the β-GalCer–coupled sensor chips tended to be two- to threefold lower than for binding to α-GalCer–coupled sensor chips containing approximately the same number of RU of coupled compound . Thus, within the limitations of our experimental set-up, it is still possible that there might be a two- to threefold difference in affinity between β-GalCer and α-GalCer. However, because there is a much greater difference in the ability of β- and α-linked compounds to stimulate mouse NK T cells, we conclude that the lack of antigenicity of β-GalCer for NK T cells is due mainly to its failure to properly contact the invariant Vα14-containing TCR expressed by these cells, as opposed to a drastically reduced binding affinity for CD1d. To determine if CD1d can bind to phospholipids, as suggested by previous studies 26 27 , we used a different biotinylated lipid, biotin-DPPE . This compound also permitted us to address the effect of having two unmodified acyl chains available for CD1d binding. Although the structure of the lipid portion of biotin-DPPE is quite different from that of galactosyl ceramides, and it is not antigenic for NK T cell hybridomas (data not shown), it is able to bind to CD1d proteins . This binding is characterized by slower association, k on 1.4 × 10 3 M −1 s −1 , and significantly slower dissociation, k off 2.7 × 10 −4 s −1 , than binding to immobilized biotin–α-GalCer. These on and off rates produce a calculated K D of 0.2 μM and a half-life of the CD1-biotin–DPPE interaction of 40 min. Therefore, like human CD1b 38 , CD1d molecules can bind to diverse lipoglycans, although for CD1d molecules, acidic pH is not required. Furthermore, while we cannot exclude a possible contribution of the phosphate group or some other portion of the biotin-DPPE to the increased stability of CD1d binding, it is most likely that availability of two uninterrupted acyl chains accounts for the increased half-life of the biotin-DPPE complexes with CD1d. It probably also accounts for the slower association rate, as interactions between the two hydrophobic acyl chains may interfere with their loading into the separate A′ and F′ pockets of the CD1d antigen-binding groove. To further demonstrate the specificity of biotinylated sphingolipid binding to soluble CD1d molecules, competition assays were carried out in which CD1d molecules were preincubated in solution with gangliosides, which are highly glycosylated sphingolipids. Gangliosides are not antigenic for the NK T cell hybridomas (data not shown), presumably on account of the β-linkage of the lipid-proximal sugar to the one carbon of the sphingosine. However, in the micromolar concentration range, gangliosides GM1 (five sugars) and GD1a (six sugars) could inhibit mCD1 or hCD1d binding either to biotin–α-GalCer or to biotin–β-GalCer. Representative data are shown for the inhibition by GM1 of mCD1 and hCD1d binding to biotin–α-GalCer in Fig. 6 A. Consistent with this finding, GM1 and GD1a could inhibit presentation of α-GalCer by either plate-bound mCD1 or hCD1d proteins (data not shown). A control hexasaccharide, disialyllacto- N -tetraose, did not inhibit presentation. This result indicates the importance of the ceramide portion of gangliosides for the competition for α-GalCer presentation. However, we cannot accurately estimate affinity constants from the CD1d–ganglioside interaction from these competition experiments because in the micromolar range, gangliosides are mostly in micelles 39 , with very few monomers present. The inhibition by the gangliosides is most likely not due to the trapping of CD1d molecules in micelles, because CD1d molecules incubated with the gangliosides bound as well to an anti-CD1d mAb coupled to a biosensor chip as did the control CD1d molecules (data not shown). Sphingolipid compounds containing only a single sugar, such as α-GalCer or β-GalCer, are much less soluble than the gangliosides, and unlike the gangliosides, must be dissolved in DMSO-containing solutions. Because of their insolubility and the adverse effect of the DMSO upon CD1d protein stability, we could not set up a reliable competition assay using α- or β-GalCer mixed in aqueous solution to compete for CD1d binding to the immobilized lipid ligands. From these data, we infer that the increased carbohydrate content of gangliosides, leading to increased solubility in aqueous solutions and therefore a higher rate of monomer formation, allows the similar lipid portions of these molecules to compete effectively for immobilized biotin-GalCer binding. Consistent with this hypothesis, we found that ceramide coupled to a highly polar polyethylene glycol moiety (PEG-ceramide) could also compete for binding to immobilized biotin–α-GalCer (data not shown). These data also underscore the importance of the ceramide group, and demonstrate that the oligosaccharide chain of the glycosphingolipid is not required for competition for CD1d binding. We and others have shown that mCD1 is able to bind long hydrophobic peptides and present them to CD8 + T cells 28 40 41 . Using equilibrium binding studies, with radiolabeled 22-mer P99 peptide and mCD1 in solution, Castaño and colleagues 28 estimated the mCD1 affinity for antigenic peptide ligands to be ∼0.9 μM, with fast association and dissociation kinetics. We examined mCD1 binding to immobilized biotinylated P99 peptide in the BIAcore and observed an association rate of 1–4 × 10 4 M −1 s −1 and a dissociation rate of 0.005–0.008 s −1 . These rates produce a calculated K D in the range of 0.2–0.8 μM, a value in good agreement with equilibrium binding studies. The mCD1 binding to biotin-P99 was readily competed by free P99 peptide . Similar to the results from peptide–MHC classical class I binding studies 42 , a peptide concentration severalfold lower than total protein concentration resulted in a 50% inhibition of mCD1 binding. This suggests that only a percentage of the mCD1 molecules in our preparations are capable of binding the peptide, either because of the conformational state of the CD1d molecules, or because of some other factors such as differential occupancy of CD1d with lipid ligands. The binding of CD1d to immobilized P99 could not be inhibited by GM1 , suggesting that GM1 and peptide do not bind to the same sites. Consistent with this, mCD1 binding to immobilized α-GalCer was not affected by soluble P99 peptide at concentrations that completely inhibited binding to immobilized peptide . In recent years, data have been generated on lipid antigen presentation by several different CD1 molecules (for reviews, see references 13 , 43 , and 44 ), but there is still very little information on the biochemistry of lipid antigen binding to CD1. The published crystallographic structure of mCD1 shows a deep, narrow, and very hydrophobic ligand-binding groove with two pockets termed A′ and F′ 45 . This analysis also shows an acyl chain containing ligand inserted into the pockets of this groove (Degano, M., and I. Wilson, personal communication). Mass spectrometric characterization of the material eluted from mCD1 molecules purified from mammalian cell lines indicated the presence of a predominant GPI ligand 26 . Our in vitro binding studies have demonstrated binding at acidic pH of phosphatidylinositol-based compounds in solution to an hCD1b-coupled chip in the biosensor 38 . However, the signal obtained in these studies is suggestive of multimeric antigen binding to the immobilized CD1b molecules, and it remains to be determined whether the type of binding we measured corresponds to the formation of antigenic glycolipid–hCD1b complexes. Here we have investigated the biochemistry of lipid antigen binding to the group II molecules hCD1d and mCD1, focusing on glycosphingolipids. These two CD1d molecules are of particular interest because they are recognized in a conserved manner by the NK T cell subset 24 25 . The ability of NK T cells to respond to CD1d is greatly augmented by the addition of α-GalCer, a compound isolated originally from the marine sponge Agelas mauritanius , based on its ability to prevent tumor metastases 46 47 . Glycosphingolipids in mammalian cells are considered to have only β-linked dextro -enantiomer sugar as the first sugar attached to ceramide. Compounds with an α-linked dextro sugar have not been found in mammalian cells, although it remains possible that low levels of such compounds are present but have not been detected. Regardless of whether α-GalCer is the natural ligand for NK T cells or merely cross-reactive with such a ligand, because of its ability to stimulate a large fraction of these cells and the availability of analogues 18 19 20 21 22 23 , α-GalCer provides a very good model for studying the CD1–glycolipid antigen interaction. Plate-bound CD1d molecules can present α-GalCer to NK T cell hybridomas. This demonstrates that some of the soluble CD1d molecules are in a native conformation, which is consistent with their ability to react with conformation-sensitive anti-CD1d antibodies. α-GalCer can be successfully loaded into the CD1d-binding groove at neutral pH, highlighting the differences in lipid binding between group I (CD1b) and group II (CD1d) molecules, at least in terms of acidification requirements for presentation. Although the loading of antigen onto CD1d molecules in acidified endosomes is not absolutely required for stable binding and antigen recognition 21 , endosomal uptake augments the presentation of α-GalCer in some circumstances 18 21 . α-GalCer–CD1d complexes formed in vitro are physiologically relevant because they can be recognized by TCRs, formally proving that the presentation of α-GalCer is carried out by CD1d. We were surprised to find that some of the Vα14 + T cell hybrids could react to a significant extent to the insect cell–derived plate-bound CD1d in the absence of exogenous ligand. Our results with soluble proteins provide one possible explanation for the CD1d autoreactivity of NK T cell hybridomas. At least some of them could be recognizing the CD1d protein directly, while the function of the endogenous or exogenous ligand could be to maintain CD1d molecules in proper conformation. Alternatively, it is possible that these T cells recognize the insect cell–derived ligand when bound to CD1d. We have used immobilized, biotinylated forms of α-GalCer and β-GalCer to demonstrate specific binding to soluble mCD1 and hCD1d molecules. This has allowed us to obtain a more quantitative estimate of the affinity of lipid CD1 binding than was possible in a previous study, since in this case we examined the monomeric interaction between CD1 molecules and glycolipids. As determined by SPR studies, the affinity of both the mCD1 and hCD1d molecules for galactosyl ceramides and for phospholipids is in the 0.1–1 μM range. The near equivalence of α-GalCer and β-GalCer binding to both mCD1 and hCD1d is established by the results from multiple experiments. This provides the first evidence for the earlier prediction that the sugar linkage does not significantly affect CD1d binding 18 but is more likely to be important for TCR contact. SPR studies also indicate that the OH groups on the 3 and 4 positions of the sphingosine 29 are likewise not required for efficient CD1d binding (data not shown). Furthermore, the compounds that compete effectively for CD1d binding to biotin-GalCer include the gangliosides, which lack the OH on the 4 position of the sphingosine , and PEG-ceramide, lacking the hexose sugar entirely. Collectively, these data are consistent with the hypothesis that the CD1d molecule is primarily contacting the hydrophobic portions of the glycolipids. This idea is additionally supported by the finding that CD1d binds strongly to a phospholipid biotin-DPPE, which has a distinctly different polar head group. Based on these data, we conclude that CD1d molecules are likely to be capable of binding a variety of lipid-containing antigens. The dissociation rate for CD1d binding to biotin–α-GalCer predicts a half-life of several minutes, faster than what was observed for MHC class I peptide dissociation 42 48 49 , although there are some exceptions from SPR experiments, such as binding of Qa-1 protein to immobilized Qdm peptide 50 . We believe that the relatively fast off rate for lipoglycan binding could be due to a combination of factors, including the method of attachment to the biosensor and structural changes in the biotinylated analogue itself. Indeed, SPR studies of MHC class I interaction with peptide have shown that the dissociation rate is significantly affected by both the amino acid through which the peptide is coupled to the sensor surface and the method of coupling 42 . The sequestration of the terminal part of the acyl chain in the streptavidin-binding pocket will prevent the acyl chain from completely filling one of the two CD1d antigen-binding pockets. Furthermore, the presence of the two hydrophilic amide bonds in the acyl chain and the presence of the biotin group at the end of it create a suboptimal antigen . Therefore, the CD1d interaction with immobilized biotin-GalCer is probably limited to a major contribution from the sphingosine only, leading to a faster off rate. This interpretation is supported by the longer lasting CD1d binding to immobilized biotin-DPPE, with two unmodified lipid chains that can insert into hydrophobic CD1d antigen-binding pockets. The dissociation of the peptide P99 for CD1d also has a relatively fast off rate, similar to the off rate for CD1d binding to the immobilized sphingolipids. This is true for measurements of CD1d peptide binding made in solution previously 28 as well as for measurements made with peptide coupled to the biosensor chip through the NH 2 -terminal biotin or through in an internal cysteine (data not shown). Because of these consistent findings, the dissociation is not likely to be due to modifications of the peptide or details of the experimental set-up. The fast off rate is consistent with the finding that peptide-reactive T cells require a relatively high dose of antigen, in the micromolar range, in order to be stimulated 28 41 . The existence of two very different kinds of mCD1-binding ligands presents a significant biological puzzle 43 . Where in the mCD1 molecule can the two types of ligands be accommodated? The results from competition studies suggest that peptides and lipids do not compete for the same site. This is consistent with our earlier finding that neither P99 nor an ovalbumin peptide could prevent α-GalCer recognition by NK T cells, while α-GalCer did not affect ovalbumin peptide recognition by mCD1-restricted CTLs 41 . Although there is evidence that lipid binds in the groove, we can only speculate about the mechanism for peptide binding. The possibilities include peptide binding outside the groove, or peptide binding to the groove of a subset of CD1d molecules. This peptide-binding CD1d subset might have a particular posttranslational modification, or it might require particular lipids in the groove, in which case the peptide might lie across the top of the groove bound to CD1d and the lipid. In conclusion, using a system in which aggregation of the antigen is minimized, we have definitively established and quantified by SPR the interaction of CD1d or group II CD1 molecules with monomeric glycosphingolipids and phospholipids. We have also demonstrated that the orientation of the sugar on the glycosphingolipid is more important for TCR than for CD1d contact, and that CD1d binds primarily to the hydrophobic acyl chains. Unlike lipid binding to CD1b, these interactions can occur at neutral pH and lead to the formation of antigenic CD1d–lipid complexes in vitro. Much still remains to be learned concerning the means by which the hydrophobic acyl chains are made available for CD1d binding in cells, the structure of the CD1d–lipid antigen complex, and the mechanism of peptide binding to mCD1. | Study | biomedical | en | 0.999996 |
10523606 | Female 6–10-wk-old BALB/c mice purchased from the Animal Resources Centre (Perth, Western Australia) were used in all experiments. Mice were anesthetized with ether or Penthrane (Abbott Australasia Pty. Ltd.) and infected intranasally with 50 μl of PBS containing 5 × 10 5 PFU of the reassortant influenza A virus Mem71 (H3/N1). The viral stock, prepared in embryonated chicken eggs, was provided by Dr. Lorena Brown (University of Melbourne, Victoria, Australia). Normal LN cell suspensions were prepared from pooled paraaortic, axillary, and inguinal LNs of uninfected mice by forcing through stainless steel mesh, and centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech). To prepare lung cell suspensions, mice were killed 7 d after influenza infection, perfused in situ with balanced salt solution to remove blood cells, and the lungs were minced and digested with collagenase and DNase as described previously 49 . Digested lung was passed through stainless steel mesh, and parenchymal cells were isolated by centrifugation over a gradient of Percoll (Amersham Pharmacia Biotech). Cells were stained for flow cytometry with PE-conjugated anti-CD8 mAb (53.6; Becton Dickinson) and biotinylated anti-CD44 mAb (IM7.8; PharMingen), followed by streptavidin-Tricolor (Caltag) and FITC-conjugated anti-CD11a mAb (Sigma Chemical Co.), then resuspended in balanced salt solution with 1 μg/ml propidium iodide. CD8 + CD44 low CD11a low and CD8 + CD44 high CD11a high cells were isolated using a FACS Vantage™ (Becton Dickinson) with Lysys II software, setting a small forward scatter/side scatter gate to collect cells from normal LNs and a large gate to include both small lymphocytes and larger activated cells from lung. Dead and damaged cells were excluded on the basis of propidium iodide uptake and low forward scatter. By reanalysis, purity of the initial bulk-sorted cells for any of the tested staining parameters was generally >96%. Cells were then resorted using the same parameters and deposited using an automated cell deposition unit directly into microwells for RNA extraction or cloning. On average in this study, CD8 + cells with the naive phenotype CD44 low CD11a low from LNs of untreated mice (“normal LN low cells”) comprised 40.5% ( n = 2) of the CD8 + LN cell pool. CD8 + cells comprised 18.1% ± 4.5 of the 15.2 ± 7.6 × 10 6 lung leukocytes recovered per mouse at day 7 after infection. Of the CD8 + fraction, 32.8% ± 4.4 were defined as CD44 low CD11a low (“influenza lung low ”) and 37.0% ± 5.3 were defined as CD44 high CD11a high (“influenza lung high ”), as illustrated previously 49 . All cultures were performed in 15 μl volumes of supplemented DME containing 5 × 10 −5 M 2-ME, 12.5% FCS, and 600 IU/ml recombinant human IL-2 (Cetus Corp.) in mAb-coated Terasaki microwells (Greiner Labortechnik) 50 . For normal LN cells, microwells were coated with purified mAb to CD3∈ (145-2C 11 ; 10 μg/ml), CD8 (53.6; 3 μg/ml), and CD11a (I21/7.7; 5 μg/ml). Antibody coating concentrations were altered to 3 μg/ml anti-CD3, 3 μg/ml anti-CD8, and 5 μg/ml anti-CD11a mAb for optimal cloning of influenza lung low cells, and to 1 μg/ml anti-CD3, 5 μg/ml anti-CD8, and 5 μg/ml anti-CD11a mAb for influenza lung high cells. For experiments where clones were generated under different conditions in parallel, all cultures were initiated with mAb and IL-2, then after 2 d, 5 μl medium was removed and replaced with 5 μl medium containing various combinations of IL-2 (final concentration 600 IU/ml), IL-4 (100 U/ml), and anti–IFN-γ mAb (supernatant of the hybridoma R4-6A2 at a concentration that reduced the activity of purified rIFN-γ by at least 30-fold in assays with WEHI-279 cells). For paired daughter analysis, cultures were initiated with mAb and IL-2, then checked microscopically for viable cells at day 2. Where a parent cell had divided one or two times, individual daughter or granddaughter cells were transferred by micromanipulation into new Terasaki wells coated with the same mAb as above: at least one cell was cultured with IL-2 and one with IL-2 plus 100 U/ml IL-4. After a total of 6 or 7 d, cultures were checked microscopically for clones or subclones, cell numbers were counted, and their RNA was extracted. Clone sizes of ≥200 cells were recorded as 200. Cells were lysed for reverse transcription (RT) 1 using NP-40 by the method of Smith et al. 51 , modified by combining the buffered saline solution and the lysis mix, and by including oligo-dT 15 (18 μg/ml final concentration; Boehringer Mannheim) as a primer instead of random hexamers. Cells were sorted directly into 11 μl of combined buffered lysing solution, or clones and subclones were lysed in microwells by replacement of culture medium with 11 μl of buffered lysing solution. Cell lysates were heated to 65°C then quick-chilled, transferred to microfuge tubes containing 14 μl RT mix comprising 90 mM KCl, 18 mM Tris-HCl, pH 8.0, 12 mM MgCl 2 , 1.4 mM dithiothreitol, 700 μM of each dNTP, 10 U RNAsin, and 2 U AMV reverse transcriptase (Promega Corp.), and incubated at 42°C for 90 min. First strand cDNA products were diluted 1:2.4 in H 2 O, and 10 μl was added to 15 μl PCR mix consisting of 2.5 μl of 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.0, 20 mM MgCl 2 ), 0.25 μl of mixed dNTPs (20 mM; Amersham Pharmacia Biotech), 1 U Ampli-Taq polymerase (Perkin-Elmer), 1 μl of each external oligonucleotide primer (10 μM), and H 2 O. PCR samples were amplified for 40 cycles in the first round, then products were diluted 1:33 in H 2 O, and 2 μl was added to 23 μl PCR mix, as above, for the second round of 30 cycles of PCR with internal intron-spanning primer pairs. PCR cycles were one cycle of 5 min at 94°C, 60 s at 60°C, and 90 s at 70°C, with subsequent cycles of 45 s at 94°C, 60 s at 60°C, and 90 s at 70°C. Primer pairs were as described previously 7 . First round amplifications were performed with the following combinations of primers: IL-4, IFN-γ, and CD3∈; IL-5 and IL-6; IL-2 and IL-10. In the second round, the respective first round products were amplified as follows: IL-4 and IFN-γ together, CD3∈ separately; IL-5 and IL-6 together; IL-2 and IL-10 separately. PCR products were separated by gel electrophoresis, visualized with ethidium bromide, and confirmed by Southern hybridization 50 . All PCR runs included a titration of cloned CD3∈ and cytokine cDNA to monitor cDNA sensitivity (at least 10 −16 g, usually 10 −17 g, with no consistent differences between cytokines) and 10 negative control samples. No PCR products were detected in the negative control samples. Clones were scored as positive for expression of a cytokine if the corresponding PCR product was identified unambiguously by electrophoretic migration at the appropriate size and Southern hybridization with a specific probe. PCR amplifications from clonal cDNA samples were repeated in several experiments. Overall, reproducibility of positive and negative results was 96% for IFN-γ ( n = 306), 94% for IL-4 ( n = 306), 100% for IL-2 ( n = 23), and 98% for IL-10 ( n = 129). Both losses and gains were observed on repetition, suggesting that variations were because the relevant cDNA was limiting in those samples. Previous experiments established that intranasal infection of BALB/c mice with the Mem71 reassortant influenza virus caused accumulation in the lung parenchyma of a population of activated CD8 + T cells that expressed mRNA for IFN-γ and other cytokines ex vivo, secreted high levels of IFN-γ in response to virus-infected cells in vitro, and contained virus-specific CTLs 46 48 49 . At the peak of the cellular response at day 7, both cytokine production and lytic activity were markedly higher in CD8 + T cells from the lung than from the draining mediastinal LNs. Separation on the basis of high CD44 and CD11a expression achieved greater enrichment of this CD8 + effector activity from both lung and LNs than any other combination of activation markers tested 49 . Therefore, these markers were used in the following experiments to distinguish the least activated (CD44 low CD11a low ) and the most activated (CD44 high CD11a high ) lung parenchymal CD8 + T cells from 7-d influenza virus–infected mice (abbreviated as influenza lung low and influenza lung high cells, respectively). Low expression of CD44 and CD11a was also used to define a control CD8 + population of naive phenotype from normal LNs (normal LN low cells). The basal patterns of expression of several cytokine mRNAs in multiple samples of 500 normal LN low , influenza lung low , or influenza lung high cells were analyzed by RT-PCR immediately after FACS ® purification , using methods previously shown to detect cytokine transcripts in single activated T cells 52 53 . No cytokine expression was detected in any of the normal LN samples, whereas almost all samples of influenza lung low and influenza lung high cells expressed IFN-γ mRNA. The influenza lung high population also frequently expressed IL-10 and occasionally one or more of the cytokines IL-2, IL-4, and IL-6. These data show the expected hierarchy of effector activity in the three populations and support earlier evidence that IFN-γ is a major product of CD8 + T cells in the response to influenza virus infection 46 47 49 . To assess the potential of activated T cells to alter their cytokine profiles in response to different signals, a culture system was required in which isolated single cells would divide and could then be subcloned under different conditions before analysis of their cytokine profiles. Ideally cultures would lack accessory cells to enable identification of individual daughter cells for subcloning and to ensure that cytokine mRNAs measured by RT-PCR at the end of culture were derived from the progeny of those daughter cells. We have previously shown that stimulation with coimmobilized anti-CD3 (adsorbed to culture wells at 10 μg/ml), anti-CD8, and anti-CD11a mAb and IL-2 activated most naive CD8 + LN cells to form clones 7 50 54 . When influenza lung high cells were cultured under these conditions, cloning frequencies and sizes were low. A series of optimization experiments showed that reduction of the plate-coating concentration of anti-CD3 mAb to 1 μg/ml increased cloning efficiencies of these cells to a mean of ∼25% (data not shown). No further improvement was obtained by varying the other mAb concentrations. Maximal cloning efficiencies of ∼75% were obtained for influenza lung low cells using 3 μg/ml anti-CD3 mAb, comparable to those achieved for normal LN CD8 + T cells using 10 μg/ml (∼80%). Average clone sizes peaked at 6–7 d of culture, after which many clones died. The ability of exogenous IL-4 to influence cytokine profile development in influenza lung high cells was tested by culturing individual cells with immobilized mAb and IL-2. After 2 d, cultures were supplemented with medium, IL-4, or IL-4 and anti–IFN-γ mAb, to mimic the protocol used in the paired daughter analyses shown below. All clones were counted and harvested on day 6 for RT-PCR analysis of their cytokine profiles . Average cloning efficiencies, clone sizes, and frequencies of IFN-γ, IL-2, and IL-10 mRNA expression were comparable among the three sets of cultures ( P > 0.05). However, addition of IL-4 at day 2 markedly increased the frequency of IL-4 expression ( P < 0.01). No further increase was observed when anti–IFN-γ mAb was also added. This and other preliminary cloning experiments suggested that a significant proportion of influenza lung high and influenza lung low cells could respond to IL-4 by expressing this cytokine, providing provisional evidence for the presence of multipotential cells in both populations. RT-PCR analyses of IL-5 and IL-6 expression by the clones in Fig. 2 and many other clones derived from influenza lung high or influenza lung low cells in the presence or absence of exogenous IL-4 rarely detected either of these cytokine mRNAs, and are therefore not presented. As shown above in Fig. 2 , IL-4 mRNA–expressing clones could be generated from influenza lung high cells in the presence of exogenous IL-4. However, this experimental protocol did not distinguish whether these clones developed de novo from multipotential cells or whether they were expanded from committed cells that had been primed to produce this cytokine in vivo. To address this question directly, paired daughter analyses were performed in which CD8 + cells from normal LNs and infected lung were cultured with antireceptor mAb and IL-2. After 2 d, when significant numbers had undergone one or two divisions, individual daughter or granddaughter cells were transferred into new cultures with or without exogenous IL-4. Subclones were analyzed for CD3∈ and cytokine mRNAs 4–5 d later. Table summarizes the mean cloning efficiencies, subclone sizes, and frequencies of CD3∈ and cytokine mRNA detection in subclones for all paired daughter analyses performed in this way with cells from normal LNs and influenza-infected lung. In general, influenza lung high cells subcloned at lower frequency and formed smaller subclones by day 6 than normal LN low or influenza lung low cells, indicating that even those influenza lung high cells that divided in the first 2 d were less able to undergo extended proliferation than the other populations. Most subclones of all cell types expressed IFN-γ mRNA, whereas IL-2 was preferentially expressed by normal LN low and influenza lung low subclones. As observed in our previous analysis of naive C57BL/6 CD8 + LN T cells 7 , some subclones of all cell types expressed IL-4 and/or IL-10 mRNA in the absence of exogenous IL-4, but frequencies were significantly higher among subclones grown with IL-4. IL-4 expression frequencies were more strongly affected by exogenous IL-4 than expression frequencies of any of the other cytokines tested. Fig. 3 shows the cytokine profiles of 12 families of subclones generated from each subset. Three or four subclones per family are illustrated in those cases where the original cell had divided twice by day 2 and more than two of its progeny yielded a CD3∈ + subclone. These examples illustrate several features of the whole data set. First, IFN-γ, IL-4, and IL-10 could be detected in almost any combination. Second, many subclones displayed identical cytokine profiles within a family regardless of culture conditions, while others displayed differences in the presence and absence of added IL-4; paired subclones grown in the same conditions also sometimes displayed different profiles. Third, IFN-γ expression was more frequently gained than lost when subclones were grown with IL-4. Finally, in many families where a subclone grown without IL-4 expressed IL-4 or IL-10, its sibling grown with IL-4 also produced this cytokine. The outcome of all paired daughter analyses for IL-4 and IL-10 expression is summarized in Table . Many families in all groups displayed a “−/−” phenotype for IL-4 expression, i.e., this cytokine was not expressed by subclones grown either in the absence or in the presence of IL-4. Several other families included subclones that expressed IL-4 in one (+/− and −/+ phenotypes) or both (+/+) types of culture. If detection of IL-4 mRNA were entirely dependent on addition of IL-4 to the cultures, the frequency of families with a −/+ phenotype would give a direct minimal estimate of the frequency of IL-4–inducible multipotential parent cells. However, in this study, there was a measurable level of IL-4 expression that occurred independently of exogenous IL-4, indicated by the frequency of +/− and +/+ families. Therefore, we estimated the frequency of multipotential parent cells by subtracting this IL-4–independent frequency from the sum of the −/+ and +/+ frequencies ( Table ). Whether these calculations are based on expression of IL-4, IL-10, or both cytokines, they indicate a loss of multipotentiality as CD8 + T cells differentiate from the naive state in normal LNs to the activated state in the infected lung parenchyma. Other analyses indicated a concomitant rise in the proportion of committed cells with differentiation from the naive to the activated state. For example, in all groups, 60% or more of subclones grown in IL-2 alone expressed IFN-γ without IL-4. Table summarizes the IFN-γ/IL-4 phenotypes of their siblings grown in IL-2 plus IL-4 and reveals that a markedly higher proportion displayed the same IFN-γ 1 IL-4 − phenotype in families derived from the most activated lung population than in families derived from normal LNs. To address this issue in another way, the frequency data in Table were subjected to contingency table testing to determine whether expression of IL-4 or IL-10 by subclones grown with added IL-4 was statistically independent of expression of the same cytokine by their siblings grown without IL-4. As shown in the table, the frequencies of each IL-4 phenotype among normal LN low and influenza lung low subclone families were close to those expected by chance, indicating that IL-4 expression was independently regulated among subclones within these families. By the same criterion, IL-10 expression was also independently regulated within families of normal LN low subclones. This was not the case for the other sets of data. The frequencies of IL-4 −/− and +/+ families of influenza lung high cells and the frequencies of IL-10 −/− and +/+ families of both influenza lung low and influenza lung high cells were significantly higher than expected by chance. This suggests that some parents of these families were committed to the expression or nonexpression of IL-4 and/or IL-10; their daughter cells were therefore unaffected by exposure to IL-4. Here we show that an activated, type 1–polarized, effector population of CD8 + T cells isolated from the lungs of influenza virus–infected mice is not terminally differentiated. Although it includes cells that appear functionally committed, it also contains a significant proportion of cells able to proliferate and express IL-4 and/or IL-10 when exposed to IL-4 in vitro. This indicates that some CD8 + T cells in an effector site can retain the potential to alter their cytokine profile in response to local stimuli. A key feature of the experimental design of this study was the analysis of paired daughter cells, in which the ability of sister cells to give rise to subclones with different cytokine profiles in different culture conditions demonstrates the multipotentiality of their parent. This protocol has several advantages over conventional bulk culture methods used by several groups to assess the stability of cytokine profiles 15 16 19 36 . First, it allows the fate of every cultured cell to be monitored, so that selective growth can be distinguished from reprogramming in response to an exogenous stimulus. Second, it enables the frequency of multipotential (programmable) cells to be estimated in heterogeneous populations. In the present study, these estimates suggested a progressive decline in the frequency of multipotential cells from normal LN low to influenza lung low to influenza lung high cells ( Table ), in parallel with the observed rise in their expression of cytokine genes in vivo . Third, culture of single parent cells and subcloning of their progeny isolate these cells from the effects of cross-regulatory cytokines from other cells, which might override the differentiative effects of IL-4 in a bulk culture 44 and mask low frequencies of multipotential cells. We have previously used paired daughter analysis to evaluate the multipotentiality of CD8 + CD44 low T cells from the LNs of normal C57BL/6 mice and found that almost 90% of cells were at least bipotential for the expression of six different cytokines; based on the expression of IL-4 alone, 50% of parent cells in that study were multipotential 7 . A similar figure (51%) was obtained here for CD8 + CD44 low CD11a low cells from LNs of normal BALB/c mice. Although it seems likely that most, if not all, naive CD8 + T cells are multipotential for expression of IL-4 and other cytokines, these minimal estimates establish a baseline for comparison with other populations. Subcloning efficiencies were high for normal LN cells in the presence and absence of IL-4, suggesting that the parent cells analyzed were representative of the larger pool and arguing against the selective expansion of committed cells by one or other culture condition. This study is, to our knowledge, the first frequency analysis of multipotentiality in T cells activated in vivo and has yielded both expected and unexpected results. Not surprisingly, the two CD8 + populations isolated from 7-d influenza virus–infected lung parenchyma differed markedly by several criteria from their counterparts of naive phenotype isolated from normal LNs. Although the influenza lung low population was selected for the same surface markers as the normal LN low cells, their frequency of IFN-γ expression ex vivo was at least three orders of magnitude higher . Their subcloning efficiency was almost as high as the LN population, but average subclone sizes and frequencies of IL-2, IL-4, and IL-10 expression were lower both in the absence and presence of exogenous IL-4. The influenza lung high population displayed greater differences from normal cells, including high expression of IL-10 as well as IFN-γ ex vivo, and yet lower subcloning efficiency, subclone size, and subclone frequencies of IL-2, IL-4, and IL-10 expression. The progressive loss of proliferative potential from normal LN low to influenza lung low to influenza lung high cells, in parallel with increased effector activity, mirrors the experience of many investigators that effector populations are more difficult to clone than naive populations, and probably reflects both senescence and increased sensitivity to activation-induced cell death 43 55 56 . While the intermediate status of influenza lung low cells implies that they are less differentiated than influenza lung high cells and therefore might be their precursors, the actual relationship between these two populations is not known. The loss of proliferative potential from the naive state to the activated effector state introduces an important consideration in interpreting the concomitant decline in multipotential cell frequency observed here. Since the assessment of multipotentiality depended on cloning and subcloning parent cells, no information is available about the fraction of influenza lung cells, especially those of CD44 high CD11a high phenotype, that failed to proliferate in these cultures. Other experiments will be necessary to determine whether individual activated T cells can change their cytokine profile without cell division. However, since prolonged survival and clonal expansion are likely to improve the chances of de novo induction of cytokine gene expression by exogenous IL-4, as recently shown for naive CD4 + T cells 26 57 , we expect multipotential cells to be found preferentially among the clonogenic cells. If this is the case, the differences in multipotentiality between influenza lung high cells and the other two CD8 + populations analyzed here would be compounded by their differences in clonogenicity and would therefore be greater than indicated by the estimates in Table . The detection of IL-4 and IL-10 mRNAs at higher frequencies in subclones grown with IL-4 than in those grown without IL-4 was not due to any detectable enhancement of subclone size. Although mean subclone sizes varied between the three populations, they were not significantly affected within each population by exogenous IL-4 ( Table ). Moreover, the minimum, median, and mean subclone sizes of IL-4 + and IL-10 + subclones were generally lower among those grown with IL-4 than those grown without IL-4 (data not shown). This suggests that IL-4 increased the probability of expression of these cytokines at the single-cell level, accelerating the onset of their expression as a clone expands 58 . In addition to demonstrating that some cells retain their multipotentiality after activation in vivo, this study provided evidence for a concomitant increase in the proportion of committed cells. Sharing of the IFN-γ + IL-4 − phenotype by siblings grown in the absence and presence of added IL-4 was progressively more common among families derived from normal LN low , influenza lung low , and influenza lung high cells. Statistical analysis also suggested that some influenza lung high cells were committed to IL-4 or IL-10 expression or nonexpression. It is not possible from this analysis to conclude whether a given family whose subclones displayed the same IL-4 or IL-10 phenotype in the absence and presence of exogenous IL-4 (−/− or +/+ phenotypes, as shown in Table ) was derived from a committed parent. However, χ 2 testing indicated that influenza lung high cells gave rise to such families at a significantly higher frequency than expected by chance. By contrast, a similar analysis of the data for normal LN low cells suggested that cytokine expression was regulated independently in related subclones, as expected if most of these cells are multipotential. The result for influenza lung low cells was mixed, indicating independent regulation of IL-4 expression but higher than expected occurrence of −/− and +/+ families for IL-10 expression. Collectively, these data indicate substantial heterogeneity within the most activated fraction of lung CD8 + cells, both in proliferative potential and in their flexibility to change cytokine profile in response to exogenous IL-4. This is despite the fact that the CD44 high CD11a high fraction contains essentially all the IFN-γ–producing effector cell activity detected in the lung CD8 + fraction at this stage of influenza infection 48 49 and might be expected to comprise terminally differentiated type 1 cells. Influenza lung high cells are likely to be heterogeneous in other ways not assessed here. Most importantly, since these experiments have not yet been done with populations selected for peptide–MHC tetramer binding, the antigen specificity of the cells we have analyzed is not known and may include a range of TCR affinities for several influenza epitopes as well as irrelevant specificities. However, in experiments by other groups with the A/HKx31 influenza virus strain, cells specific for the immunodominant nucleoprotein epitope comprised 3–12% of CD8 + cells in the bronchoalveolar lavage at day 7 59 and ∼20% of CD8 + T cells and 94% of CD44 high CD8 + T cells isolated from lung tissue at day 8 60 . Virus-specific cells are therefore likely to be a substantial fraction of the CD44 high CD11a high CD8 + T cells isolated from lung tissue at the peak of the response in our study. When methods become available to monitor both antigen specificity and relative avidity in polyclonal systems, it will be possible to determine whether multipotentiality persists among the most reactive cells or is limited to those receiving the weakest antigenic signals. In addition, isolation of antigen-specific T cells will allow multipotentiality to be analyzed in the small populations of cells that persist in the lung after the peak of the primary response and in secondary lymphoid organs as memory cells. The proliferative history of the cells isolated from infected lung is also unknown. Others have shown marked heterogeneity in cell division number among responding CD4 + T cells in draining LNs in the first few days after immunization 61 . We expect that both the influenza virus–specific and other activated T cells found in the lung 7 d after infection would also vary in their history of division before and after recruitment to the lung. It is not yet known whether there is any relationship between division history and multipotentiality. Previous studies of peptide-stimulated transgenic CD4 + T cells have generally shown a loss of flexibility at the population level by the second or third cycle of in vitro stimulation, but proliferative responses and division numbers were not reported and these findings cannot be readily extrapolated to the CD8 + T cell response to a replicating antigen in vivo where clonal burst sizes are likely to be higher. However, our previous observation that multipotential cells persisted in some type 1 CD8 + T cell clones through as many as 13–15 cell divisions over 7 d in vitro raises the possibility that even the most responsive, influenza virus–specific CD8 + T cells activated over the same time frame in vivo might also retain some flexibility in their cytokine profiles. The molecular mechanisms underlying cytokine profile commitment in T cells are beginning to be unraveled. Loss of responsiveness to the counter-regulatory cytokines IL-12 and IL-4 is one mechanism that perpetuates existing cytokine profiles 21 22 23 24 , but it is not yet known whether the reported changes in IL-12 receptor expression and IL-4 receptor function are causally or temporally associated with commitment. Similarly, while certain signal transducers and transcriptional activators selectively activate or antagonize type 1 or type 2 cytokine expression 29 30 31 32 , their relationship to the loss of multipotentiality is not clear. Changes in CpG methylation and chromatin conformation of cytokine and other regulatory gene promoters are likely to be important events in enabling transcription 25 27 28 . Recent work showing that demethylation of the IFN-γ promoter after primary activation of CD8 + T cells can be faithfully inherited through at least 16 cell divisions 27 suggests demethylation as a stable marker of gene activation. Again, however, its relationship to commitment is unknown. Key issues are whether this or any other candidate commitment event is irreversible and whether it can be permanently prevented from activating a silent cytokine gene. In conclusion, we have found that the activated effector CD8 + T cell population in the lungs of influenza virus–infected mice contains both committed and multipotential cells. The presence of multipotential cells indicates that cytokine profiles are not irreversibly programmed during priming in the draining LN. Until now, it has been unclear whether differences in T cell cytokine profiles sometimes noted between tissue sites during an immune response were entirely due to preferential migration of differentiated effector cells 48 62 63 64 . Our finding that some activated T cells can proliferate and express new cytokine genes in response to exogenous signals after recruitment to an effector site allows the possibility of substantial reshaping of the T cell response by the local environment. It remains to be seen whether this flexibility can be exploited to alleviate pathological cytokine responses. | Study | biomedical | en | 0.999999 |
10523607 | The SCB29 cell line, which expresses the pre-TCR on the cell surface, was described previously 23 . Single-cell suspensions from thymi were prepared by compression between ground glass slides followed by centrifugation on a density gradient of Lympholyte-M (Cedarlane Labs., Inc.). Streptavidin–Cy-Chrome, streptavidin–PE, and mAbs to the following mouse antigens were purchased from PharMingen: CD3∈ (clone 145-2C11), CD4 (L3T4), CD8α (53-6.7), CD25 (7D4), CD44 (IM7), Thy1.2 (53-2.1), Mac-1 (M1/70), Gr-1 (RB6-8C5), and TCR-β (H57-597). Anti-CD25 mAb (3C7) conjugated to PE and anti-CD44 mAb conjugated to FITC were purchased from Sigma Chemical Co. Anti-pTα mAb 2F5 was raised against the extracellular (Ig-like) domain as described previously 24 . In the case of pre-TCR surface detection, thymocytes were enriched for the CD4 − CD8 − subset by depletion of CD4/CD8-positive cells using Dynabeads (Dynal). pTα (2F5) and TCR-β (H-57) biotinylated antibodies were revealed with streptavidin–PBXL-3 (Martek Biosciences). NK and NK-T cells were gated out with DX-5, NK1.1, or 2B4 antibodies (PharMingen). Cytoplasmic staining for TCR-β was performed as previously described 25 . Cells were surface stained and analyzed on a FACSCalibur™ flow cytometer (Becton Dickinson) as previously described 26 . Intracellular staining for TCR-β was performed on CD25 + sorted cells as previously described 12 . Data on 5 × 10 5 –2.0 × 10 6 viable, nonerythroid cells (as determined by forward versus side scatter) were collected for each sample. FACS™ analysis was performed on cells from groups of at least three mice aged 2–4 wk. The generation of TCR-α −/− , pTα −/− , RAG-2 −/− , and SLP-76 −/− mice was previously described 2 4 17 27 . Mice transgenic for the TCRVα13 and TCRVβ8.2 chains of the OVA-specific mouse T cell hybridoma DO11.10 were a gift of Dr. Dennis Loh (Hoffmann-LaRoche, Inc., Nutley, NJ; reference 28). Screening for expression of the transgenic TCR was done by PCR analysis of tail DNA, using primer sequences provided by Dr. K. Murphy (Washington University, St. Louis, MO), and by FACS™ analysis, using the antiidiotypic mAb KJ126, provided by Dr. K. Murphy. Screening for disruption of the SLP-76 gene was done as previously described 17 . Genomic DNA was isolated according to published procedures 29 . Vβ to DJβ rearrangements were assessed as described in detail in reference 16. In brief, upstream primers were located on the Vβ segments 5, 8, and 10, and the downstream primer was located 3′ of Jβ2.7. After amplification of the rearrangements by PCR, the products were separated on agarose gels, transferred to nylon membranes, and hybridized with a Jβ2.7-specific, 32 P-labeled oligonucleotide probe. Hybridizing bands were scanned using a PhosphorImager (Molecular Dynamics). Amplification of a fragment of the nonrearranging Cμ gene served as a loading control. TCR-β + ic CD25 + small single cells from SLP-76 −/− mice were sorted using a FACSVantage™ equipped with an automatic cell deposition unit (Becton Dickinson). DNA from single cells was prepared as previously described 12 . TCR-β gene rearrangements were amplified by a seminested two-step PCR protocol 12 30 . In the first step, both alleles were amplified simultaneously by addition to each tube of 35 μl of a mixture containing dNTPs, buffer, and Taq polymerase at 0.5 U/sample (Perkin-Elmer Corp.). 18 5′ primers (3 pmol of each) homologous to 16 Vβ gene families and Dβ1 and Dβ2 genes, and 2 3′ primers (3 pmol of each) that primed downstream of the Jβ1 and Jβ2 cluster sequences, respectively, were used 12 . In addition to the previously described primers, 5′ primers specific for Vβ3 (5′-ACGattctctgctgagtgtcctcc-3′), Vβ9 (5′-gaacagg-gaagctgacacttttgag-3′), and Vβ17 (5′-gtcctgaaaaagggcacactgcct-3′) were used. The first round of amplification was performed in a final volume of 60 μl for five cycles, in which the annealing temperature decreased from 68 to 60°C, followed by 25 cycles of amplification (30 s at 94°C, 1 min at 58°C, 1 min at 72°C), and finally 5 min at 72°C. For the second round of amplification, 1 μl of the first PCR product was transferred into separate tubes, each containing a single 5′ primer in combination with the nested Jβ2 or Jβ1 3′ primer (10 pmol of each), dNTP, reaction buffer, and 1 U of Taq polymerase in a final volume of 20 μl. Amplification was then carried out for 35 cycles, following the procedure of the first PCR. Vβ and Jβ were identified by migration of the total PCR product on a 1.5% ethidium bromide–stained agarose gel, and positives were purified using Geneclean III (Bio 101). Direct sequencing of the PCR products was performed using the Ready Reaction DyeDeoxy Terminator Cycle sequencing kit (Applied Biosystems, Inc.) and sequenced by automated sequencing (Applied Biosystems, Inc.). Surface expression of the pre-TCR is required for TCR-β allelic exclusion (for review see reference 31 ). In the analysis of allelic exclusion in SLP-76 −/− mice, it was therefore important to ascertain expression of the pre-TCR by SLP-76–deficient thymocytes. In previous experiments, we have detected low levels of expression of CD3 and TCR-β on SLP-76 −/− CD4 − CD8 − DN thymocytes, suggesting that the pre-TCR is expressed on the cell surfaces of these immature thymocytes 17 . To extend these observations, we repeated these experiments with a fluorochrome reagent, streptavidin–PBXL-3 (Martek Biosciences), that gives much higher signal intensity than conventional reagents (i.e., streptavidin–FITC or –APC) when used in combination with biotinylated antibodies. This can be clearly seen by staining the SCB29 cell line, which expresses the pre-TCR on the cell surface 23 , with biotinylated mAb to TCR-β (H57) or pTα (2F5), followed by incubation with either streptavidin–FITC or –PBXL-3 . Incubation with irrelevant mAbs of the same Ig class produces some slight background that, however, is clearly distinct from the staining obtained with antibodies specific for cell surface–expressed proteins . In subsequent experiments, CD4 − CD8 − DN thymocytes from wild-type (WT), TCR-α −/− , SLP-76 −/− , pTα −/− , and RAG-2 −/− mice were examined for TCR-β and pTα expression. Thymocyte development in SLP-76 −/− mice is blocked at the stage of CD25 + CD44 − DN cells. CD25 − CD44 − cells, which represent a more mature stage of DN thymocytes, are absent in SLP-76 −/− mice 17 . We therefore focused our analysis on CD25 + CD44 − DN cells. DN thymocytes were stained with directly labeled antibodies against CD44 and CD25, as well as biotinylated antibodies against either TCR-β or pTα followed by streptavidin–PBXL-3, and gated populations of CD25 + CD44 − cells were analyzed for TCR-β and pTα expression. The histograms in Fig. 2 show that, as expected, mAb to TCR-β did not stain thymocytes from RAG-2 −/− mice and mAb to pTα did not stain thymocytes from pTα −/− mice when compared with control antibodies. This further confirms the specificity of the staining. There was negligible, if any, staining of RAG-2 −/− thymocytes with anti-pTα mAb or of pTα −/− thymocytes with anti–TCR-β mAb. CD25 + CD44 − DN thymocytes from SLP-76 −/− mice stained with both antibodies to the same or slightly higher level as CD25 + CD44 − DN cells from TCR-α −/− mice . DN thymocytes from TCR-α −/− mice were used as controls to eliminate the possibility of staining TCR-β proteins that are transported to the cell surface as α/β TCRs. The surface expression of TCR-β and pTα chains on CD25 + CD44 − as well as CD25 − CD44 − thymocytes from TCR-α −/− mice and WT controls was identical (data not shown). We next examined intracellular TCR-β protein expression in SLP-76 −/− thymocytes. Fig. 3 shows that SLP-76–deficient CD25 + thymocytes expressed lower levels of intracellular TCR-β protein than WT CD25 + thymocytes . Decreased levels of expression of intracellular TCR-β protein were also observed in CD25 + thymocytes from pTα −/− mice, which are also blocked at the CD25 + CD44 − DN stage 2 . The CD25 − TCR-β + cells in SLP-76 −/− mice may represent γ/δ cells that express cytoplasmic TCR-β chain, as has been shown recently in pTα −/− mice 32 . These results suggest that SLP-76 is not required for assembly and surface expression of TCR-β chains. However, it is required for the generation of CD25 + DN cells that express high levels of TCR-β protein intracellularly. Introduction of a TCR-β transgene into SCID or RAG-deficient mice drives the development of DN thymocytes into DP cells 33 34 . To examine the effect of introduction of TCR transgenes on thymic development in SLP-76 −/− mice, they were bred with mice transgenic for the TCRVα13 and TCRVβ8.2 chains of the OVA-specific mouse T cell hybridoma DO11.10. The transgenes are concordantly expressed early in thymocyte development at the DN stage 28 , and their introduction into a RAG-deficient background drives T cell development to the DN and single-positive stage 33 34 . SLP-76 −/− TCR-α/β–transgenic (SLP-76 −/− -tg) F2 mice were identified by PCR analysis of tail DNA. The effect of introduction of the DO11.10 TCR-α/β transgenes on thymic development in SLP-76 −/− mice was assessed by examining the thymi of SLP-76 −/− -tg mice for cellularity, expression of CD3, TCR-β, CD4, and CD8, and distribution of CD25 + and CD44 + DN subsets. As previously described, thymic cellularity in SLP-76 −/− mice was severely reduced to ∼1% of that of heterozygous littermates, and there were no detectable DP nor single-positive thymocytes 17 . Introduction of the transgenic TCR into the SLP-76 −/− background did not increase thymic cellularity (1.30 ± 0.18 × 10 6 cells in SLP-76 −/− -tg mice versus 1.62 ± 0.75 × 10 6 cells in SLP-76 −/− mice; n = 4 in each group), but resulted in an increase in CD3 + TCR-β + thymocytes, consistent with surface expression of the transgene . The transition from DN to DP cells remained severely impaired, as we could detect no or very few DP thymocytes in SLP-76 −/− -tg mice . Analysis of the B220 − Mac-1 − Gr-1 − CD3 − CD4 − CD8 − DN compartment for the expression of CD25 and CD44 revealed that introduction of the transgenic TCR failed to overcome the block in thymic development at the CD25 + CD44 − stage present in SLP-76 −/− mice . These results suggest that the failure of DN cells from SLP-76 −/− mice to progress to the DP stage is due to deficient SLP-76–mediated signaling and cannot be overcome by surface expression of the transgenic TCR. Having established that the pre-TCR is expressed on DN thymocytes from SLP-76 −/− mice, we proceeded to address the issue of whether TCR-β allelic exclusion takes place in the absence of SLP-76. There are two ways in which feedback inhibition by the pre-TCR resulting in TCR-β allelic exclusion has been analyzed: one is the inhibition of endogenous Vβ rearrangements by TCR-β transgenes, and the other is the analysis of both TCR-β alleles in single cells. We initially examined inhibition of Vβ rearrangements by TCR transgenes. In normal thymocytes, introduction of functionally rearranged TCR-β transgenes leads to inhibition of rearrangements in the endogenous TCR-β locus at the V to DJ step 35 . Furthermore, introduction of a TCR-β transgene in pTα −/− and p56 lck−/− also leads to inhibition of endogenous TCR-β rearrangements 13 36 . To examine if introduction of the transgenic TCR into SLP-76–deficient thymocytes could also inhibit endogenous Vβ to DJβ rearrangements, we used a semiquantitative PCR assay as previously described 16 37 . In these experiments, Vβ to DJβ2 rearrangements involving representative Vβ segments (Vβ5, Vβ8, and Vβ10) were amplified from genomic DNA samples isolated from DN thymocytes of TCR-transgenic or nontransgenic SLP-76 +/− or SLP-76 −/− mice. The identity of the resulting DNA fragments corresponding to rearrangements between a particular Vβ and one of the Jβ segments was confirmed by Southern blot analysis with a Jβ2.7-specific probe. In agreement with our previously published observations 17 , Vβ to DJβ rearrangements were readily detectable in both SLP-76 +/− and SLP-76 −/− thymocytes . As expected, introduction of the TCR-α/β transgene into SLP-76 +/− thymocytes almost completely blocked Vβ to DJβ rearrangements of the endogenous TCR-β genes . In contrast, rearrangements of the endogenous TCR-β genes occurred at comparable levels in both TCR-transgenic and nontransgenic SLP-76 −/− thymocytes . These results suggest that SLP-76 is essential for transgene-mediated inhibition of TCR-β locus rearrangement. Introduction of a TCR-β transgene into pTα −/− mice 13 inhibits rearrangements of endogenous Vβ segments, although single-cell PCR analysis of Vβ rearrangements reveals a failure of TCR-β allelic exclusion in pTα −/− mice 12 . This suggests that expression of the transgene provides signals for inhibition of TCR-β rearrangement that differ quantitatively and/or qualitatively from physiologic signals delivered via the pre-TCR and may not reflect authentic allelic exclusion. To assess the status of TCR-β allelic exclusion in unmanipulated SLP-76 −/− mice, we examined Vβ rearrangements in single CD25 + CD44 − thymocytes from these mice. In WT mice, only ∼40% of CD25 + TCR-β + thymocytes rearrange Vβ on both alleles due to the feedback inhibition by the first productively rearranged allele that prevents further rearrangement on the other allele 12 . In pTα −/− mice, this feedback inhibition fails, and hence a greater population of cells (60%) exhibits two completely rearranged VDJβ alleles 12 . When single CD25 + CD44 − DN cells from SLP-76 −/− mice that expressed cytoplasmic TCR-β chains were sorted and analyzed, a similar proportion of the cells (60%) was found to contain rearranged VDJβ on both alleles ( Table ). T cells with two productive Vβ rearrangements are a rare event in WT mice; <5% of normal cells with two Vβ rearrangements contain two productive alleles 12 . When the alleles from SLP-76 −/− thymocytes with two Vβ rearrangements were sequenced, it was found that 31% of the cells with two Vβ rearrangements had two productive alleles ( Table ). The Vβ usage and the sequences of the two in-frame alleles are shown in Table and Table . They provide no indication for the selection of certain Vβ alleles in allelically included cells, even though it is difficult to entirely rule out this possibility due to the relatively small sample size. Taken together, the data obtained by the single-cell PCR analysis indicates that allelic exclusion at the TCR-β locus requires the SLP-76 adaptor protein. The results of this study indicate that the adaptor protein SLP-76 is not only essential for thymocyte expansion and maturation from the DN to the DP stage but is also essential for TCR-β allelic exclusion. Despite expression of surface pre-TCR, thymocytes from SLP-76 −/− mice, which are blocked at the CD25 + CD44 − DN stage, failed to exhibit TCR-β allelic exclusion, as evidenced by single-cell PCR. Neither the defect in the feedback inhibition of endogenous TCR-β locus rearrangements nor the block in thymic development could be overcome by introduction of a functional TCR-α/β transgene. We have ascertained that CD25 + CD44 − DN thymocytes from SLP-76 −/− as well as WT mice express the pTα and TCR-β chains of the pre-TCR complex . As the 2F5 antibody we used is directed against the extracellular Ig-like domain of pTα, it is also clear from our studies that it is the form of pTα that expresses the extracellular Ig-like domain that reaches the cell surface. DN thymocytes from SLP-76 −/− mice expressed equal or slightly higher amounts of TCR-β chains on their surfaces compared with TCR-α −/− or WT mice (data not shown). The fact that DN thymocytes from both SLP-76 −/− and pTα −/− mice express lesser amounts of intracellular TCR-β chains than WT mice raises the possibility that signals through the pre-TCR mediated by SLP-76 may be important in upregulating TCR-β chain expression. The presence of a very small population of CD25 − TCR-β + DN thymocytes in SLP-76 −/− thymocytes (∼2%) is consistent with the presence of a very small population of CD25 − CD44 − triple-negative cells in these mice . The equal or slightly higher surface expression of TCR-β and pTα in SLP-76 −/− CD25 + CD44 − DN thymocytes may then be accounted for by the developmental arrest that results in the accumulation of small, noncycling CD25 + CD44 − DN cells, allowing these cells to bring a relatively higher amount of the pre-TCR to the cell surface. Alternatively, in the absence of SLP-76, internalization and subsequent degradation of the pre-TCR, which is dependent on activation of serine/threonine and tyrosine kinases 38 , may be retarded. Introduction of TCR-β transgenes into rearrangement-deficient mice drives proliferative expansion and differentiation of DN to DP cells 33 34 . In contrast, introduction of functional TCR-β transgenes into SLP-76 −/− mice failed to increase thymic cellularity and failed to overcome the block in the development of CD25 − CD44 − DN cells and in the transition from DN to DP cells . We had previously shown that treatment of SLP-76 −/− mice with anti-CD3∈ mAb induced the appearance of only a few DP thymocytes 17 , suggesting that an SLP-76–independent pathway for DN to DP transition may exist. Such a pathway could involve linker for activation of T cells (LAT)-Grb2-Sos–mediated activation of Ras, as a transgene encoding an active form of Ras (Ras V12 ) has been shown to drive the development of DP cells in the RAG −/− background 39 . It appears from our data that expression of a transgenic TCR may not be sufficient to activate an SLP-76–independent pathway of thymocyte maturation. Introduction of TCR-α/β transgenes into the SLP-76 background failed to suppress endogenous TCR-β rearrangement . It has been suggested that inhibition of endogenous TCR-β rearrangement by TCR-β transgenes in pTα −/− mice might be artifactual and might reflect activation of downstream pathways not ordinarily engaged by the endogenous pre-TCR 12 . Indeed, whereas introduction of a TCR-β transgene into the pTα −/− background shuts off endogenous TCR-β rearrangement, single-cell analysis in pTα −/− mice reveals failure of TCR-β allelic exclusion 12 13 . As introduction of TCR-β transgenes into pTα −/− mice also drives the maturation of DN cells into DP cells and their proliferation, premature transgene-generated signals might accelerate development of normal cells through stages at which the endogenous TCR-β genes are assembled, leading to a block in rearrangement 40 . The failure of TCR-α/β transgenes to suppress endogenous TCR-β rearrangement and induce maturation and expansion of DN cells in SLP-76 −/− mice suggests that transgene-generated signals that lead to maturation and expansion of DN cells and to feedback inhibition of endogenous TCR-β rearrangement are strictly dependent on SLP-76. Normally, 60% of surviving thymocytes carry only a single Vβ to DJβ rearrangement, whereas the remaining 40% carry one nonproductive and one productive Vβ to DJβ rearrangement 35 . The presence of >40% of cells with Vβ to DJβ rearrangement on both alleles indicates a violation of TCR-β allelic exclusion. Single-cell PCR analysis revealed that 60% of CD25 + CD44 − TCR-β + DN thymocytes from SLP-76 −/− mice have undergone Vβ to DJβ rearrangement on both TCR-β alleles ( Table ). This indicates that TCR-β allelic exclusion is defective in the absence of SLP-76 and suggests that SLP-76 is essential for the transduction of the physiologic pre-TCR signal that inhibits V to DJ rearrangement on the second TCR-β allele. Due to feedback inhibition of TCR-β locus rearrangement by a productively rearranged allele 41 , <3% of normal T cells carry two productive Vβ gene rearrangements 12 . In the absence of feedback inhibition, it is expected that in 20% of cells that rearrange two alleles, both alleles are rearranged productively. When the alleles from SLP-76 −/− thymocytes with two Vβ rearrangements were sequenced, it was found that in 31% of the cells, both rearrangements were productive, i.e., in frame ( Table ). This is almost exactly the same percentage that was found in pTα −/− mice 12 . The discrepancy between the observed and the predicted fraction of cells with two productive Vβ rearrangements may be due to selection against cells that make a productive rearrangement on the second allele only, due to lower TCR-β staining intensity, when cells are sorted for expression of cytoplasmic TCR-β chains. There was no indication for the selection of specific Vβ alleles in allelically included cells in either SLP-76 −/− mice ( Table and Table ) or pTα −/− mice 12 . The presence of a significant proportion of SLP-76 −/− thymocytes with two productively rearranged TCR-β alleles documents an essential role of SLP-76 in allelic exclusion at the TCR-β locus. The fact that similar percentages of cells with two in-frame alleles were found in SLP-76 −/− and pTα −/− mice suggests that the pre-TCR is the most important receptor that mediates feedback inhibition of Vβ rearrangement under physiological conditions. Recent data suggests that activation of Ras results in differentiation and expansion of DN thymocytes but not in TCR-β allelic exclusion 16 . Thus, suppression of TCR-β gene rearrangements appears to require the activity of an additional and/or complementary pathway. As SLP-76 −/− thymocytes are arrested at the DN stage and also fail to suppress endogenous Vβ to DJβ rearrangements, even after introduction of TCR-α/β transgenes, SLP-76 is likely to be critical for TCR-mediated activation of both Ras-dependent and Ras-independent pathways. | Study | biomedical | en | 0.999996 |
10523608 | CV1 African green monkey kidney cells were used for propagation and titration of the virus. For studies of K b expression and T cell activation, NIH 3T3 murine fibroblast cells or the murine mastocytoma cell line P815 and derived transfectants were employed, namely: P815-K b expressing the gene for the heavy chain of the murine MHC class I molecule K b 21 , P815-B7 expressing the human B7.1 22 , and P815-K b -B7, which express both molecules on the cell surface 21 . Human embryonic lung fibroblasts (HELs), the human lymphoblastoma cell line Jurkat, and human PBMCs isolated from units of buffy coat supplied from the University of Heidelberg blood bank were used for investigation of HSV1 infection of human cells. CV1, NIH 3T3, P815, and HEL cells were maintained in DMEM supplemented with 10% FCS, 100 IU penicillin, 100 μg/ml streptomycin, and 4 mM l -glutamine (DM medium). Transfectants of P815 cells were maintained in the same medium with a supplement of 0.4 mg/ml G418 (GIBCO BRL). Murine splenocytes and Jurkat cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 IU penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 10 mM Hepes, and 50 μM 2-ME (RP medium). Human PBMCs were propagated in the same medium, except that FCS was replaced with 10% heat-inactivated pooled human AB serum (RP-h medium). F-US5MHC (recombinant HSV1 expressing murine β2-microglobulin and H-2K b ) and F-US5β (expressing β-galactosidase), which are identical with respect to HSV1 genes other than the transgenes, were gifts of D.C. Johnson (McMaster University, Hamilton, Ontario, Canada; 13). HSV1 strain F, from which both recombinant viruses were derived, was obtained from American Type Culture Collection (ATCC). A mutant HSV1 strain F that lacks the transporter associated with antigen processing blocking protein ICP47 (F-ICP47Δ) was a gift of R.L. Hendricks (University of Illinois, Chicago, IL; 23 ). For production of virus stock supernatant, HSV1-infected CV1 cell cultures were centrifuged at 10,000 g and then filtered with a 0.45-mm mesh filter to remove cellular debris before being pelleted for 1.5 h at 43,000 g . The viral pellet was resuspended in RPMI with 10% heat-inactivated FCS and then frozen at −80°C to give a concentrated viral stock. Frozen viral stocks were titrated using the endpoint dilution method, tested for the presence of mycoplasma using the mycoplasma detection kit (Boehringer Mannheim), and used within 4 mo of manufacture. To infect cells, virus was added to a minimal quantity of the medium appropriate for various cell types and then cultured for 1 h at 37°C. Subsequently, cells were washed three times with PBS to remove unadsorbed virus before adding fresh medium. K b -specific TCR (Des.TCR)-transgenic mice have been previously described 24 . These transgenic animals express the rearranged TCR-α and -β genes derived from CTL clone KB5.C20, which is allospecific for the K b antigen 25 . Perforin-deficient mice 26 were provided by Dr. H. Hengartner (University of Zürich, Zürich, Switzerland). Des.TCR and perforin-deficient mice were crossed and kept under pathogen-free conditions in the animal facility of the German Cancer Research Center (Heidelberg, Germany). Female DBA/2 and C57Bl/6 mice, 4–6 wk old, were purchased from Charles River Labs. and maintained in the animal facility of the University of Heidelberg. Gamma irradiation of 8,000 rads was found to be equally effective in preventing either cellular (P815 cells and T cell blasts) proliferation or HSV1 replication. Mononuclear cells were isolated from murine splenocytes or human peripheral blood by separation over Ficoll-Paque™ columns (Amersham Pharmacia Biotech). To generate murine T cell blasts by polyclonal stimulation, cells were incubated at a density of 2.5 × 10 6 cells/ml in RP medium with 2.5 μg/ml Con A for 2 d. As CD8 + T cells derived from Des.TCR splenocytes are >90% positive for the transgenic K b -reactive TCR, and because Des.TCR CD4 + T cells are functionally inert, polyclonal stimulation of Des.TCR splenocytes yields K b -reactive CD8 + T cells without further purification. For generation of CTLs specifically activated against K b , mononuclear cells from Des.TCR-transgenic mice were incubated with irradiated P815-K b -B7 cells at a ratio of 5:1 for 3 d in RP medium. To generate pure activated murine CD8 + cells, Con A–stimulated mononuclear cells were initially depleted of professional APCs by incubation for 1 h on a Sephadex G-10 column (Amersham Pharmacia Biotech). For removal of B cells, the remaining nonadherent cells were incubated with anti–mouse IgG magnetic beads (Paesel & Lorei). Murine CD8 + cells were then isolated by depletion of other cell populations by passing over a CD8 negative selection column (R & D Systems, Inc.). The remaining cells were >95% CD8 + cells. Murine mononuclear cells were prepared from the spleens of DBA/2 mice infected 6 wk before with 2.5 × 10 6 PFU of HSV1 strain F injected intraperitoneally in 100 μl PBS. Human mononuclear cells were prepared from the peripheral blood of HSV1-positive donors, and aliquots were frozen at −80°C, one aliquot being retained and cultured in RP-h medium with 4 μg/ml PHA for 24 h to generate activated T cells. Murine splenocytes from DBA/2 mice were activated with 2.5 μg/ml Con A. Stimulator cells were then prepared by infecting murine DBA/2 or human T cell blasts with HSV-1 F-ICP47Δ mutant at a multiplicity of infection (MOI) of 2 and incubating for 24 h before irradiation. Stimulator cells were then incubated with responder cells at a ratio of 1:5 in RP or RP-h medium for 72 h before half of the medium was changed and recombinant IL-2 (human or murine) added at a concentration of 40 (human) or 90 (murine) U/ml. Medium replenishment was repeated every 3 d. On day 9, cells were removed by Ficoll-Paque™ purification and, when required, CD4 + or CD8 + cells positively selected by biotinylated anti-CD4 or anti-CD8 antibody and streptavidin-coated magnetic particles using the MACS™ system (Miltenyi Biotec). The purified HSV1-reactive T cells were incubated with fresh stimulator cells in the relevant medium with IL-2 for a further 48 h before being used in JAM assays. Polyclonally stimulated human or mouse T lymphocytes were generated using PHA (4 μg/ml) or Con A (2.5 μg/ml), respectively, as the initial stimulus. Polyclonally activated T cells were cultured as described for HSV1-reactive T lymphocytes except that they were restimulated with PHA and Con A, respectively, in combination with uninfected irradiated stimulator cells. For preparation of HCMV-reactive T cells, PBMCs were isolated from HCMV-positive donors and frozen. Monocytes were isolated by adhesion to tissue culture dishes, harvested, and then mixed with mononuclear cells at a ratio of 3:1 in RP-h medium supplemented with 4 μg/ml PHA. After 4 d of cultivation, the stimulated monocytes/macrophages were infected with a clinical strain of human (H)CMV (HCMV-N) at an MOI of 5, washed, and recultivated for a further 3 d before irradiation. Thereafter, these cells were used to stimulate autologous T cells essentially as described for HSV1-reactive T lymphocytes. Standard procedures were used for flow cytometric analysis 27 . In brief, for surface immunofluorescence, cells in suspension were washed once with ice-cold wash solution (PBS with 2% BSA and 0.05% sodium azide) before being resuspended with the first antibody in ice-cold blocking solution (PBS with 10% heat-inactivated FCS and 0.2% sodium azide) for 45 min. The cells were then washed in ice-cold wash solution and the staining repeated with a secondary antibody (where relevant). After the final staining step, the cells were washed in ice-cold PBS and then either resuspended in 200 μl blocking solution for measurement or fixed in 4% ice-cold paraformaldehyde for 30 min. Fixed cells were washed twice with ice-cold PBS before being stained by the TUNEL (TdT-mediated dUTP-biotin nick-end labeling) method. For TUNEL staining, fixed cells were permeabilized by ice-cold 0.1% Triton X-100 with 0.1% sodium citrate for 2 min before being washed twice with ice-cold PBS. Fixed permeabilized cells were then incubated for 1 h at 37°C with TUNEL solution (TUNEL staining kit; Boehringer Mannheim) before being washed twice with room temperature PBS. Flow cytometry was performed on a FACScan™ (Becton Dickinson) linked to an Apple Macintosh Quadra 650 using CELLQuest™ software (Becton Dickinson) for data analysis. The following reagents were employed for FACS staining: FITC-coupled Désiré-1 as a clonotype-specific mAb to monitor the transgenic TCR 24 28 , and antibodies against murine CD8α (clone Ly-2), H-2K b (clone AF6-88.5), CD95 (clones DX2 and JO2), CD95L (ligand; clone MFL3), human CD8 (clone RPA-T8), and human CD4 (clone RPA-T4), purchased from PharMingen. Antibody against viral gD was obtained from Advanced Biotechnologies, Inc. The hybridoma producing antibody W6/32, specific for MHC class I, was obtained from ATCC. Secondary antibodies and streptavidin-linked fluorochromes were obtained from Southern Biotechnology Associates. Chimeric proteins consisting of the extracellular portion of murine CD95, human TNF-related apoptosis-inducing ligand (TRAIL)R2, and human TNFR2 combined with the Fc portion of human IgG were produced by transient transfection of COS7 cells with expression plasmids encoding the various proteins in pcDNA3.9 (Invitrogen Corp.) and subsequent purification on protein G–Sepharose columns (Amersham Pharmacia Biotech). P815 cells or CD8 + cells from DBA/2 mice were infected with HSV strain F, F-US5β, or F-US5MHC at an MOI of 10 and incubated for 4 h. Cells were then used as target cells in a 51 Cr-release assay as described elsewhere 29 . 51 Cr was obtained from New England Nuclear. The JAM assay 30 was performed to measure the degree of cell death. For this purpose, target cells are labeled with tritiated thymidine. If apoptosis occurs in the labeled cell population, DNA fragments will be washed through glass fiber filters during cell harvesting. In contrast, DNA from surviving target cells remains intact and will be captured by the filters. The percent cell death can be calculated by comparing the amount of [ 3 H]thymidine bound to filters in the presence and absence of the apoptosis-inducing event. In brief, proliferating lymphocytes were pulsed overnight with tritiated thymidine at a concentration of 5 μCi/ml (New England Nuclear). Cellular debris was removed by Ficoll-Paque™ purification, and the cells were washed twice with wash medium (RPMI with 5% heat-inactivated FCS) and counted before being used as target cells. Infected or uninfected target cells were incubated for 7 h at a density of 100,000 cells in 200 μl proliferation medium in round-bottom 96-well plates before being harvested and counted. When required, blocking antibodies or chimeric proteins were added 2 h after infection at the specified concentrations. All measurements given represent the mean of six or eight wells, and all experiments were replicated at least once on separate occasions. The highest MOI used was 10 to avoid inhibition of cytotoxic activity by HSV1 as has been previously reported 31 . PBMCs were stimulated with staphylococcal enterotoxin B (SEB) at a concentration of 1 mg/ml for 24 h. Cells were then either infected with HSV1 strain F at an MOI of 10 or mock infected and then incubated for a further 18 h with apoptosis-inducing anti-CD95 mAb. Cells were then stained for CD8 or CD4 and used in the TUNEL assay. We hypothesized that infection of T lymphocytes by HSV1 could pose a severe risk for the antiviral immune response because the infected cells could present viral antigen to and thereby kill each other (fratricide). Previous studies have demonstrated that HSV1 efficiently interferes with antigen presentation in human fibroblasts due to its ability to block transport of MHC class I molecules 13 14 . This effect, however, can vary in different cell types. Therefore, we investigated the efficiency of HSV1's blocking of the transport of MHC class I molecules in human T cells compared with human fibroblasts. For this purpose, the human fibroblast cell line MRC-5 and activated human CD8 + lymphocytes were infected with a recombinant HSV1 strain coding for K b , a murine MHC class I molecule (F-US5MHC). Infection of both cell types was efficient, as shown by FACS™ analysis of gD, a strongly expressed HSV1 glycoprotein . Expression of endogenous MHC class I molecules and of virus-encoded K b was analyzed in parallel. Human fibroblasts infected with F-US5MHC illustrated the effective viral block of MHC class I expression : virus-encoded K b could not be detected, and the level of endogenous MHC class I molecules was reduced by ∼50% 5 h after infection. By contrast, we could detect the viral K b on the cell surfaces of human CD8 + lymphocytes and, in addition, we observed no inhibitory effect on the expression of endogenous MHC class I molecules 5 h after infection. These results demonstrate that, in contrast to its effect on human fibroblasts, HSV1 does not inhibit MHC class I expression by human T cells. It has been demonstrated that freshly activated T cells are resistant to apoptosis and, in addition, that HSV1 itself confers resistance to apoptosis in certain cell types 15 32 . To study the consequences of viral antigen presentation in antiviral T cell populations, we generated different HSV1-reactive T cell lines from seropositive human donors and from mice previously inoculated with HSV1 strain F. As a control, polyclonally stimulated T cells and HCMV-specific T cell lines were employed. HSV1-reactive and control cells expressed comparable levels of CD95 (data not shown). Rapid apoptosis occurred after infection with the HSV1 strain F (MOI of 10) in both murine and human HSV1-reactive T lymphocytes. By contrast, significant cell death was absent in polyclonally stimulated murine and human T lymphocytes and in HCMV-specific human T cell lines . Infection-mediated apoptosis of the specific antiviral T cell lines suggests that HSV1 infection renders antiviral T cells susceptible to apoptosis and that recognition of viral antigen may be necessary to initiate the cascade of events that lead to fratricide. Like T cell clones or T cell hybridomas, however, T cell lines are especially susceptible to induction of apoptosis and might not reflect the situation in vivo, where freshly activated antiviral CTLs eliminate the invading pathogen. Therefore, we developed a system to investigate the above effects in mice. Molecular analysis of freshly activated antiviral CTLs is hampered by the fact that T cells reactive to a particular virus occur only at a low frequency in the peripheral T lymphocyte pool. To circumvent this problem, we employed a transgenic mouse model of HSV1 infection for further analysis of HSV1-induced fratricide. In this model, TCR-transgenic mice that express a T cell repertoire strongly skewed toward K b (Des.TCR mice) serve as the hosts and F-US5MHC virions encoding K b as a viral neoantigen are used as infectious particles. First, we tested whether the transgenic TCR can recognize viral K b on murine cells as a target structure. For this purpose, we infected the murine mastocytoma cell line P815 (MHC haplotype H-2 d ) with F-US5MHC. As demonstrated by analysis of gD, the P815 cells were efficiently infected with F-US5MHC . However, these cells showed only weak expression of K b in comparison to stable transfectants constitutively expressing K b under the control of a strong promotor (P815-K b cells) . Nonetheless, the K b -reactive CTLs from Des.TCR mice could recognize and kill F-US5MHC–infected P815 cells . The obtained lysis was relatively weak in comparison to lysis of P815-K b cells, reflecting the weak K b expression of F-US5MHC–infected P815 cells. In contrast, we observed no lytic activity against target cells infected with a recombinant HSV1 strain encoding lacZ (F-US5β) as a control. Thus, the transgenic TCR recognizes virus-encoded K b as a target structure on infected cells. We then investigated whether fratricide also applied to F-US5MHC–infected T lymphocytes from Des.TCR mice. After F-US5MHC infection, we detected viral K b on the cell surfaces of activated murine T cells . Consistent with this observation, we detected rapid apoptosis in the JAM assay in activated T cells from Des.TCR mice after infection with F-US5MHC but not after F-US5β infection . As a further negative control, we used Con A–activated T lymphocytes from DBA/2 mice. In these cells, expression of HSV1-encoded genes was as efficient as in activated Des.TCR lymphocytes (data not shown), but the cells did not show an enhanced rate of DNA fragmentation after F-US5MHC infection . In addition, we analyzed whether expression of the K b antigen by means other than F-US5MHC infection results in a significant increase in death of Des.TCR lymphocytes. For this purpose, activated but uninfected Des.T cells were incubated with P815-K b transfectants that express much higher levels of K b than F-US5MHC–infected cells. After 7 h, only a slight increase in DNA fragmentation (i.e., 3%) occurred in comparison to that in activated Des.TCR cells that had been incubated with K b -negative P815 cells (data not shown). Thus, the observed fratricide was dependent on HSV1 infection and occurred not only in long-term–cultured T cell lines but also in freshly activated CTLs, which comprise a high proportion of antigen-reactive T cells. These results demonstrate that Des.TCR mice, in combination with F-US5MHC, represent a suitable model with which to analyze the underlying mechanisms of HSV1-induced fratricide. There are two known main antigen-dependent pathways by which CTLs can kill virus infected-cells: killing by the perforin/granzyme system and killing by molecules that belong to the TNF cytokine family 33 . Nearly all of the cytotoxicity mediated by CD8 + CTLs is due to the perforin/granzyme system. Therefore, we analyzed whether perforin was involved in HSV1-induced fratricide. For this purpose, CTLs from either Des.TCR mice or Des.TCR mice lacking perforin (Des.TCR × perforin −/− ) were infected with F-US5MHC. Surprisingly, we detected identical levels of apoptosis in the JAM assay, indicating that HSV1-induced fratricide was perforin independent . To further dissect the molecular events underlying HSV1-induced apoptosis, we investigated whether apoptosis-inducing members of the TNF cytokine family, namely TNF, CD95L, and TRAIL, play a role in HSV1-induced fratricide by blocking the activity of the respective ligands with receptor–Fc fusion proteins. In addition, we determined whether antigen recognition is necessary to trigger the apoptogenic signaling cascade. To this end, we used an anti-K b mAb to inhibit the interaction between the transgenic K b -reactive TCR and the K b molecule . We found that upon infection of activated Des.TCR CTLs with F-US5MHC, inhibition of the CD95–CD95L interaction substantially prevented apoptosis, blocking of TRAIL had virtually no effect, and inhibition of TNF partially abrogated apoptosis. Interestingly, blocking of the TCR–K b interaction also prevented apoptosis after F-US5MHC infection of activated T lymphocytes from Des.TCR mice. Given the importance of the CD95–CD95L interaction in HSV1-induced fratricide, we next examined the expression of CD95L and its receptor on infected CD8 + lymphocytes . Interestingly, CD95L was upregulated on activated Des.TCR CD8 + T cells only after productive infection with F-US5MHC but not after infection with F-US5β, implying that triggering of the transgenic TCR by K b resulted in higher surface expression of CD95L on activated CTLs. Supporting these data, levels of CD95L remained unchanged on HSV1-infected nontransgenic murine T cells, which are not antigen specific. Thus, HSV1 infection per se does not induce higher expression of CD95L on activated T lymphocytes 5 h after HSV1 infection. In contrast to CD95L, the expression of its receptor was not significantly altered, regardless of whether the HSV1-infected T cells recognized antigen or not. Thus, an enhanced level of CD95L expression is relevant for HSV1-induced fratricide, whereas upregulation of CD95 expression is not required. Altogether, these findings demonstrate that the CD95 system is the main effector system of HSV1-induced fratricide and, in addition, that the virus-induced increase of CD95L expression is dependent on TCR–antigen interaction. Virus-induced upregulation of CD95L is necessary but not sufficient for induction of fratricide, as freshly activated T cells are normally resistant to CD95 signaling. Therefore, we determined whether HSV1 renders activated T cells susceptible to CD95 triggering after infection. For this purpose, we used the superantigen SEB to stimulate PBMCs. SEB activation is similar to normal activation with antigen because it also requires APCs and a CD28 costimulation signal and yields activated T cells that are resistant to apoptosis induction via CD95 34 . After SEB activation, PBMCs were infected with HSV1 or, as a control, left uninfected and subsequently challenged with apoptosis-inducing anti-CD95 mAb . We found that HSV1-infected CD8 + and CD4 + T cells are highly susceptible to CD95-mediated apoptosis, whereas the majority of uninfected T lymphocytes and T lymphocytes treated with UV-inactivated virus particles (mock infection) remained resistant to CD95 ligation. Similar data were obtained when PHA was used instead of SEB to stimulate T lymphocytes (data not shown). These results demonstrate that triggering of CD95 alone is not sufficient for induction of fratricide. Only in the course of productive HSV1 infection is an additional viral competence-to-die signal generated that renders short-term–activated T cells sensitive to CD95-mediated apoptosis. In this paper, we analyzed the functional implications of HSV1 infection of activated T cells. We observed that, in contrast to human fibroblasts, MHC class I expression on HSV1-infected human T lymphocytes is not disrupted. This supports the notion that the efficiency of HSV1's interference with transport of peptides into the lumen of the endoplasmic reticulum varies not only in different species 13 35 36 but also in different cell types of the same species 13 . As a consequence of unaltered presentation of viral antigens by MHC class I molecules, antiviral CTL populations infected with HSV1 are rapidly eliminated. In theory, this could either be due to suicide of individual CTLs or, alternatively, fratricide (killing of each other). We prefer the latter because antigen recognition by infected T cells was required for induction of apoptosis, and it has been shown that TCR molecules are unable to interact with MHC–peptide complexes on the same cell 19 . To identify the molecules involved in virus-induced fratricide, we used a transgenic mouse model of HSV1 infection. CTLs from mice expressing a K b -reactive transgenic TCR (Des.TCR mice) were infected with a recombinant HSV1 strain coding for K b (F-US5MHC). Blocking experiments revealed that HSV1-induced fratricide is mainly triggered by the CD95–CD95L system. Intriguingly, blocking CD95 did not totally prevent cell death, as TNF also contributed to fratricide, although to a lesser extent. In contrast, TRAIL, another member of the TNF family, was not involved. Similarly, perforin did not play a role in HSV1-induced fratricide, because T lymphocytes from Des.TCR × perforin −/− mice were as susceptible to apoptosis as T cells from Des.TCR mice. Blocking of the TCR–antigen interaction reduced fratricide to the same extent as blocking CD95. As an explanation, we found that activated CTLs from Des.TCR mice further upregulated CD95L molecules on the cell surface within a few hours after infection with K b -encoding HSV1. Such a rapid upregulation may be due to recruitment of preformed CD95L molecules rather than to de novo synthesis 37 38 . This result, together with the fact that rapid cell death was not induced in human T cell lines reactive with CMV, suggests that signaling through the TCR complex after recognition of viral antigen is a prerequisite of HSV1-induced fratricide and may be the basis of viral immune evasion. In this aspect, it resembles FasL- and TNF-mediated mechanisms that induce peripheral tolerance and maintain physiological lymphocyte homeostasis 39 . It has been shown that uninfected T cells acquire a transient state of resistance to CD95-mediated signaling after activation lasting several days 32 . Therefore, we have compared the susceptibility of HSV1-infected versus uninfected T lymphocytes to CD95-mediated apoptosis after SEB stimulation. As previously reported, SEB stimulates both CD4 + and CD8 + T cells 40 and confers resistance to CD95-induced apoptosis in freshly activated T cells 34 . We found that subsequent HSV1 infection renders SEB-activated CD4 + and CD8 + T cells highly susceptible to induction of apoptosis through engagement of CD95 by mAbs. In contrast, abortive infection with UV-inactivated HSV1 did not confer susceptibility to CD95 signaling. This result implies that viral proteins expressed in the course of productive infection facilitate activation-induced cell death (AICD). Such a competence-to-die signal 41 has recently been demonstrated by stimulating uninfected T lymphocytes with variant peptide ligands that do not induce T cell effector functions (e.g., cytokine secretion). This does not result in upregulation of death receptors on “competent” T lymphocytes but renders them susceptible to death ligands on neighboring cells. However, the molecular basis of this phenomenon has yet to be defined, although tyrosine phosphorylation of intracellular proteins could play a role 42 . Taken together, these data suggest that apoptosis of activated T cells requires two distinct signals: one that leads to upregulation of CD95/CD95L and another that removes the block of CD95-signaling pathways at the time of CD95 triggering. AICD is triggered by repeated antigenic stimulation 43 44 45 . Accordingly, HSV1 proteins could lower the AICD threshold, the critical number of triggered TCRs during TCR–peptide–MHC interaction. It is unlikely that the viral proteins target expression or function of antiapoptotic proteins of the Bcl family like Bcl-x L or Bcl-2, because it has been reported that AICD is not prevented by these molecules 46 . Alternatively, HSV1 proteins could restore the formation of the death-inducing signaling complex (DISC), which has been shown to lack FLICE (FADD-like IL-1β–converting enzyme, now referred to as caspase-8; reference 47 ) in freshly activated T cells that are resistant to AICD 48 . This could be accomplished by downregulation of the antiapoptotic cellular FLIP (FLICE-inhibitory protein), which competes for caspase-8 recruitment to the DISC 49 50 51 52 53 54 . Thus, the viral proteins involved in facilitating AICD and the molecular basis of this effect remain to be elucidated. Fratricide has also been implicated as an important factor in the pathogenesis of AIDS 55 . T cells from HIV-1–infected individuals show upregulation of CD95/CD95L and enhanced susceptibility to CD95-mediated killing 56 57 58 59 . Moreover, HIV-1–derived proteins (Tat, gp120) can enhance CD95L expression 55 60 . Recently, it has been demonstrated that Nef, another HIV-1 protein, forms a signaling complex with the TCR. This results in T cell activation and upregulation of CD95L expression, thereby circumventing the need for antigen recognition 60 . In this way, HIV may cause global immunodeficiency, which finally results in opportunistic infections and death of the infected individuals. We propose a different mechanism underlying fratricide in active HSV1 infections . In HSV1 lesions, which usually remain focused to an area of epithelial cells, free virions can infect invading antiviral CTLs by binding to herpesvirus entry mediator (HVEM) molecule. Subsequently, viral antigens are efficiently presented on infected antiviral CTLs. Neighboring CTLs that recognize viral antigen upregulate CD95L. In addition, HSV1 lifts the block for AICD. As a consequence, infected antiviral CTLs could be better targets than infected epithelial cells, in which antigen presentation via MHC class I molecules is prevented by viral proteins. Such a cell type–specific regulation of viral defense mechanisms could turn antiviral CTLs into decoy targets, thereby enhancing local spread of the virus in epithelial cells. The elimination of antiviral CTLs by antigen-dependent fratricide could be a general immune evasion mechanism employed by viruses that can infect and replicate in activated T cells, although they mainly propagate in nonlymphoid cells. In contrast, viruses that are dependent on T cells for virion production (e.g., human herpesviruses 6 and 7) may have to prevent T cell death to yield enough infectious particles. | Study | biomedical | en | 0.999998 |
10523609 | CD4 CD62L high cells were prepared from BALB/c mice aged 6–12 wk by MACS ® (Miltenyi Biotec), as described elsewhere 11 . CD4 T cells which were consistently >95% pure for CD4 and CD62L high expression were activated in 24-well plates (Nunc) previously coated with 10 μg/ml anti–murine CD3 mAb (2C11; PharMingen). The cultures were incubated with or without a 10% final concentration of a supernatant of anti-CD28 mAb (clone 37.51; a gift of Dr. Jim Allison, University of California at Berkeley, Berkeley, CA). BALB/c, CD11c-OX40L, and CTLA4-Ig tg experimental animals were bred and maintained in accordance with animal house guidelines. For in vivo studies of mRNA expression, footpad immunizations were carried out using 50 μg alum-precipitated chicken γ-globulin resuspended in 200 μl normal saline, or heat-killed Bordetella pertussis (5 × 10 8 ) resuspended in 200 μl normal saline as indicated. Immunized mice were killed 3 and 7 d after immunization by CO 2 asphyxiation. RNA and cDNA samples were prepared from draining popliteal LNs as described 13 . cDNA prepared from the tissue sections was diluted to a final volume of 100 μl. The PCR β-actin signal was used to correct for differences in the amount of starting cDNA from each sample. The specific primers were: β-actin (Stratagene), 5′-agcgggaaatcgtgcgtg and 5′-CAGGGTACATGGTGGTGCC; IL-4 14 , 5′-GAATGTACCAGGAGCCATATC and 5′-CTCAGTACTACGAGTAATCCA; IFN-γ 14 , 5′-AACGCTACACACTGCATCTTGG and 5′-GACTTCAAAGAGTCTGAGG; murine OX40, 5′-TGTATGTGTGGGTTCAGCAGCC and 5′-ccctcaggagtcaccaaggtggg; and murine OX40L, 5′-atggaaggggaaggggttcaacc and 5′-TCACAGTGGTACTTGGTTCACAG. For semiquantitative reverse transcription PCR of OX40, OX40L, IL-4, and IFN-γ mRNA, ∼10 fewer cycles than were required to detect PCR product using ethidium bromide gels were performed, so that quantitation could be performed by PCR Southern blot or Southern dot blot analysis. For each set of primers, three different numbers of PCR cycles were performed, to ensure that amplification was logarithmic and that the conditions were not saturating. The PCR product was separated on a 1.5% agarose gel and transferred onto prewetted Hybond-N+ membrane (Nycomed Amersham plc) by capillary transfer under alkaline conditions. The membrane was hybridized with a 32 P-labeled purified PCR product used as a probe and imaged using a PhosphorImager ® (Molecular Dynamics). Using ImageQuant ® software (Molecular Dynamics), a grid was laid over PCR bands with individual fields covering the central 50% of a band. The signal in each field was calculated and these figures transferred to spreadsheet software to sort the randomized files to the correct order. The average of the three PCRs with different cycle number for each gene was taken and divided by the average of the three corresponding β-actin PCRs. These values represent the relative amount of mRNA for each gene per cell. This value was multiplied by the size of the section area (determined by microscopy on adjacent sections to those taken for cDNA using the point counting technique ) to give mRNA amount per section. A polyclonal rabbit antiserum prepared by immunizing with CXCR5 peptides was a gift of Dr. Jason Cyster, University of California San Francisco, San Francisco, CA 16 . The specificity of this Ab was confirmed in cross-blocking studies with a rat mAb against mouse CXCR5. OX40-Ig was prepared by using protein A purification of supernatant cultures from a hybridoma expressing this fusion protein 11 . Control Ig was prepared in a similar way. Anti-CD28 mAb was prepared by protein G purification of supernatants of clone 37.51. These proteins were sterilized by filtration through 0.2-μm filters, and frozen in aliquots before use. Mice expressing soluble CTLA4-Ig or littermate controls were immunized in the intraperitoneal cavity with 50 μg alum-precipitated 4-hydroxy-3-nitrophenyl acetyl (NP)-KLH resuspended in 200 μl of saline. KLH (Sigma Chemical Co.) was haptenated as described elsewhere 17 . Assuming a molecular weight of 3 million to KLH, there are ∼400 molecules of NP conjugated to each KLH molecule. Anti-CD28 mAb (100 μg) was injected intraperitoneally where indicated 24 h after antigen administration. OX40-Ig or control Ig (100 μg) was injected intraperitoneally on days 1, 2, and 3. Animals were killed after 9 d, then bled, and their spleens were taken for frozen section analysis and flow cytometry. mAbs used for flow cytometry analysis were FITC-conjugated rat anti–mouse CD4 (Southern Biotechnology), PE-conjugated rat anti–mouse OX40 (Serotec), biotin-conjugated rat anti–mouse CD62L (Southern Biotechnology), and rabbit anti–mouse Burkitt's lymphoma receptor 1 (BLR1 [CXCR5]). Secondary step reagents were streptavidin-cychrome (PharMingen) and anti–rabbit PE (Jackson ImmunoResearch Labs). Animals were killed by CO 2 asphyxiation, and their spleens were removed, snap-frozen in liquid N 2 , and stored at −70°C until use. 5-μm cryostat sections from these tissues were mounted onto 4-spot glass slides that were air dried for 1 h, fixed in acetone at 4°C for 20 min, dried again, and stored in sealed polythene bags at −20°C. Tissue sections were double-stained for IgD (sheep anti–mouse IgD; Binding Site) with CD3 (KT3), CD4 (GK1.5), or CD8 (2.43) (American Type Culture Collection). Slides were stained as described elsewhere 4 . In brief, second step reagents were donkey anti–sheep peroxidase (Binding Site) and biotinylated rabbit anti–rat (DAKO) followed by StreptABComplex alkaline phosphatase (DAKO). NP-binding cells were identified using NP conjugated to rabbit IgG. Secondary Abs were biotinylated swine anti–rabbit and biotinylated rabbit anti–rat. Sheep anti–mouse IgD was detected using peroxidase-conjugated donkey anti–sheep Ig (Binding Site). Substrate for peroxidase was 3-3′ diaminobenzidine (Sigma Chemical Co.), and for alkaline phosphatase the substrate Naphthol AS-MX phosphate and chromogen Fast Blue BB salt/levamisole (Sigma Chemical Co.) were used to block endogenous alkaline phosphatase activity. For quantitative analysis, GC size was assessed by counting the number of graticule intercepts (units) at 400× magnification of IgD − B cell areas within B follicles 15 . The numbers of CD3 + T cells were counted in each GC. To test for the CD28 dependence of OX40 expression, naive CD62L high CD4 T cells were purified from mouse spleens 11 and activated with anti-CD3 mAb, with and without agonistic CD28 mAb. Unstimulated CD4 cells did not express detectable levels of OX40 compared with a control isotype-matched mAb . In addition, the specificity of staining was checked by cold competition with unlabeled anti-OX40 mAb. This could not inhibit fluorescence on unstimulated CD4 T cells, indicating undetectable levels of OX40 protein as assessed by flow cytometry. By 24 h after activation through CD3, naive CD4 T cells with and without agonistic CD28 mAb were equivalently enlarged in size (data not shown) but it was already clear that CD28 costimulation augmented OX40 expression (32 vs. 4% positive CD4 T cells). The differences were even more pronounced after 48 h of culture . Specificity of staining was shown by the capacity of unlabeled OX40 mAb to inhibit binding of OX40-PE–specific mAb (data not shown). These results appear to contradict reports that OX40 expression in vitro is relatively independent of signaling through CD28 10 18 . Because differences in strength of signaling through CD3 with mAb could account for this discrepancy, we went on to investigate the induction of OX40 expression in vivo. To test for the CD28 dependence of OX40 expression in vivo, we compared OX40 induction in CTLA4-Ig tg mice and nontransgenic littermates. Mice were immunized in the left footpad with the Th2-inducing antigen, alum-precipitated, chicken γ-globulin 13 , and the draining popliteal nodes were examined 3 or 7 d later. Levels of mRNA for OX40 and its ligand, together with the cytokines IL-4 and IFN-γ, were quantified in the draining LN of transgenic and normal littermate mice . Previous studies have shown that upregulation of OX40L occurs on day 2, and IL-4, IFN-γ, and OX40 are induced by day 3 11 13 . There was less mRNA for IL-4, IFN-γ, and OX40 in CTLA4-Ig tg mice compared with control littermates on day 3, although OX40L expression was comparable. In particular, in CTLA4-Ig tg mice, median levels of day 3 OX40 and IL-4 mRNA were similar to those in day 0 unimmunized controls (data not shown). After 7 d, CTLA4-Ig tg mice made comparable amounts of IFN-γ as has been reported elsewhere 8 , but there was still much less mRNA for IL-4 and OX40, and less OX40L in transgenic animals. CTLA4-Ig mice immunized with B . pertussis , which evokes IL-4 and IFN-γ, and a combination of IgG1 and IgG2a Abs in normal mice 13 had comparable levels of IFN-γ at day 3 (data not shown). These data show there is a relative defect in IL-4 compared with IFN-γ, and this is correlated with deficient OX40 expression in CTLA4-Ig tg mice. This suggests that in vivo OX40 expression is CD28 dependent, and that poor OX40 induction correlates with reduced IL-4 levels. Previously, we reported that ligation of OX40 on CD4 T cells upregulates mRNA for CXCR5 11 , a chemokine receptor which appears to direct lymphocytes to B cell follicles 19 20 . Using mAb and polyclonal rabbit antiserum to CXCR5, we confirm previous reports 19 that a subset of memory CD4 T cells, which are low in expression of L-selectin (CD62L), expresses this chemokine receptor . We also found a proportion of CD62L high cells expressing CXCR5 as has been reported in humans 19 . This population may represent an intermediately activated CD4 T cell population. Alternatively, some CD62L low cells may revert to a CD62L high phenotype. Mice which constitutively express OX40L on their DCs (CD11c-OX40L tg mice) are characterized by increased numbers of CD4 T cells in B cell follicles 12 . Here we show that this phenotype is linked to an expansion of CXCR5 + CD62L low CD4 cells . In contrast, immunized CTLA4-Ig tg mice, which lack CD28 costimulation, have some CD62L low CD4 cells, but these are strikingly deficient in expression of CXCR5 . However, if the CD80/CD86 blockade is bypassed by injection of an agonistic CD28 mAb, then this subset is rapidly restored . These data link CD28 and OX40 signaling with CXCR5 expression on CD4 T cells. CD11c-OX40L tg mice, which constitutively express OX40L on DCs, have increased numbers of CXCR5 + CD62L low CD4 cells and increased numbers of follicular T cells. To test the CD28 dependence of this phenotype, we crossed CTLA4-Ig tg mice (heterozygous for the transgene) with CD11c-OX40L tg mice (heterozygous for the transgene). On average, 25% of the offspring of this pairing are normal (nontransgenic), 25% are transgenic for CTLA4-Ig, 25% transgenic for CD11c-OX40L, and 25% express both CTLA4-Ig and CD11c-OX40L. CXCR5 expression was compared in all four groups of animals immunized with alum-precipitated protein antigens. Normal mice form GCs by day 10 (data not shown) and have CXCR5 + CD62L low CD4 T cells . CTLA4-Ig tg mice lack CXCR5 + CD62L low CD4 cells , and do not form GCs 7 . CD11c-OX40L tg mice have increased CXCR5 + CD4 cells and form GCs 12 . However, double transgenic mice have the phenotype of their CTLA4-Ig tg littermates: they lack CXCR5-expressing CD62L low CD4 T cells , and do not develop GCs (data not shown). This clearly indicates that the phenotype in CD11c-OX40L tg mice requires CD28 signaling, and is consistent with the expression of OX40 being CD28 dependent in vivo. However, this does not exclude the possibility that other CD28-dependent signals induce CXCR5 expression on CD4 T cells. The lack of GC formation in CTLA4-Ig tg mice can be reversed by a single injection of agonistic anti-CD28 Ab . These mice develop large GCs, unlike CTLA4-Ig tg mice given control hamster Ig (data not shown). Production of specific IgG Ab is also restored, and there is evidence of affinity maturation (data not shown). Blocking OX40 interactions with a fusion protein between murine OX40 and human IgG1 (OX40-Ig) allowed us to test our hypothesis that the restoration of GC formation by anti-CD28 treatment is dependent on OX40 signaling. CTLA4-Ig tg mice were immunized with alum-precipitated protein intraperitoneally, and 1 d later were given anti-CD28 mAb (100 μg) with either OX40-Ig (100 μg in three doses on days 1, 2, and 3) or a control Ig fusion protein. Because CTLA4-Ig tg mice have no GCs, injection of mAb to CD28 allows accurate timing of their onset. Also, because CTLA4-Ig tg mice are tolerant to fusion proteins between human IgG1 and self-proteins, blockade is more likely to persist and be effective. The presence of CTLA4-Ig, which binds to CD80 and CD86 on activated B cells, controls for potential artifacts due to OX40-Ig binding to B cells, independent of its effect on OX40/OX40L blockade. CTLA4-Ig tg mice treated with anti-CD28 mAb and control Ig had GCs with a mean size of 30.6 U with 67.3 T cells per GC . In marked contrast, CTLA4-Ig tg mice treated with OX40-Ig had much smaller but not absent GCs with a mean size of 8.1 U , and a mean number of T cells per GC of 18.7 . Although GC size was diminished in OX40-Ig–injected mice, the extrafollicular anti-NP plasma cell responses were consistently increased (data not shown). These data link CD4 T cell OX40 signaling with GC development, but not with the extrafollicular plasma cell response. We have investigated the sequence of molecular instructions that control CD4 T cell migration and differentiation to provide GC T cell help. We recently published evidence that OX40 signaling was linked with induction of IL-4 expression 11 , and studies in humans have yielded a similar conclusion 10 . We also found that OX40 ligation was linked with upregulation of CXCR5 on CD4 T cells, a chemokine receptor linked with lymphocyte migration to B cell areas. This idea was supported by the phenotype of CD11c-OX40L tg mice, which have increased CD4 T cell numbers within GCs 12 . CD28-deficient mice are also deficient in their capacity to provide B cell help: they have a relatively selective deficit in Th2 development 8 , and lack GCs 7 21 . In keeping with the idea that CXCR5 + CD4 cells are implicated in GC development, we report here that CTLA4-Ig tg mice have a selective deficit in these cells. In contrast, CD11c-OX40L tg mice, which have greatly increased CD4 T cells in B cell areas, have the opposite phenotype, with an increased proportion of CXCR5 CD4 + CD62L low cells. To understand the relationship between CD28 and OX40 signaling in development of CXCR5 + CD4 T cells and GCs, we investigated the CD28 dependence of OX40 expression. Although it has been reported that OX40 expression in vitro is independent of CD28 costimulation 10 18 , in our hands we found that levels of OX40 in vitro and in vivo were greatly augmented by CD28 signaling. We hypothesized that poor expression of OX40 in CD28-deficient mice was linked with the lack of GC development 7 8 . To test this, we acutely reversed the CD28 signaling defect by injection of agonistic CD28 mAb. GCs reappear in Ab-treated mice, and this is correlated with the appearance of CXCR5 + CD62L low CD4 T cells. If, however, OX40 interactions were blocked by OX40-Ig, GC formation was greatly reduced, although significantly the extrafollicular plasma cell response was augmented (data not shown). These data strongly support the idea that CD28-costimulated CD4 T cells primed in the outer T zone by DCs upregulate OX40, which allows them to respond to OX40L expressed by either CD40-activated DCs 12 22 or activated B cells 23 . This sequential signal then activates a program of events that includes upregulation of IL-4, CXCR5 expression, and migration into follicles to help B cells form GCs. This model predicts that the enhanced accumulation of CD4 T cells in B cell follicles in CD11c-OX40L tg mice would be dependent on CD28 signaling, which indeed proved to be the case in our study. Although our data implicate ligation of OX40 on T cells in CD4 migration into B follicles, injection of OX40-Ig reduced rather than abolished GC formation. Preliminary experiments show OX40-deficient mice can develop GCs albeit at a slower tempo (our unpublished observations), indicating that other molecules can substitute for OX40. CD70 (CD27L), whose receptor is expressed on resting CD4 T cells 24 , is a good candidate, as in vitro it has similar effects on CD4 T cell differentiation and, like OX40L, is expressed on both CD40-activated B cells and DCs (our unpublished observations). This situation is analogous to CD80 and CD86, which can both costimulate T cells through CD28, but perhaps because of temporal differences in expression, exert subtle differences on T cell immune responses. Our model of the role of OX40 in CD4 T cell differentiation is different from that proposed elsewhere 23 25 , which suggests that ligation of B cell OX40L by CD4 T cell OX40 promotes the extrafollicular plasma cell response, but does not influence GC development. In the in vivo experiments described 25 , a polyclonal rabbit antiserum to OX40 was used to block OX40/OX40L interactions. We speculate that this antiserum might signal OX40-expressing CD4 T cells to migrate into B follicles, as in the CD11c-OX40L tg mice 12 . In our experiments, injection of OX40-Ig would allow signaling through B cell OX40L and the development of the extrafollicular plasma cell response, but by blocking OX40 ligation of CD4 T cells, would inhibit T cell migration and reduce GC formation. Much controversy exists in the literature concerning the role of CD28 signaling in immunosuppression strategies. In general, mice rendered deficient from birth in CD28 signaling, by either gene deletion of CD28 6 or constitutive expression of CTLA4-Ig 7 , make surprisingly normal immune responses. They can reject grafts ( 26 ; and our unpublished observations), resist intracellular infections such as Leishmania 27 , and mount many normal viral immune responses 6 28 . The one consistent immune deficit is the capacity to make B cell GCs 7 21 , and this is linked to selective deficiency in Th2 responses 8 29 . However, if CD28 is blocked acutely in normal mice by injecting CTLA4-Ig, very different results can be obtained 30 . CD4 T cell responsiveness is regulated by a balance between CD28 (positive) and CTLA4 (negative) signaling 26 , and the differences observed with acute and chronic blockade of CD28 by CTLA4-Ig could be due to individual T cells tuning their responsiveness to the costimulatory environment in which they mature. In this paper, we found that acute reversal of CD28 signaling in CTLA4-Ig tg mice led to massive GC formation, which is in keeping with the idea that CD4 T cells in these mice are hyperresponsive when CD28 blockade is reversed 31 32 . However, our data also provide a potential explanation for why the timing of CD28 blockade may have very different consequences for the subsequent immunological response. We have shown that CD28 signaling is permissive for other costimulatory signals such as OX40. We speculate that delaying CD28 blockade allows OX40 signaling to occur at the onset of immune responses, which allows Th2 CD4 T cells to develop 30 33 , resulting in less damaging autoimmune and transplant responses. It could also explain why blocking CD86 aggravates experimental allergic encephalitis, a model in which pathology is largely Th1 mediated 34 . CD86 is constitutively expressed on DCs and could provide crucial early CD28 signals required for OX40 expression. Evidence that OX40 blockade modifies the course of inflammatory disorders 35 36 indicates roles for OX40 other than in development of GCs. In addition to its expression on activated DCs and B cells, OX40L is expressed on vascular endothelium 37 . An intriguing possibility is that ligation of OX40 here is involved in recruitment of activated T cells to inflammatory sites. Both CTLA4-Ig 38 and OX40-Ig 35 abrogate susceptibility to experimental allergic encephalitis. However, CTLA4-Ig does not block priming of encephalogenic T cells 38 . This might be explained if CTLA4-Ig blocked OX40 expression, hence inhibiting migration into the central nervous system. Therefore, the effects of OX40 ligation may depend on the context in which CD4 T cells find themselves. | Study | biomedical | en | 0.999996 |
10523610 | Six 10-wk-old Balb/cAnN mice were obtained from Charles River Laboratories. C57BL/6 (B6), MRL/MpJ (MRL), and MRL/Mp- lpr/lpr (MRL-lpr) mice were from The Jackson Laboratory. DO11.10 TCR-transgenic mice 32 on the Balb/c background, and aged and control B6 mice for memory T cell analysis were maintained in the University of California at San Francisco animal care facility. Eight 10-wk-old, specific pathogen-free, male B10.BR mice (The Jackson Laboratory) were housed under barrier conditions at the Duke University Vivarium. HIS 6 -tagged murine BLC was prepared by PCR-based insertion of six histidine codons preceding the BLC stop codon. The BLC-his6 construct was inserted into the CMV-based mammalian expression vector pRK5 33 and stably transfected into HEK-293 cells using the Lipotaxi Mammalian Transfection kit (Stratagene) according to the manufacturer's instructions. HIS 6 -BLC was purified from tissue culture supernatants using an NiNTA column (Qiagen). The protein was eluted in 100 μM imidazole (Fisher Scientific Co.). SDS-PAGE separation of the eluate revealed a band representing >90% of total protein corresponding to the recombinant HIS 6 -BLC. HIS 6 -ELC was produced in bacteria and purified as described 23 . A similarly constructed vector for bacterial HIS 6 -SLC production was a gift from M. Gunn (Duke University, Durham, NC). Stromal cell–derived factor (SDF)1α (N33A) produced by chemical ligation (Gryphon Sciences), HIS 6 -BLC, -ELC, and -SLC were used in all chemotaxis assays, except for MRL-lpr chemotaxis where, due to availability at the time of the experiments, non–HIS-tagged murine SLC (gift of M. Gunn) and human ELC (R&D Systems) were used. We have not observed significant differences in the response of mouse cells to mouse or human ELC. Adoptive transfer and immunization of recipients were carried out essentially as described 13 . Lymphocytes were isolated from LNs or spleen of DO11.10 donor mice, and the percentage of OVA 323–339 peptide/I-A d –specific CD4 + T cells was determined by flow cytometric analysis of an aliquot of cells stained with FITC-conjugated clonotypic mAb KJ1-26 and anti-CD4–PE (Caltag). 2.5 × 10 6 KJ1-26 + CD4 + cells were adoptively transferred into sex-matched Balb/c recipients by intravenous injection. The day after cell transfer, mice were immunized with 300 μg OVA 323–339 peptide either emulsified in CFA (Sigma Chemical Co.) and injected subcutaneously in a total volume of 0.1 ml distributed over three points on the back, or in sterile PBS by intravenous injection. Recipients were killed and dissected 2, 3, 5, 7, 10, 14, or 25 d after immunization. In a second immunization protocol 34 , mice were injected subcutaneously with 2 mg OVA protein (Sigma Chemical Co.) mixed in 0.2 ml of 250 μg/ml LPS (Sigma Chemical Co.). For subcutaneously injected mice, lymphocytes were isolated from axillary and brachial LNs. For intravenously injected mice, cells from mandibular, cervical, axillary, brachial, inguinal, and in some cases mesenteric nodes were pooled. The remaining peripheral LNs were frozen in OCT (Miles, Inc.) for sectioning. Flow cytometric analysis was performed using affinity-purified anti-CXCR5 rabbit antiserum 35 , followed by biotinylated goat anti–rabbit IgG (PharMingen) with normal mouse and rat serum (1:100 dilution), and then streptavidin-Cychrome (PharMingen), KJ1-26–FITC, anti-B220–PE, and anti-CD8–PE (Caltag). Whole PCC (Sigma Chemical Co.) was diluted into PBS and mixed with the Ribi adjuvant system (RAS; Ribi Immunochem Research). B10.BR mice were immunized with 400 μg of PCC in 200 μl of adjuvant emulsion in two 100-μl doses by subcutaneous injection on either side of the base of the tail. Animals were killed at 3, 5, 7, and 9 d after immunization, and the draining LNs were harvested as described previously 17 . In brief, inguinal and periaortic nodes were collected, and using 0.17 M NH 4 Cl solution for erythrocyte lysis were made into single cell suspensions. Cells were incubated with anti-CXCR5 rabbit antiserum, followed by anti–rabbit IgG-biotin (Santa Cruz Biotechnology). After blocking with normal rabbit and mouse serum (1:100 dilution) for 5 min, staining was completed using streptavidin-PE (PharMingen), anti-Vα11–FITC (PharMingen), anti-Vβ3–allophycocyanin, anti-B220–Cy5PE (PharMingen), anti-CD8–Cy5PE (PharMingen), anti-CD11b–Cy5PE (Caltag), and anti-CD44–Texas red. Finally, cells were resuspended in 2 μg/ml propidium iodide (for dead cell exclusion) in PBS with 5% FCS. The cells were analyzed using a dual laser modified FACStar PLUS™ (Becton Dickinson Immunocytometry Systems; an argon laser as the primary, a tunable dye laser as the secondary) capable of seven-parameter collection. Files were acquired using CELLQuest™ software (Becton Dickinson) and analyzed using FlowJo software (Tree Star, Inc.). Chemotaxis assays were performed as described 23 using 10 6 total cells per 5-μm transwell (Corning Costar Corp.). To identify migrating populations, a fraction of transmigrated cells was stained and analyzed by flow cytometry. Transmigrated LN cells from Balb/c recipients of OVA-specific T cells were stained with KJ1-26–FITC and anti-CD4–TriColor (Caltag), or with anti-CD4–PE (Caltag) and KJ1-26–biotin followed by streptavidin-Cychrome (PharMingen). Because BLC causes reversible internalization of CXCR5 36 , transmigrated splenocytes from aged B6 mice were washed twice and incubated in RPMI plus 0.5% BSA for 1 h at 37°C, 5% CO 2 to allow CXCR5 reexpression before staining with anti-CXCR5 rabbit antiserum/goat anti–rabbit IgG-biotin/streptavidin-Cychrome, anti-CD4–FITC (Caltag), and anti-CD62L–PE (PharMingen). LN suspensions from 5–8-mo-old MRL-lpr mice were stained with anti-B220–PE, anti-Thy1.2–biotin (Caltag), and anti-CXCR5 rabbit antiserum followed by goat anti–rabbit IgG-FITC (Caltag) and streptavidin-Cychrome. To provide an internal control, MRL-lpr splenocytes (70% Thy1 + B220 + , 9% Thy1 + B220 − ) were mixed 3:1 with B6 splenocytes (<1% Thy1 + B220 + , 27% Thy1 + B220 − ) for chemotaxis assays. Transmigrated cells were stained with anti-Thy1.2–FITC (Caltag), anti-B220–PE, and anti-CD21–biotin/streptavidin-Cychrome. DN T cells were purified from LNs of 5–8-mo-old MRL-lpr mice. Total LN cells were incubated with biotinylated mAbs against CD22 (PharMingen), CD4, and CD8 (Caltag) followed by streptavidin-coated magnetic beads, and then passed over a MACS ® column (Miltenyi Biotec). Memory phenotype splenocytes from 14-mo-old mice were enriched by MACS ® depletion with biotinylated mAbs against CD8, B220, and CD11b (Caltag). For all transfers, 2 × 10 7 cells were labeled with 5- (and 6-)carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes) as described 35 and transferred by intravenous injection into appropriate syngeneic (MRL or B6) recipients. After ∼24 h, recipients were killed, and spleen, LNs, and Peyer's patches were frozen in OCT for sectioning. For immunohistochemistry, cryostat sections (7–8 μm) were fixed and stained as described previously 35 with the following reagents: biotinylated or FITC-conjugated KJ1-26, rat anti-CD4 and anti-CD8 (Caltag), and biotinylated peanut agglutinin (PNA; Sigma Chemical Co.). Biotinylated reagents were detected with avidin–alkaline phosphatase (Sigma Chemical Co.), rat mAbs with horseradish peroxidase–conjugated goat anti–rat IgG (Southern Biotechnology Associates), and KJ1-26–FITC with horseradish peroxidase–conjugated antifluorescein (NEN). Enzyme reactions were developed with conventional substrates for peroxidases (diaminobenzidine/H 2 O 2 ; Sigma Chemical Co.) and alkaline phosphatase (Fast Red/Naphthol AS-MX; Sigma Chemical Co.). Endogenous alkaline phosphatase activity was blocked with levamisole (Sigma Chemical Co.). Some sections were counterstained with hematoxylin (Fisher Scientific Co.). Sections were mounted in crystal mount (Biomeda Corp.). For immunofluorescence microscopy, unfixed sections were air-dried and incubated with biotinylated mAbs against CD8, CD4, or Thy1.2 (Caltag) and CD3∈ (PharMingen) followed by streptavidin-Cy3 (Jackson ImmunoResearch Labs). Three-color staining of spleen sections was achieved by costaining with rat anti–MOMA-1 37 followed by goat anti–rat IgG-aminomethylcoumarin (Jackson ImmunoResearch Labs). Sections were mounted in Fluoromount G (Southern Biotechnology Associates), viewed, and photographed as described 35 . In situ hybridization analysis was performed as described 23 using a BLC probe spanning nucleotides 27–1042 of mouse BLC 18 . Mice that had received an inoculum of OVA-specific TCR-transgenic (DO11.10) T cells were injected with OVA 323–339 peptide either subcutaneously in CFA to promote T cell trafficking to follicles, or intravenously in PBS to promote transient T cell activation without migration to follicles 13 . At days 2, 3, 5, and 10 after immunization, LNs were isolated and analyzed by flow cytometry with a clonotypic antibody, KJ1-26, that recognizes the transferred OVA-specific T cells, and with an antiserum specific for CXCR5 35 36 . Before immunization, transferred OVA-specific T cells were uniformly CXCR5 lo/− . However, within 2 d of immunization with peptide in CFA, when OVA-specific T cell numbers in the draining LNs start to increase 13 , a subpopulation of CXCR5 + cells could be identified . By day 3, when the OVA-specific T cell frequency had increased ∼30-fold, as in previous studies 13 , most of the antigen-specific cells expressed high levels of CXCR5 (fluorescence intensity at least 10-fold greater than the staining control), and after 5 d the cells were uniformly CXCR5 hi . Appearance of T cells in follicles followed similar kinetics to the CXCR5 upregulation . Consistent with previous reports 13 34 , KJ1-26 + OVA-specific T cells began appearing in follicles by day 3 after immunization and reached maximal numbers by day 5 . An enlargement of draining LN B cell areas occurred over this time period, and by day 5 many of the follicles contained nascent GCs . Immunization of mice with OVA protein in LPS, a protocol that has been shown to promote T cell trafficking to follicles 34 , also led to increased expression of CXCR5 on CD4 T cells . In contrast to these effects, intravenous injection of OVA peptide in saline led to only weak induction of CXCR5 on OVA-specific T cells in LNs and spleen (data not shown), and did not promote KJ1-26 + T cell migration into follicles . When mice immunized with OVA peptide in CFA were followed for longer times, a decline in CXCR5 expression was found to occur, although a significant proportion of KJ1-26 + cells remained CXCR5 hi at day 10 after immunization , and CXCR5 hi cells could still be detected at day 25 (data not shown). By day 10, many B cell areas had become secondary follicles, comprising a well-developed GC and a surrounding mantle of small resting B cells . Significant numbers of KJ1-26 + T cells were detectable in the secondary follicles, with many residing in the mantle zone and smaller numbers being associated with the outer zone of GCs . BLC in situ hybridization analysis of LNs containing well-developed GCs showed that BLC was highly expressed in the follicular mantle zones . Within GCs, only occasional cells could be identified that hybridized with the BLC probe, and these cells tended to be most frequent in the area of the GC distal to the T zone . This is also the region of the GC most enriched for CD4 T cells . To test whether CXCR5 was upregulated on nontransgenic T cells under conditions not involving T cell transfer, the phenotype of PCC-specific Vα11Vβ3-expressing CD4 T cells was followed in mice immunized subcutaneously with PCC protein in adjuvant. Vα11Vβ3-expressing T cells responding to PCC were detected in immunized, but not unimmunized, animals by upregulation of CD44 . CXCR5 expression became detectable on a subset of PCC-responsive Vα11 + Vβ3 + T cells by day 3 after immunization . This subpopulation grew in frequency through day 9 , reaching maximal total numbers by day 7 . These kinetics of CXCR5 expression are in close accord with the kinetics of Vα11Vβ3-expressing T cell accumulation in follicular mantle zones and GCs during the response to PCC 9 17 . A subpopulation of the responding CD44 + Vα11Vβ3-expressing T cells did not upregulate CXCR5 . Such bimodality was not observed in the response of the monoclonal DO11.10 T cells to OVA peptide and may indicate that T cells with differing affinity for peptide/MHC differ in their propensity to upregulate CXCR5. The rapid upregulation of CXCR5 on T cells after injection of antigen in adjuvant and the migration of a fraction of the cells into follicles suggested that these cells had acquired responsiveness to BLC. This was tested directly ex vivo in transwell migration assays. OVA-specific T cells from draining LNs of adoptive transfer recipients immunized 7 d earlier with OVA peptide subcutaneously in CFA showed that a strong dose-dependent response to BLC . By contrast, OVA-specific cells from recipients given the antigen intravenously in the absence of adjuvant did not respond to BLC . Kinetic analysis using cells from mice immunized with OVA peptide in CFA showed the BLC response was detectable by day 2 and was maximal between days 5 and 10 . In addition, flow cytometric analysis of transmigrated OVA-specific cells on day 3 revealed that while most input cells expressed CXCR5 , there was an enrichment for cells expressing the highest levels of CXCR5 in the responding population . These findings provide evidence for a direct relationship between CXCR5 expression on T cells and acquisition of BLC responsiveness, although they do not establish whether the activation state of the cells also affects their ability to respond. To investigate the BLC responsiveness of CXCR5-expressing T cells with a resting phenotype, we took advantage of previous observations that a subset of memory CD4 T cells expresses CXCR5 20 21 . CD4 T cells from aged (≥1 yr) mice were characterized as a source of memory phenotype T cells, and a great majority of the L-selectin lo CD4 T cells from these animals were found to express CXCR5 . In addition to low L-selectin expression, the majority of CXCR5 hi T cells in young and old mice expressed high levels of CD44 and reduced amounts of CD45RB (data not shown). Most of the CXCR5 hi cells were also negative for the activation markers CD69 and CD25, further supporting their designation as memory cells. Interestingly, though a significant proportion of memory phenotype T cells in young mice expresses CXCR5, this proportion was consistently increased in aged mice . In in vitro chemotaxis assays, the CXCR5 hi memory T cells showed a very similar dose-sensitive BLC response to the activated OVA-specific T cells characterized above . These results establish that both resting and activated CXCR5 hi T cells respond to BLC. Interestingly, although the magnitude of the T cell response to BLC was lower than that observed for B cells, the T cells responded maximally to lower concentrations of BLC than did B cells . This finding is similar to that made previously with transfected Jurkat T cells 18 , suggesting that T cells are intrinsically more sensitive than recirculating B cells to CXCR5 signaling. CD4 T cells express severalfold less surface CXCR5 than B cells , demonstrating that higher surface chemokine receptor expression does not equate to higher chemokine sensitivity. T cells have been shown to respond strongly to ELC and SLC in in vitro chemotaxis assays 22 23 24 27 28 29 30 . Since these chemokines are expressed in the T zone and might be able to counteract the ability of a cell to respond to a chemokine made in follicles, we tested whether CXCR5-expressing T cells were altered in their responsiveness to ELC and SLC. In striking contrast to the elevated ELC and SLC responsiveness of in vitro–activated T cells 28 29 30 , OVA-specific T cells activated in vivo by subcutaneous peptide/CFA injection showed a significant downregulation in responsiveness to these T zone chemokines . The time course of this decreased responsiveness to ELC was similar to the time course over which the cells became responsive to BLC . In contrast, OVA-specific T cells from mice immunized intravenously with peptide in saline maintained their ability to respond to ELC throughout the 10-d time course . Responsiveness to SDF1 was also decreased in the in vivo–activated T cells , consistent with the recent in vitro finding that anti-CD3 stimulation reduces responsiveness to SDF1 38 . CXCR5-expressing memory phenotype cells from aged mice showed similarly low responsiveness to ELC and SLC . Interestingly, in these cells the responsiveness to SDF1 was elevated . The studies above demonstrate a tight relationship between CD4 T cell upregulation of CXCR5 expression, acquisition of BLC responsiveness, and migration into lymphoid follicles during an immune response. However, they do not establish whether intrinsic changes in the T cell are sufficient to direct these cells to follicles or whether additional (extrinsic) changes that accompany the adjuvant-induced immune response are also needed. Although many of the CD4 T cells in aged mice express CXCR5 , immunohistochemical analysis did not reveal a significantly greater number of T cells in follicles in aged mice compared with young mice (data not shown). When CXCR5 hi CD4 cells were transferred from aged to young mice, they were found to localize within the T zone, with only occasional cells migrating into follicles . These findings indicate that expression of CXCR5 is not sufficient to direct all types of T cells into B cell follicles. However, in contrast to aged normal mice, aged MRL-lpr mice contain very large numbers of T cells in a follicular distribution 39 40 . These CD3 + T cells are unusual in lacking CD4 and CD8 and in expressing B220 39 40 . Flow cytometric analysis showed that they also express high surface CXCR5 , and in in vitro chemotaxis assays they demonstrated a robust response to BLC and a reduced response to ELC and SLC . Importantly, the BLC dose–response curve of the DN T cells was typical of CXCR5 + CD4 T cells and not of B cells . Although the follicular location of the DN T cells in MRL-lpr mice suggests these cells have acquired the intrinsic ability to migrate to follicles, MRL-lpr mice have multiple immunological abnormalities, and it was possible that homing of DN T cells to follicles was dependent on extrinsic changes in the lymphoid tissues. To test this directly, DN T cells were purified from LNs of MRL-lpr mice, labeled with CFSE, and transferred to normal syngeneic MRL mice. Strikingly, the transferred T cells migrated to regions proximal to B cell areas in all secondary lymphoid tissues of the recipient mice . Differences in the distribution of the cells were noted in the different tissues. In the spleen, the T cells homed to the outer rim of the follicles, especially near the marginal zone bridging channels, and often the cells appeared in contact with marginal metallophilic macrophages . In LNs the T cells homed to perifollicular and interfollicular locations , and in Peyer's patches the cells were seen to circle the whole follicular area . These observations demonstrate that T cells can acquire the intrinsic ability to migrate to the boundaries of lymphoid follicles. The findings above establish that immunization with antigen in adjuvant causes antigen-specific T cells to upregulate CXCR5 expression and acquire responsiveness to the follicular chemokine, BLC, while simultaneously becoming less responsive to the T zone chemokines, ELC and SLC. We propose that this reprogramming of responsiveness to B and T zone chemokines is part of the mechanism by which antigen-activated T cells home to follicles to help initiate T-dependent antibody responses. In the adoptive transfer studies of Jenkins and co-workers, it was observed that antigen needed to be injected in adjuvant for activated T cells to migrate to follicles 13 34 . When antigen was injected in the absence of adjuvant, T cell activation was transient and the activated cells failed to home to follicles. Our results provide a basis for understanding the different trafficking patterns of the activated cells as they show that CXCR5 upregulation and acquisition of BLC responsiveness only occurs after injection of antigen in adjuvant. Many studies have indicated that the effectiveness of adjuvants is through their potent activation of dendritic cells (DCs 41 ), and it is therefore reasonable to suggest that effective induction of CXCR5 expression on T cells requires interaction with appropriately activated antigen-presenting DCs within the lymphoid tissue. OX40L is expressed by a subset of activated DCs 42 , and recent studies by Lane and co-workers provide evidence that stimulation of T cells through OX40 can promote upregulation of CXCR5 43 and homing of T cells to follicles 44 . Further studies are needed to define whether additional costimulatory molecules can regulate CXCR5 expression on T cells. In vivo activation by antigen in adjuvant decreases T cell responsiveness to ELC and SLC at the same time as increasing responsiveness to BLC. This contrasts with findings in vitro, where PHA- and IL-2–activated T cells responded more strongly than unactivated cells to SLC and ELC 28 29 30 and again indicates that the mode of T cell activation can strongly influence chemokine responsiveness. Recently, it has been shown that plt/plt mice, which exhibit defective homing of T cells to splenic T zones and LNs 45 , have a compound defect that causes a deficiency in SLC expression and markedly reduced ELC expression 31 . This finding provided strong evidence that SLC and ELC are needed for T cell homing to lymphoid T cell areas. Therefore, reduced responsiveness of CXCR5 hi T cells to ELC and SLC may allow the cells to more readily leave the T zone and enter follicles. Reciprocally, the failure of T cells activated after intravenous injection of antigen to downregulate their SLC and ELC response might contribute to their inability to migrate to follicles. Since SLC appears important for cells to enter LNs via high endothelial venules or lymphatics 22 31 46 , decreased responsiveness to this chemokine is also likely to influence the recirculation pattern of the cells. Our studies provide strong evidence that altered responsiveness to constitutively expressed chemokines is part of the mechanism by which antigen-activated CD4 T cells migrate towards and into B cell follicles. This conclusion is also supported by the transfer experiments showing that DN T cells from MRL-lpr mice are intrinsically capable of migrating to areas proximal to follicles . However, the failure of CXCR5 hi CD4 T cells to migrate to follicles after adoptive transfer suggests that additional factors might normally help guide antigen-activated CD4 T cells. The migration of only a subset of OVA-activated T cells to follicles also suggests that CXCR5 hi cells may be heterogeneous in their responsiveness to these factors. Several studies have shown that B cell receptor–stimulated B cells upregulate expression of chemokines, including MIP-1α, MIP-1β 47 48 , and macrophage-derived chemokine (MDC 49 ), that can attract subsets of activated T cells 50 51 52 53 . Since antigen-activated B cells move to the boundary of B and T zones 3 , it is likely that chemokines produced by activated B cells work together with constitutively expressed chemokines to bring antigen-activated T cells to the outer T zone. Whether further cues are needed to direct cells from the outer T zone into follicles remains unclear, although the failure of MRL-lpr DN T cells to migrate to the inner regions of follicles suggests this is the case. Possibilities include changes in the responsiveness of the T cells to other unknown chemokines, and changes in the relative adhesiveness of the cells for features of the B or T cell compartments. Several examples of cell sorting occurring as a result of differential adhesiveness of cells have been reported 54 55 56 . A major role of T cells inside follicles is to support the GC response 1 2 . 9 d after immunization with PCC in adjuvant, a large majority of the PCC-responsive Vα11Vβ3-expressing T cells express CXCR5 , and at this time point, ∼75% of the cells are localized within GCs 17 . Therefore, at least a subset of PCC-responsive CXCR5 hi T cells acquires the ability to enter GCs. Similarly, in the DO11.10 adoptive transfer system, 10 d after immunization with OVA peptide in CFA, the majority of KJ1-26 + T cells were CXCR5 hi , and many cells were found in follicular mantle zones and GCs . The strong expression of BLC in primary follicles 18 and in follicular mantle zones of secondary follicles is consistent with BLC playing a role in attracting T cells to these sites. The presence of only small numbers of BLC-expressing cells within GCs, predominantly in the region distal to the T zone (most likely corresponding to the GC light zone ) suggests that while BLC/CXCR5 might have a role in helping position cells within GCs, additional cues are likely to be needed. These findings also establish that there is substantial heterogeneity among follicular stromal cells in terms of BLC expression levels, with GC follicular DCs expressing relatively little of this chemokine. Perhaps by being concentrated predominantly outside the T zone–distal pole of the GC, BLC helps polarize the GC light and dark zone compartments. The notion that cues other than BLC play important roles in GC organization is supported by the finding that GCs are able to form in CXCR5-deficient mice 21 . Furthermore, although spleens of CXCR5-deficient mice lack polarized follicles and contain aberrantly located GCs, the follicular disruption in LNs appeared to be minimal 21 . Although this suggests that the role of CXCR5/BLC in B and T cell homing to LN follicles is redundant to other chemokine/receptor systems, studies in mice lacking lymphotoxin or TNF have shown that effects on follicular organization in LNs are more difficult to detect than in spleen or Peyer's patches 57 58 , yet these effects can still be substantial 59 60 . As we have shown here, BLC is expressed in LN follicles, and CXCR5 is strongly upregulated on activated T cells in LNs. Future studies of BLC-deficient mice should help further dissect the contribution of BLC and CXCR5 to follicular organization and GC formation in LNs. T cell homing to follicles may be important not only for providing help to B cells, but also for providing activated T cells with growth and survival signals. This possibility is suggested by the finding that antigen injected in the absence of adjuvant fails to promote T cell migration to follicles, and also fails to promote survival of activated or memory T cells 13 34 . The selective accumulation of the CXCR5-expressing subset of CD4 T cells in aged mice and in HIV-infected humans during disease progression 61 is consistent with the notion that trafficking through B cell areas plays a role in long-term survival of memory T cells. This possibility is also supported by the finding of a defect in CD4 T cell memory in B cell–deficient mice 62 . Although we did not observe trafficking of CXCR5 hi memory T cells to follicles in short-term transfer experiments, studies in rats have suggested that memory T cells migrate through follicles at greater frequency than naive T cells 63 . In summary, our findings suggest a model for how helper T cells home to follicles. After engagement of peptide/MHC complexes on appropriately activated T zone DCs, CD4 T cells upregulate CXCR5, acquire responsiveness to the follicular chemokine BLC, and simultaneously downregulate responsiveness to the T zone chemokines ELC and SLC. Together with additional presently undefined changes, this reprogrammed chemokine responsiveness helps propel T cells toward B cell areas. Further cues, such as those emanating from activated B cells and from GC cells, can then act upon these T cells to more precisely control their positioning and facilitate their ability to act as B cell helpers. | Study | biomedical | en | 0.999999 |
10523611 | Rats (male Sprague-Dawley) were purchased from The Jackson Laboratory, and islets were isolated as described previously 23 . Human islets were a gift from Dr. C. Ricordi (Diabetes Research Institute, University of Miami School of Medicine, Miami, FL). Both rodent and human islets were cultured in RPMI 1640, 10% FCS with 2 mM l -glutamine, 5 mM d -glucose, and 50 U/ml of penicillin and streptomycin, at 37°C with 5% CO 2 . Total mRNA was isolated from human and rodent islets (RNeasy Mini Protocol; Qiagen), and cDNA was synthesized using random hexamers (Superscript Preamplification System for First Strand cDNA Synthesis; GIBCO BRL). PCR reactions were performed with the following primers: rodent β-actin: sense, 5′-CCTGACCGAGCGTGGCTACAGC-3′, and antisense, 5′-AGCCTCAGGGCATCGGAAC-3′; A20: sense, 5′-TTTGAGCAATATGCGGAAAGC-3′, and antisense, 5′-AGTTGTCCCATTCGTCATTCC-3′; rat iNOS: sense, 5′-TGACCTGAAAGAGGAAAAGGAC-3′, and antisense, 5′-CCAGTTTTTGATCCTCACGTG-3′. The PCR reaction was optimized for each primer pair. PCR was performed over a range of cycles 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 to ensure that amplification occurred in the linear range, and equal starting amounts of each sample were used. The rAd vector expressing A20 (rAd.A20) was a gift from Dr. V. Dixit (Department of Molecular Oncology, Genentech, Inc., South San Francisco, CA); the control vector expressing β-galactosidase (rAd.β-gal) was a gift from Dr. R. Gerard (Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, TX). Islets were infected with rAd vectors immediately after isolation as described previously for other cell types 28 . After infection, islets were cultured for an additional 24 h before being used for further experiments. For all experiments (unless otherwise stated), 200 islets were cultured in 500 μl of media in 24-well tissue culture plates. Expression of A20 protein after rAd.A20 gene transduction in islets was determined by Western blotting using standard techniques. A20 protein expression was detected with a polyclonal A20 antiserum (A20-NT) raised against an NH 2 terminus peptide sequence of human A20 (IRERTPEDIFKPTN). Islet viability after viral infection was assessed by staining with propidium iodide (10 μg/ml) and calcein-AM (2 μM; Molecular Probes), then determined by two-color fluorescence microscopy. Islet cultures were stimulated with recombinant murine IL-1β (10 U/ml) and recombinant rat IFN-γ (300 U/ml) (R&D Systems) for 40 h. Islets were then harvested, dispersed, fixed in 70% ethanol, and suspended into DNA staining buffer (PBS, pH 7.4, containing 0.1% Triton X-100, 0.1 mM EDTA, 50 μg/ml propidium iodide, 50 μg/ml RNase A). Islet DNA content was analyzed on a FACScan™ using CELLQuest™ acquisition software (Becton Dickson Immunocytometry Systems). Islets with a normal DNA content (≥2 N) were scored as viable, whereas islets with a hypodiploid DNA content (<2 N, termed A°) were scored as apoptotic. To exclude debris and apoptotic cell-free fragments, all events with an FL-2 area profile below that of chicken erythrocyte nuclei were excluded from analysis. To determine the effects of A20 expression on iNOS protein induction, islets were stimulated with IL-1β (10 U/ml) for 24 h. iNOS protein expression was determined by Western blotting using the polyclonal anti-iNOS Ab, N-20 (Santa Cruz). Culture media were analyzed for NO levels (measured as nitrite) by adding 50 μl of Griess reagent (equal volume of 1% sulfanilamide in 0.1 M HCl and 0.1% N -[-1-naphthyl-ethylenediamine dihydrochloride]) to 50 μl of culture media. Nitrite concentration was determined by spectrophotometry (560 nM) from a standard curve (0–200 μM) derived from NaNO 2 . NO data are expressed as mean ± SD [nitrite] in μM per 200 islets. To examine whether NO could directly induce apoptosis, islets were treated for 24 h with the NO donors S -nitrosoglutathione (GSNO) or N -(2-aminoethyl)- N -(2-hydroxy-2-nitrosohydrazino-1,2-ethylenediamine (NONOate) over a range of concentrations (0.001–10 mM). To determine the role of NO in cytokine-induced apoptosis, islets were treated with IL-1β (10 U/ml) and IFN-γ (300 U/ml) in the presence or absence of the NOS inhibitor l - N 5 -(1-iminoethyl) ornithine, dihydrochloride (L-NIO) used at the optimal concentration of 500 μM. The extent of islet apoptosis and NO generation was determined as described above. β-TC 3 cells 29 were plated at a density of 1.5 × 10 6 cells/well into 6-well tissue culture plates and transfected 24 h later using the Lipofectamine-Plus reagent (GIBCO BRL) with 1 μg total DNA. Specifically, β-TC 3 cells were transfected with 0.6 μg of the iNOS reporter (pGLH/H2; containing 1,755 bp of the murine iNOS promoter linked to a luciferase gene ), a gift of Dr. W.J. Murphy (Wilkinson Laboratory of the Kansas Cancer Institute, University of Kansas Medical Center, Kansas City, KS); 0.3 μg of an expression plasmid containing the human A20 gene (pcDNA 3 /HA-A20) or the control empty plasmid pcDNA 3 ; and 0.1 μg of a β-gal reporter (driven by the CMV promoter), used to correct for transfection efficiency. 24 h after transfection, cells were stimulated with IL-1β (100 U/ml) for 36 h. These conditions were shown to be optimal in preliminary experiments (data not shown). Luciferase and β-gal activity were assessed as described 27 . Data are expressed as relative luciferase activity according to the formula: luciferase light units/β-gal light units × 100. To determine the effect of A20 overexpression on the transcription factor NF-κB, islets (1,000 islets/1 ml media in 24-well tissue culture plates) were stimulated with IL-1β (100 U) for 1 h. Islet nuclei were recovered by an isoosmotic/NP-40 lysis procedure, and nuclear proteins were extracted as described 31 . DNA binding reactions were performed by incubating 5 μg of nuclear proteins with 1 μg of poly(dI-dC) and 10 5 cpm of radiolabeled NF-κB consensus oligonucleotide, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′ (Promega Corp.). For competition assays, 1.75 pmol of either unlabeled NF-κB or an unrelated oligonucleotide was added to the reaction mixture. Supershift analysis was conducted by adding 0.1 μg of Ab specific for p50/NF-κB1, p65/RelA, Rel-B, c-Rel, or Ets-1 (Santa Cruz) to the reaction 1 h before the addition of radiolabeled oligonucleotide. The DNA binding reactions were resolved on a 6% polyacrylamide gel and analyzed by autoradiography. The effect of A20 expression on IκBα protein degradation was determined by Western blot analysis, after treatment with IL-1β (100 U/ml) for 0, 15, and 60 min. IκBα protein expression was detected using the polyclonal anti-IκBα Ab, C-20 (Santa Cruz). All statistical analysis was conducted using the alternate Welch's method. We first examined if A20 was expressed constitutively in islets and whether A20 expression could be induced by cytokine stimulation. No or weak constitutive A20 mRNA was detected in rat and human islets as analyzed by reverse transcription (RT)-PCR . A20 mRNA was rapidly induced (within 1–2 h) in both rat and human islets after IL-1β stimulation . Rat β insulinoma cells (Rin5F) could also be induced to rapidly express A20 mRNA after IL-1β stimulation, indicating that β cells specifically express A20 . The identity of the A20 PCR product was confirmed by sequence analysis (data not shown). Our data demonstrate that A20 is an early response gene in cytokine-activated islets. To study the function of A20 in islets, we overexpressed A20 by rAd-mediated gene transfer. Islets infected with a recombinant β-gal adenovirus (rAd.β-gal) were used as controls. Islets infected in vitro with a rAd carrying the A20 transgene (rAd.A20) expressed high levels of A20 protein . In vitro–infected islets showed normal morphology and viability in culture . Infection with greater multiplicity (MOI; e.g., ≥30:1) led to significant toxicity in our system (data not shown) and hence MOIs in the range of 1–20:1 were used. To test the function of islets after infection with rAd, 500 freshly isolated islets were infected in vitro with rAd.β-gal (MOI 10:1) for 1 h at 37°C. Islets were then washed and transplanted under the kidney capsule of B6AF1 mice rendered diabetic by intraperitoneal injection of streptozotocin (160 mg/kg) 7–14 d before the day of transplantation 32 . All transplanted animals were normoglycemic (glucose levels 76–116 mg/dl) by day 4–5 after transplantation. This result indicates that adenoviral infection of islets per se does not alter their function. Previous work has demonstrated that A20 is an early response gene that protects cells against cytokine-mediated cytotoxicity 25 26 . The proinflammatory cytokine IL-1β is cytotoxic to β cells and represents a significant mediator of β cell apoptosis in IDDM, especially in combination with IFN-γ 10 . Therefore, we examined whether A20 would protect islets against IL-1β– and IFN-γ–mediated toxicity. IL-1β and IFN-γ used at the optimal dose of 10 and 300 U/ml, respectively, induced a significant percentage of apoptosis in rat islets after 40 h in culture . This percentage (mean ± SD, n = 4 independent experiments) reached 57.58 ± 16.51 and 55.08 ± 18.35% in both noninfected and control rAd.β-gal–infected islets, respectively ( P < 0.01, n = 4), as evaluated by FACS ® analysis of DNA content . In contrast, rAd.A20-infected islets were protected from IL-1β– and IFN-γ–mediated apoptosis; the percentage of apoptosis in these islets was not significantly different ( P = 0.714, n = 4) from that observed in non–cytokine-activated control islets . These data demonstrate that A20 protects islets from cytokine-mediated apoptosis. There is substantial evidence that free radical generation, such as release of NO and peroxynitrites, mediates the proapoptotic effects of cytokines on islets 9 13 14 . Therefore, we examined the levels of NO released in the culture medium of noninfected, rAd.β-gal–, and rAd.A20-infected islets 40 h after cytokine stimulation. Noninfected and rAd.β-gal–infected islets produced equally high levels of NO after stimulation with IL-1β and IFN-γ . In contrast, NO production in rAd.A20-infected islets was totally suppressed compared with noninfected and rAd.β-gal–infected islets and was not significantly different ( P = 0.099, n = 4) from background levels observed in non–cytokine-activated groups . Thus, the percentage of islets undergoing apoptosis for each treatment correlated with their production of NO. Our data demonstrate that A20 can protect islets from cytokine-induced apoptosis. Furthermore, they show that the antiapoptotic effect of A20 correlates with suppression of cytokine-induced NO production, suggesting that A20 is protecting islets through effects on NO generation. This hypothesis is in accordance with data from the literature showing that NO is a key mediator of cytokine-induced islet cytotoxicity 9 14 33 . To determine whether the antiapoptotic effect of A20 was a direct result of its ability to suppress NO production, we examined the role of NO in cytokine-induced apoptosis of islets. We first examined if NO could directly induce apoptosis in rat islets. Rat islets were cocultured with one of two NO donors, NONOate or GSNO, at various concentrations ranging from 0.01 μM to 10 mM. 16 h later, islets were examined for induction of apoptosis . Both NONOate and GSNO, in a dose-dependent manner, induced significant levels of apoptosis in rat islets. However, NONOate was 10-fold more potent than GSNO due to its higher release of NO in the medium . Given that NO is able to directly induce apoptosis in rat islets, we next examined whether NO was the agent responsible for islet apoptosis after cytokine stimulation. The NOS inhibitor L-NIO (500 μM) was added to cytokine-stimulated islets. Islets stimulated with IL-1β and IFN-γ underwent apoptosis and generated high levels of NO . In contrast, islets stimulated with IL-1β and IFN-γ in the presence of L-NIO were completely protected from apoptosis ( P < 0.001, n = 3), and NO generation was suppressed to below background levels . Taken together, these data demonstrate that NO is the central mediator of cytokine-induced islet apoptosis. To clarify the mechanism(s) by which A20 was suppressing NO production, we examined the effects of A20 overexpression on iNOS protein expression, steady state mRNA levels, and regulation of gene transcription. For these and subsequent experiments, islets were stimulated with IL-1β alone, as IFN-γ by itself had little or no effect on NO induction (data not shown). We examined whether A20 overexpression would modulate the induction of iNOS protein after cytokine stimulation. Noninfected and rAd.β-gal–infected islets expressed high levels of iNOS protein 24 h after activation with IL-1β . These data are in accordance with previous studies demonstrating that in islets, cytokine treatment results in de novo production of iNOS mRNA and protein 34 . In contrast, IL-1β–mediated upregulation of iNOS protein was totally suppressed in A20-expressing islets . Accordingly, NO generation after IL-1β stimulation was highly suppressed (≥90%) in A20-expressing islets compared with the significant NO levels detected in noninfected and rAd.β-gal infected islets (data not shown). To determine the underlying mechanism by which A20 was suppressing iNOS protein upregulation, we examined, by RT-PCR analysis, iNOS steady state mRNA levels after IL-1β activation. No iNOS mRNA was detected in nonstimulated islets, whereas iNOS transcript was induced 5 h after IL-1β stimulation in both noninfected and rAd.β-gal–infected islets . In contrast, no iNOS mRNA was detected in rAd.A20-infected islets . It has been established that induction of iNOS mRNA expression by IL-1β is regulated at the transcription level 30 34 35 . Therefore, we questioned whether the inhibitory effect of A20 on inos gene upregulation occurred at the level of gene transcription. To address this possibility, β-TC 3 cells were cotransfected with a murine iNOS reporter 30 and a human A20 expression plasmid or the control plasmid, pcDNA 3 . β-TC 3 cells were stimulated with IL-1β (100 U/ml) for 36 h after transfection, and luciferase values were calculated as described in Materials and Methods. As shown in Fig. 6 c, IL-1β stimulation resulted in a significant two- to threefold induction of the iNOS reporter in the pcDNA 3 -transfected β-TC 3 cells . In contrast, IL-1β induction of the iNOS reporter in A20-expressing β-TC 3 cells was totally suppressed to the extent that there was no difference relative to background levels in pcDNA 3 -transfected β-TC 3 cells ( P = 0.75, n = 5). Interestingly, A20 overexpression also significantly reduced the basal (nonstimulated) iNOS reporter activity by ∼50% ( P < 0.005, n = 5) compared with β-TC 3 cells transfected with pcDNA 3 . Our data indicate that A20 can suppress the IL-1β–dependent activation of the inos gene. Previous reports have implicated the transcription factor NF-κB as an essential component of this activation 34 36 . Therefore, we examined whether A20 was suppressing inos transcription via modulation of NF-κB activation. To check whether A20 expression was altering NF-κB translocation to the nucleus, we performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts isolated from noninfected, rAd.β-gal–, and rAd.A20-infected islets after IL-1β stimulation . A slow migrating complex, binding to an NF-κB consensus sequence, was observed in noninfected and rAd.β-gal–infected islets 1 h after stimulation with IL-1β . In contrast, this complex was not detected in nuclear extracts from rAd.A20-infected islets after IL-1β stimulation. This complex was resolved by supershift analysis to comprise the p50 and p65 NF-κB subunits . The fastest migrating band was not affected by any treatment and most likely represents a nonspecific protein interaction. These data show that A20 inhibits, in islets, the translocation of NF-κB to the nucleus. Degradation of the natural inhibitor of NF-κB, IκBα, in response to IL-1β is a prerequisite for NF-κB translocation 37 38 . We next examined whether expression of A20 in islets was affecting the degradation of IκBα in response to IL-1β. Western blot analysis of cytoplasmic extracts from noninfected and rAd.β-gal–infected islets showed that IκBα was rapidly degraded within 15 min after IL-1β stimulation . In contrast, expression of A20 in islets totally inhibited the degradation of IκBα observed after IL-1β stimulation . To ascertain that expression of A20 in islets was not simply delaying IκBα degradation, we examined IκBα levels at several time points after IL-1β stimulation (e.g., 20, 30, 45, and 60 min). No IκBα degradation was observed at any of these time points . IDDM is an autoimmune disease characterized by the specific destruction of β cells in islets of Langerhans 3 . Cumulative evidence suggests that apoptosis of the β cell is a critical component of IDDM at both the initiation and effector phases of the disease 5 . Transplantation of islets of Langerhans represents a potential cure for IDDM, but here again the success of this treatment is hampered by destruction of the islets and loss of β cells to apoptosis 23 . β cell apoptosis can be triggered by both nonspecific inflammatory reactions and specific immune responses 3 21 . One potential solution to overcome the susceptibility of β cells to apoptosis is the use of gene therapy to express genes that may impart protective properties on islets, thus enabling successful transplantation 24 39 . Little is currently known about the expression of cytoprotective genes in β cells and the molecular basis of their susceptibility to apoptosis. Recent reports demonstrated that islets constitutively express the prototypic antiapoptotic molecule Bcl-2, the stress-related heat-shock protein HSP70, and several free radical scavenging enzymes such as manganese superoxide dismutase (MnSOD) and catalase 40 . Despite expression of these proteins, β cells remain particularly sensitive to apoptosis when challenged with additional cellular stress 41 . This is in part explained by their lower expression of constitutive cytoprotective genes 41 . With this perspective in mind, we questioned whether islets are able to mount a protective response to inflammation. In this report, we examined whether β cells could be induced to express the antiapoptotic protein A20. A20 was originally described as a TNF-α–inducible 7-Zn finger protein in endothelial cells 25 . Its expression can also be induced in response to a variety of inflammatory stimuli, such as LPS, CD40 ligation, the LMP1 protein of EBV, and the Tax protein of HIV 42 43 44 45 . The rapid induction of A20 mRNA by these diverse stimuli requires the activation of the transcription factor NF-κB. Two κB binding elements map within the A20 promoter and are essential for its expression 46 . Here we show that expression of A20 is rapidly induced in β cells in response to IL-1β. This is the first report showing the induced expression of the antiapoptotic gene A20 in β cells. Further, our data show that IL-1β induces the activation of NF-κB in islets, which concurs with its ability to upregulate the expression of A20. The rapid kinetics of A20 expression in islets suggests that, as in endothelial cells, it may be a component of their physiological protective response to injury 47 . Having established that A20 is a rapid response gene in β cells, we examined whether A20 maintained its antiapoptotic function in islets. Expression of A20 in islets by means of an rAd protects them from apoptosis induced by IL-1β and IFN-γ. The protective effect of A20 against IL-1β– and IFN-γ–induced apoptosis is critical given the central role of IL-1β in β cell dysfunction and destruction during IDDM 9 48 . IL-1β inhibits glucose-dependent insulin secretion, impairs glucokinase synthesis, and induces cell death by apoptosis 49 50 . Inhibition of IL-1β using neutralizing mAbs prevents diabetes progression in NOD mice 51 . The pathway by which IL-1β mediates β cell destruction and toxicity has recently been clarified. IL-1β is produced by activated resident macrophages within the islets 48 21 52 53 . Once produced, IL-1β acts directly and selectively upon β cells to induce iNOS, leading to the production of high and sustained levels of NO and to a lesser extent superoxide 12 54 . NO directly induces apoptosis of β cells and is the mediator of the multiple toxic effects of IL-1β on β cells 55 56 57 . We confirmed the apoptotic potential of NO in our system with the NO donors GSNO and NONOate, which rapidly induced apoptosis of rat islets. Furthermore, the addition of the NOS inhibitor L-NIO to cytokine-activated islets prevented both NO production and apoptosis. These data demonstrate that endogenously generated NO is the mediator of cytokine-induced islet apoptosis in our system. The central role of NO in cytokine-mediated β cell toxicity prompted us to examine whether the protective effect of A20 in islets was associated with modulation of NO levels. We found that expression of A20 in islets abrogated NO production in response to cytokines. Taken together with our data showing that pharmacologic suppression of NO production also protects from cytokine-induced apoptosis, these data establish the suppression of NO production as one mechanism by which A20 protects islets 58 . The suppression of NO production by A20 could also impact on T cell–dependent β cell destruction. Indeed, NO facilitates T cell–dependent killing via upregulation of Fas on human islets 15 59 . Ongoing work in our laboratory is aiming at determining whether expression of A20 in islets will also protect β cells against T cell–mediated cytotoxicity via the perforin/granzyme or the Fas/FasL pathway. The mechanism by which A20 suppresses cytokine-induced NO production is shown to be via inhibition of IL-1β–induced iNOS mRNA and protein expression. Expression of iNOS protein in islets is regulated by de novo transcription of the inos gene 30 34 35 . We reasoned that the absence of iNOS protein and mRNA after cytokine stimulation points to a blockade at the level of transcription. Indeed, we found that A20 suppresses IL-1β–induced activation of a murine iNOS reporter, indicating that A20 was regulating iNOS expression at the level of gene transcription. Since NF-κB is the major transcription factor responsible for de novo activation of inos transcription by inflammatory stimuli including IL-1β, we examined the effect of A20 overexpression on NF-κB activation 60 . We found that A20 suppresses the activation of the transcription factor NF-κB in islets. Expression of other NF-κB–dependent proinflammatory genes involved in IDDM, such as intercellular adhesion molecule 1 (ICAM-1), are also expected to be blunted by A20, thereby adding to the beneficial effect of A20 as a gene therapy tool 61 62 . We have previously shown that A20 has a dual antiapoptotic and antiinflammatory function in primary endothelial cells 28 . This dual function is clearly maintained in islets, suggesting that inhibition of NF-κB activation by A20 is an important component of the natural physiological role of A20. The effect of A20 seems specific to NF-κB and is not a result of a toxic effect of A20 on the transcription machinery. Indeed, A20 overexpression had no effect on IFN-γ–mediated MHC class I upregulation (data not shown), a process requiring the activation of the transcription factors signal transducer and activator of transcription 1α (STAT-1α) and IFN regulatory factor 1 (IRF-1) 63 64 . NF-κB is a ubiquitous transcription factor constitutively expressed in the cytoplasm in an inactive form associated to an inhibitory protein termed IκBα 37 38 . Cellular activation by inflammatory stimuli such as IL-1β results in the phosphorylation and subsequent degradation of IκBα, thus allowing NF-κB to translocate into the nucleus and activate target genes such as inos 37 38 . Therefore, we examined what effect A20 had on IκBα degradation. Our data demonstrate that A20 interferes with NF-κB activation at a level upstream of the kinase cascade leading to IκBα degradation, as no IκBα degradation was observed in A20-expressing islets after IL-1β stimulation. Several potential targets for A20 within the IL-1β–stimulated cascade leading to NF-κB activation have been reported. Yeast double hybrid studies have demonstrated that A20 interacts with TNF receptor–associated factor (TRAF)-1/2, TRAF-6, and the adapter proteins 14-3-3 65 66 66a . The interaction of A20 with 14-3-3 proteins is interesting given the potential involvement of 14-3-3 (via their interaction with c-raf) in multiple signaling cascades leading to NF-κB activation 67 . In addition, IL-1β–mediated activation of NF-κB requires TRAF-6 and the IL-1 receptor–associated kinase IRAK 68 69 70 . Therefore, TRAF-6 is also a likely point where A20 intercepts the IL-1β signaling cascade. Interactions between A20 and TRAF-6 or 14-3-3 in islets are currently being studied in our laboratory. In addition, data in the literature show that IL-1β–induced NF-κB activation and inos mRNA induction can be suppressed in islets by antioxidants such as pyrrolidine dithiocarbamate (PDTC) 34 . Moreover, NF-κB is a redox-sensitive transcription factor, as indicated by the fact that NF-κB activation can be induced by H 2 O 2 or, conversely, NF-κB nuclear translocation is blocked by antioxidants such as PDTC 71 72 . The potential for A20 to interfere at the oxidative step in NF-κB activation is currently being tested. Interestingly, several studies have addressed the protective potential of antioxidants in islets by overexpressing free radical scavenging enzymes 41 73 74 75 . The overexpression of MnSOD in an engineered β cell resulted in selective protection from IL-1β–induced cytotoxicity as well as a reduction in cytokine-induced NO generation 75 . In addition, transgenic expression of the antioxidant thioredoxin in β cells of NOD mice reduced the incidence of spontaneous diabetes and protected from streptozotocin-induced diabetes 76 . Interestingly, thioredoxin has been shown to inhibit NF-κB by interfering with a redox-sensitive step required for its activation 77 78 . Thus, in the model of Hotta et al. 76 , the protective effect of thioredoxin may involve inhibition of NF-κB activation, given the role of NF-kB activation in NO generation and islet destruction 36 54 79 . Together, these data illustrate a novel concept whereby protection of the target (in this case, β cells) would offer a potent therapeutic strategy to inhibit disease occurrence even in the presence of the effector mechanisms (cellular and soluble mediators). This approach might constitute an alternative to systemic modulation of the immune system as currently practiced using diverse immunosuppressants, such as costimulation blockade 80 81 82 83 . Along with this approach, other antiapoptotic genes such as bcl-2 have been proposed as gene therapy tools to protect islets from cytokine-mediated apoptosis. Expression of Bcl-2 in a murine β cell line did provide modest protection from cytokine-mediated apoptosis 84 85 . Interestingly, bcl genes have, like A20, antiinflammatory properties through blockade of transcription factors, such as NF-κB in endothelial cells 86 87 88 . We are currently testing whether they maintain this dual function in islets and could synergize with A20 to protect β cells. However, in contrast to A20, Bcl-2 is expressed constitutively in islets and is not induced upon cytokine activation (data not shown). We propose that constitutively expressed antiapoptotic proteins such as Bcl-2 may function to protect cells from baseline cellular stress, whereas induced cytoprotective proteins such as A20 protect cells from greater stress caused by inflammatory reactions 47 . We suggest that A20 could be a more relevant gene therapy candidate for protection of β cells against the additional stress encountered in the setting of transplantation and autoimmunity. Future experiments will determine the efficacy of A20 in both islet transplant and autoimmune diabetes models. | Study | biomedical | en | 0.999996 |
10523612 | House dust mite–specific, Japanese cedar pollen Cryptomeria japonica 1–specific, and purified protein derivatives of tuberculosis (PPD)-specific T cell clones were established by a standard procedure using limiting dilution technique 26 . All of the clones expressed TCR-α/β, CD3, CD4, and CD45RO but were negative for CD8. To induce cytokine production, the clones were stimulated with the relevant Ag plus irradiated autologous PBMCs and, in some experiments, irradiated autologous purified monocytes as APCs for 18–24 h. IL-2 (BioSource International), IL-4 (R & D Systems, Inc.), and IFN-γ (BioSource International) levels of culture supernatants were measured using ELISA kits. We assigned Th0, Th1, and Th2 as follows 26 : Th1 clones that produce IFN-γ and undetectable IL-4 (<10 pg/ml); Th2 clones that produce IL-4 and undetectable IFN-γ (<10 pg/ml); and Th0 clones that produce both IL-4 and IFN-γ. PBMCs were separated into sheep red blood cell (SRBC)-rosetted cells and unrosetted cells. CD3 + T cells were purified from SRBC-rosetted cells by magnetic bead depletion (MBD) of CD11a, CD14, CD19, and CD56 cells 27 . CD4 + CD3 + and CD8 + CD3 + T cell subsets were similarly purified by the MBD technique. The resulting cell populations were always >97% pure for cells of the relevant phenotype. Human Txk cDNA in λ phage was provided by Dr. G.W. Litman (University of South Florida, St. Petersburg, FL) 11 . Full length Txk cDNA was amplified by PCR using a sense primer (CGGAATTCATGATCCTTTCCTCCTATAACA) and an antisense primer (TTCTCTAGATCACCAGGTTTCCGCAATCTC), and the product was ligated into a mammalian expression vector, pME18S (SR-α promoter; provided by Dr. K. Maruyama, Tokyo Medical and Dental University, Tokyo, Japan) 28 . The vector pME18S-Txk carries the full length wild-type human Txk cDNA. The Txk mutant was created using the QuickChange site-directed mutagenesis kit (Stratagene Inc.). In brief, pME18S-Txk was used as a template. The primers containing the desired mutation were employed for PCR amplification using PfuTurbo DNA polymerase (Stratagene Inc.). The amplification cycle consisted of 1 cycle of denaturation (95°C) for 1 min, followed by 18 cycles of denaturation (95°C) for 30 s, annealing for 1 min (55°C), and polymerization for 10 min (68°C). After PCR cycling, the PCR product was treated with DpnI, which is specific for methylated and hemimethylated DNA, and the synthesized nonmethylated DNA containing the desired mutation was recovered. The resultant mutant vector was used for transformation of Escherichia coli DH5α. A part of the hypothetical nuclear localization sequence of Txk, KRKP, was deleted from the wild-type Txk, and the rest of Txk cDNA was kept intact 16 17 29 . The primers used for constructing the deletion mutant were as follows: KRKP-deletion, 5′-CGGGCCGTGTGCAGCCGTCACTGCCTCCCCTCCCACCCTC-3′ and 5′-GAGGGTGGGAGGGGAGGCAGTGACGGCTGCACACGGCCCG-3′. Fidelity of all the constructs was confirmed by DNA sequencing. Purified plasmids were transfected into Jurkat and Raji cells by electroporation as described 28 . After a 48-h incubation, cells were collected, counted, and stimulated with PHA (1 μg/ml) plus PMA (10 ng/ml) or PMA (10 ng/ml) plus ionomycin (1 μg/ml) for 24 h to induce lymphokine production. 5 μg of plasmid (p)IFN-γ (-538)-luciferase (provided by Dr. C.B. Wilson, University of Washington, Seattle, WA), 5 μg pRSV (Rous sarcoma virus)–chloramphenicol acetyl transferase (CAT), and 10 μg of pME18S-Txk (Txk transfection) or pME18S (empty vector; mock transfection) was cotransfected into Jurkat cells. pIL-2(-568)-luciferase (provided by Dr. C.B. Wilson) with pRSV-CAT and pIL-4(-265)-CAT (provided by Dr. S.N. Georas, Johns Hopkins University, Baltimore, MD) with pGL-3 control vector (SV-40 promoter; Promega Corp.) were similarly transfected. 48 h after transfection, the Jurkat cells were stimulated with PHA plus PMA for 8 h. Thereafter, luciferase assay and CAT-ELISA (Roche Diagnostics) of the cells were carried out 30 . Txk expression of transfected cells and normal lymphocytes was studied by immunoblotting 28 , immunocytochemical staining 31 , and immunofluorescence analysis using goat anti–Txk Ab (Santa Cruz Biotechnology). Fluorescence-conjugated anti–IFN-γ mAb (Immunotech) was used for intracytoplasmic IFN-γ staining. Immunofluorescence staining of intracytoplasmic proteins was carried out by a modification of the method of Sander et al. 32 . In brief, the cells were fixed by using 4% paraformaldehyde and permeabilized by 0.1% saponin (Sigma Chemical Co.) in PBS with 0.01 M Hepes buffer solution. Thereafter, intracytoplasmic antigens were stained with purified first Abs, biotin-conjugated second Abs, and streptavidin–fluorochrome. Thereafter, the cells were analyzed by flow cytometry. Appropriate control Abs were included to define the background immunofluorescence of the cells in this study. T cells or T cell clones were recovered and cytospin preparations of them were made. The samples were fixed with cold acetone for 15 min and were blocked with 2% skim milk for 30 min. The samples were incubated with first Abs overnight at 4°C. All subsequent procedures were performed using an LSAB kit (DAKO JAPAN). IFN-γ mRNA expression of Jurkat cells was estimated by reverse transcription (RT)-PCR using limiting dilutions of cDNA to accurately estimate the relative amounts of mRNA expression in different samples as previously reported 31 . Txk-transfected and mock-transfected Jurkat cells were cultured for 48 h and then stimulated with PHA plus PMA for 8 h. Total RNA was extracted from these cells, and was cDNA synthesized. The sequences of IFN-γ, IL-4, and β-actin primers and PCR conditions were reported previously 33 . For amplification of Txk cDNA, the following primers were used: Txk sense, TTGCTGTTCAGTGCAGAA; Txk antisense, GCA- CCTTCTTTAGACTCT; 475-bp product. Sense (GGGCTACCATGAGGTTTC) and antisense (GAAACCTCATGGTAGCCC) oligodeoxynucleotides (ODNs) specific for Txk were synthesized as a sulfonylated form. Peripheral blood T cells and Ag-specific cloned T cells (10 6 cells/ml) were incubated in the presence of various concentrations of ODNs for several hours. Thereafter, peripheral blood T cells were stimulated with PHA (1 μg/ml) and Ag-specific cloned T cells with irradiated autologous PBMCs plus the optimal concentration of Ag 26 . We first studied Txk expression of various cell types. Jurkat and MOLT-4 cells (T cell lines) but not Raji (a B cell line)- nor EBV-transformed B cells expressed Txk. Both peripheral blood CD4 + and CD8 + T cells expressed Txk; peripheral blood B cells and monocytes, however, never expressed it. We next asked whether Txk expression is restricted to a certain T cell subpopulation, such as Th1 cells. Various Ag-specific T cell clones that had been cultured for 7 d after the last stimulation with irradiated autologous PBMCs plus the relevant Ags were recovered. RT-PCR analysis revealed that all 20 Th1 cell clones and all 20 Th0 clones tested expressed Txk mRNA, whereas none of the 20 Th2 clones expressed it . Immunocytochemical staining confirmed that all Th1 and Th0 clones with various Ag specificities from several donors expressed Txk; all of these clones have IFN-γ producing potential . However, Th2 clones, regardless of Ag specificity or the donor, did not express Txk at all . Thus, there is an intimate association between Txk expression and Th1/Th0 clones with IFN-γ producing potential. We next studied the effects of Txk overexpression on cytokine production by T cells. To this end, Jurkat cells were transfected with pME18S-Txk. We used Jurkat cells in these experiments because they produce relatively low levels of Th1 cytokines upon mitogen activation. Overexpression of Txk by the transfection was confirmed by immunoblotting with anti-Txk Ab . In parallel experiments, Jurkat cells were stimulated with PHA plus PMA. When we used ELISA to detect cytokine secretion, Txk transfection resulted in several-fold more IFN-γ production as compared with the mock transfection . We also confirmed that Txk transfection of Jurkat cells enhances IFN-γ production by using an IFN-γ–specific ELISpot assay (data not shown). Similarly, intracytoplasmic IFN-γ staining of the Jurkat cells was carried out. As shown in Fig. 2 c, Txk transfection of Jurkat cells led to a several-fold increase of intracytoplasmic IFN-γ–positive cells; in mock-transfected Jurkat cells stimulated with PHA plus PMA, 13% were intracytoplasmic IFN-γ–positive cells; in Txk-transfected Jurkat cells stimulated with PHA plus PMA, 36% were intracytoplasmic IFN-γ–positive cells . Txk transfection did not affect IL-2 production of the Jurkat cells . Txk-transfected and mock-transfected Jurkat cells did not produce detectable levels of IL-4 upon stimulation . Txk transfection of Raji cells did not induce IFN-γ, IL-2, or IL-4 production, even upon stimulation with PMA plus ionomycin . These results suggest that Txk has a key role in IFN-γ production by T cells. We next studied the IFN-γ mRNA expression of the Jurkat cells. Txk transfection led to enhanced IFN-γ mRNA expression by the Jurkat cells as compared with the mock transfection . We also examined whether Txk transfection positively affects IFN-γ gene transcription by enhancing IFN-γ promoter/enhancer activity. To this end, we cotransfected pIFN-γ(-538)-luciferase and pME18S-Txk into Jurkat cells. We found that Txk transfection induced several-fold more luciferase activity in the cells than the mock (pIFN-γ[-538]-luciferase and pME18S)-transfected cells . As control cytokine-promoter plasmids, we used pIL-2(-568)-luciferase and pIL-4(-265)-CAT. We found that Txk transfection did not affect activities of pIL-2 promoter-luciferase– and pIL-4 promoter-CAT–transfected Jurkat cells, regardless of the presence or absence of mitogenic stimulation. The results revealed that Txk acts specifically on IFN-γ promoter/enhancer (-538) and upregulates IFN-γ gene transcription. Because Txk has a hypothetical nuclear localization signal sequence 16 17 29 , we examined nuclear translocation of the Txk protein in response to activation signals. Jurkat cells were stimulated with either PHA or IL-12, and subsequent localization of Txk was assessed by immunocytochemical staining . Unstimulated Jurkat cells showed cytoplasmic localization of Txk. Txk protein accumulated in the nuclei of Jurkat cells after treatment for 1 h with PHA. The nuclear accumulation of Txk was specific for PHA, because Txk protein remained in the cytoplasm of Jurkat cells treated with IL-12. The results suggest that Txk itself translocates into nuclei and enhances IFN-γ gene transcription in T cells. To study the role of nuclear translocation of the Txk protein upon activation, we constructed a pME18S-mutant Txk vector expressing Txk protein that lacked a nuclear localization signal sequence (KRKP-deleted) 16 17 29 . Jurkat cells were transfected with either wild-type or mutant Txk expression vector and cultured for 48 h. In the mutant Txk–transfected Jurkat cells, a vast majority of the (mutant) Txk protein stayed in cytoplasm and did not translocate into nuclei, even after stimulation with PHA plus PMA. However, very small amounts of endogenous Txk in the Jurkat cells translocated into nuclei upon activation. In contrast, in the wild-type Txk–transfected Jurkat cells, Txk protein translocated into nuclei in response to the stimulation . We measured IFN-γ production of the transfected Jurkat cells in parallel experiments. We found that wild-type Txk did enhance IFN-γ production, whereas nuclear localization signal sequence (KRKP)-deleted Txk did not affect IFN-γ production by the transfected Jurkat cells. These results indicate that nuclear localization of Txk is obligatory for its effect on cytokine expression . To confirm the involvement of Txk in IFN-γ production by human T cells, we tested inhibition of IFN-γ production by Txk antisense ODN. Peripheral blood T lymphocytes were cultured in the presence of sense or antisense ODN corresponding to the original translation start site of Txk 16 for several hours and were then stimulated with PHA. Antisense ODN, but not sense ODN, specifically inhibited cytoplasmic expression of Txk ; IFN-γ production of T cells was specifically inhibited by the antisense ODN . IL-2 and IL-4 production were not modulated by either the sense or antisense ODNs . To further confirm that Txk antisense ODN inhibits IFN-γ production, Ag-specific T cell clones were cultured with ODNs and then stimulated with Ag plus irradiated autologous PBMCs. Again, IFN-γ production by the Ag-specific Th1 and Th0 clones , but not IL-4 production by the Ag-specific Th0 and Th2 clones (data not shown) was inhibited by the Txk antisense ODN. We also studied whether Txk expression of human T cells is under the influence of Th1 cytokines. Normal peripheral blood CD4 + T cells were cultured with various concentrations of Th1 and Th2 cytokines. Intracytoplasmic Txk protein in T cells was subsequently assessed by flow cytometric analysis with anti-Txk Ab . IL-2 treatment of the CD4 + T cells did not affect Txk expression levels. IL-4 markedly reduced Txk expression of the CD4 + T cells . In contrast, IL-12 treatment for 4 h was sufficient to enhance Txk expression of the CD4 + T cells . This further supports an intimate association between Th1 cells and Txk expression. IL-12 may be involved in the polarization toward Th1 cells of T cells via Txk but is not affected by the outcome of IFN-γ production via Txk. It has been shown that Txk protein includes two isoforms that arise by alternative initiation of translation from the same cDNA 16 . We confirmed that Txk protein has two isoforms in COS cells when overexpressed by transfection (data not shown). However, Jurkat cells and normal PBLs almost exclusively express a longer isoform of Txk . In normal PBLs, antisense ODN, which primes with the original translation start site (the longer isoform), almost completely abolished Txk staining , suggesting that a longer isoform of Txk mainly mediates regulation of IFN-γ production in T cells. In summary, Txk expression is restricted to Th1/Th0 cells with IFN-γ producing potential and is significantly involved in IFN-γ gene transcription and subsequent IFN-γ protein production in human T cells. We believe that this is the first description of Txk involvement in IFN-γ production by human Th1 cells. More recently, we have found that Txk is phosphorylated and translocates into nuclei upon activation, and Txk or a protein complex including Txk binds to the IFN-γ promoter sequence (Nagafuchi, H., N. Suzuki, and T. Sakane, unpublished observation). Thus, we are currently investigating whether Txk itself or a protein complex including Txk acts as a Th1 cell–specific transcription factor for the IFN-γ gene. | Study | biomedical | en | 0.999996 |
10523613 | Reagents and sources were as follows: GM-CSF and leucine zipper (LZ)-CD40L (100 ng/ml; Immunex Corp.); IFN-α and -γ (100 ng/ml; Genzyme Corp.); LPS (5 ng/ml; Difco Labs., Inc.); MOPC-21, nonspecific IgG1 isotype control; M181, IgG1 anti-TRAIL (Immunex Corp.); 7G3, IgG2a anti–IL-3Rα–biotin; G155-178, IgG2a–biotin isotype control (PharMingen); 3.9, IgG1 anti-CD11c–PE; TUK4, IgG2a anti-CD14–FITC; 4D3, IgG2b anti-CD33–FITC; TU39, IgG2b anti–HLA-DR–FITC; IgG1–PE isotype control; IgG2a–FITC isotype control; IgG2b–FITC isotype control; and IgG2b–biotin isotype control (Caltag Labs., Inc.). HB-15a, IgG2b anti-CD83 (a gift of Dr. Thomas F. Tedder, Duke University Medical Center, Durham, NC). The soluble fusion proteins TRAILR2–Fc, Fas–Fc, and TNFR–Fc were produced at Immunex Corp. The LZ-huTRAIL expression plasmid and the production and purification of LZ-huTRAIL (TRAIL) have been previously described 26 . 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 prostate carcinoma cell line PC-3 was obtained from Dr. Michael Cohen (University of Iowa, Iowa City, IA). The human melanoma cell lines WM 793 and 164 were obtained from Dr. M. Herlyn (Wistar Institute, Philadelphia, PA). The Jurkat cell line was purchased from American Type Culture Collection. All tumor cell lines were cultured as directed. Normal lung fibroblasts, lung microvascular endothelial cells, and skeletal muscle cells were purchased from Clonetics Corp. and cultured as directed. Peripheral blood DCs were enriched using countercurrent elutriation. Cells from leukopheresis packs obtained from healthy volunteers were loaded onto a JE-5 elutriator (Beckman Instruments, Inc.), and 50-ml fractions were collected while increasing the flow rate from 65 to 85 ml/min at 2,000 rpm. Fractions containing the highest blood DC percentages were further enriched using magnetic bead selection. The DC-positive fractions were pooled and incubated with anti-CD3 (OKT3), anti-CD14 (MY23), anti-CD16 (3G8), anti-CD19 (B43), and anti-CD56 (B159) mAbs for 10 min at 20°C. Cells binding these mAbs were then removed using Dynal goat anti–mouse Ig–coated magnetic beads. Remaining cells were recovered and incubated with anti-CD7 (T3-3AI), anti-CD8 (OKT8), anti-CD11b (OKM1), anti-CD34 (MY10), and antiglycophorin A (10F7MN). Cells binding these mAbs were removed using the goat anti–mouse Ig–coated magnetic beads. To separate CD11c + DCs from IL-3Rα + DCs, the remaining cells were stained with a PE-labeled anti-CD11c and biotin-labeled anti–IL-3Rα, followed by APC-labeled streptavidin, and sorted on a FACStar PLUS™ (Becton Dickinson) into CD11c + and IL-3Rα + populations. Peripheral blood Mφ were enriched in elutriated fractions generated from a flow rate >75 ml/min. The fractions were >93% CD14 + Mφ, as assessed by flow cytometric analysis. Complete medium used for DC and Mφ culturing and functional assays consisted of RPMI 1640 (GIBCO BRL) supplemented with penicillin–streptomycin–glutamine and 10% pooled human serum (Immunex Corp.). Cell analysis was performed on a FACScan™ (Becton Dickinson), with >5,000 cells analyzed per sample. For multicolor cell analysis, samples consisting of 20 μl cells were combined in a 96-well flat-bottom plate (Costar Corp.) with 20 μl human IgG (12 mg/ml; Sigma Chemical Co.) to block Fc binding of the mAbs and 20 μl each of the direct PE-labeled, FITC-labeled, and biotin-labeled mAbs (60 μg/ml). Cells were then incubated on a rotator at 4°C for 1 h. After three washes with 200 μl PBS containing 2 mg/ml BSA, 40 μl of APC-labeled streptavidin (1:100 dilution; Molecular Probes, Inc.) was added for an additional 1 h. Cells were analyzed immediately after staining or fixed in 1% paraformaldehyde until analysis. Cytospins were performed by centrifuging 2 × 10 5 sorted DCs at 500 rpm for 5 min onto slides. The DCs were stained with a Hemacolor Stain Set (EM Diagnostic Systems), and photomicrographs were recorded using a Nikon Diaphot microscope with a 40× objective (Carl Zeiss, Inc.). DCs were cultured for 12 h in medium alone, GM-CSF, LZ-CD40L, LPS, 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 cells/well) were incubated with varying numbers of DC effector cells for 8 h. As a positive control, soluble TRAIL was added to the target cells. Mφ used for comparison were cultured and stimulated under conditions identical to those described for DCs. In some cultures, TRAILR2–Fc, Fas–Fc, or TNFR–Fc (20 μg/ml) was added to the DCs 15 min before adding tumor cell targets. All cytotoxicity assays were performed in 96-well round-bottom plates, and the percent specific lysis was calculated as: 100 × (experimental cpm − spontaneous cpm)/(total cpm − spontaneous cpm). Spontaneous and total 51 Cr release values were determined in the presence of either medium alone or 1% NP-40, respectively. The presence of TRAILR2–Fc, Fas–Fc, or TNFR–Fc during the assay had no effect on the level of spontaneous release by the target cells. Apoptotic cell death of tumor cells was measured by flow cytometry using FITC-conjugated annexin V and propidium iodide (apoptosis detection kit; R & D Systems, Inc.) as per the manufacturer's protocol. Light scatter characteristics were used to distinguish the tumor cells from the DCs. Total RNA was isolated from DCs 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 Corp.) 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′); and human TRAIL (forward: 5′-CAACTCCGTCAGCTCGTTAGAAAG-3′, reverse: 5′-ttagaccaacaactatttctagcact-3′), giving products of 219 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 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. The enriched “bulk” DCs comprised two distinct subsets distinguishable by the surface expression of CD11c and IL-3Rα . The ratio of these two cell populations varied by donor, with some donors demonstrating as high as 70% CD11c + DCs and 30% IL-3Rα + pre-DCs, whereas others demonstrated the reverse percentages. Neither CD11c + DCs nor IL-3Rα + pre-DCs expressed CD14, but they did differentially express CD33 and HLA-DR. After 12 h of culture in complete medium, the levels of CD11c and CD33 remained relatively constant, whereas CD83 expression was detected on the majority (>90%) of CD11c + DCs . In contrast, the IL-3Rα + pre-DCs expressed lower levels of CD33 and HLA-DR and failed to express CD83 after 12-h incubation under identical conditions . Distinct morphological differences were also observed between the two DC subsets. The CD11c + DCs exhibited a characteristic multilobulated nucleus, in contrast to the IL-3Rα + pre-DCs, which displayed an immature appearance consisting of an oval nucleus . Highly pure CD11c + DCs and IL-3Rα + pre-DCs were rapidly obtained using this method for functional analysis of TRAIL. Previous reports have demonstrated that a variety of lymphoid and myeloid cells (T cells, NK cells, Mφ) can express TRAIL and kill TRAIL-sensitive target cells under certain circumstances 27 34 35 . To determine if DCs also exhibit tumoricidal activity, unsorted “bulk” DCs were cultured for 12 h with IFN-γ, IFN-α, GM-CSF, CD40L, or LPS and cultured for an additional 8 h in the presence of the TRAIL-sensitive human tumor cell lines OVCAR3, an ovarian carcinoma cell line, and WM 793, a melanoma cell line, at a 50:1 DC/target ratio. Whereas unstimulated, GM-CSF-, CD40L-, and LPS-stimulated DCs demonstrated little or no tumoricidal activity toward the tumor cell targets, DCs stimulated with IFN-γ or -α were potent killers of each of these tumor cells . The cytotoxic activity of unstimulated, IFN-γ-, and IFN-α-stimulated Mφ is presented for comparison. This cytotoxic activity was seen with IFN-stimulated DCs from multiple donors and with other TRAIL-sensitive tumor cells but not with the TRAIL-resistant melanoma cell line WM 164 or several normal primary cell types ( Table ). No cytotoxic effect was observed when normal human lung fibroblasts, microvascular endothelial cells, or skeletal muscle cells were used as targets. Once it was determined that bulk DCs can kill TRAIL-sensitive tumor cell targets, we examined the tumoricidal activity of the CD11c + DC and IL-3Rα + pre-DC subsets. Sorting the bulk DCs into these subsets revealed that nearly all of the cytotoxic activity of the IFN-γ–stimulated DCs was attributable to the CD11c + DC subset . Furthermore, the observed tumoricidal activity was dependent upon the number of IFN-γ–stimulated DCs, as decreasing the E/T ratio decreased the amount of target cell death. The tumoricidal activity of the IFN-γ–stimulated CD11c + DCs was nearly equivalent to that of IFN-γ–stimulated Mφ. Similar results were observed with IFN-α–stimulated DCs and Mφ (data not shown). To confirm that the observed cytotoxicity of the CD11c + DCs was specific to TRAIL and not other apoptosis-inducing molecules (i.e., FasL and TNF), IFN-γ–stimulated CD11c + DCs were treated with recombinant soluble receptors for TRAIL (TRAILR2–Fc), Fas (Fas–Fc), or TNF (TNFR–Fc) before incubation with the tumor cell targets. Pretreating the IFN-γ–stimulated CD11c + DCs with TRAILR2–Fc reduced target cell death to control (unstimulated DC effector cells) levels, whereas Fas–Fc or TNFR–Fc did not alter the cytolytic ability of the IFN-γ–stimulated DCs . These results demonstrate that the TRAIL expressed on the CD11c + DCs induces the killing of TRAIL-sensitive targets. The results presented in Fig. 3 Fig. 4 Fig. 5 clearly demonstrate that IFN-stimulated CD11c + DCs kill tumor cells via a TRAIL-dependent mechanism. However, these experiments only measure the release of 51 Cr-labeled intracellular proteins into the culture supernatant, which is an event that can occur with either necrotic or apoptotic cell death. Previous reports have shown that TRAIL induces apoptotic death, as measured by DNA fragmentation, caspase activation, and annexin V binding 23 24 28 . Thus, to demonstrate that the TRAIL-expressing CD11c + DCs were inducing the apoptotic cell death of the target tumor cells, the binding of FITC-conjugated annexin V to the tumor cells was measured 36 37 . Light scatter characteristics were used to distinguish the tumor cells from the DCs, such that only the tumor cells were counted in the analysis. After 8-h incubation, only those tumor cells incubated with soluble TRAIL or IFN-stimulated DCs (E/T ratio, 4:1) were positive for annexin V binding . This apoptosis-inducing, tumoricidal activity of the IFN-stimulated DCs was seen with DCs from multiple donors and with other tumor cell targets (data not shown). These results demonstrate that TRAIL-expressing human DCs can kill TRAIL-sensitive tumor cells by inducing apoptosis. The results obtained thus far functionally describe the tumoricidal activity of IFN-stimulated CD11c + DCs to be via a TRAIL-dependent mechanism. However, we wanted to correlate this functional activity with TRAIL protein expressed on the surfaces of these cells. Thus, DCs were cultured in medium or stimulated with GM-CSF or IFN-γ for 12 h and then analyzed for TRAIL expression using flow cytometry. Whereas Mφ express significant levels of TRAIL on their surfaces after stimulation with IFN-γ 27 , lower levels of TRAIL expression were detected on CD11c + DCs after IFN-γ stimulation . TRAIL was undetectable on unstimulated or GM-CSF–stimulated CD11c + DCs , as well as on cells stimulated with CD40L or LPS (data not shown). In contrast, TRAIL was not detected on the unstimulated or cytokine-stimulated IL-3Rα + pre-DCs. Analysis of the IFN-γ–stimulated CD11c + DCs by reverse transcriptase (RT)-PCR revealed that TRAIL mRNA levels increased during the culture period as compared with unstimulated or GM-CSF–stimulated CD11c + DCs . Conversely, whereas TRAIL mRNA was detected in unstimulated IL-3Rα + pre-DCs, the levels were unaltered after GM-CSF or IFN-γ stimulation . The data presented here demonstrate that human blood DCs express the apoptosis-inducing molecule TRAIL after stimulation with either IFN-γ or IFN-α and acquire the ability to kill TRAIL-sensitive target cells. The TRAIL-specific activity was restricted to CD11c + DCs and correlated with the increased levels of TRAIL mRNA and protein after IFN-γ stimulation. The low number (<8 × 10 6 ) of blood CD11c + DCs obtained after enrichment from each donor restricted our ability to examine TRAIL expression and cytotoxicity after stimulation with the various cytokines. The 12-h stimulation time used in these studies was based on previous studies where TRAIL was maximally expressed on monocytes 27 . Although the TRAIL surface expression was lower in comparison to that on IFN-γ–stimulated human Mφ, the IFN-stimulated CD11c + DCs were able to mediate apoptosis with comparable efficiency, suggesting that even low levels of membrane-bound TRAIL result in potent tumoricidal activity. Moreover, the cytotoxic activity of the IFN-γ–stimulated CD11c + DCs was completely inhibited by soluble TRAILR2–Fc and not by Fas–Fc or TNFR–Fc. These blocking studies provide additional evidence that the cellular apoptosis elicited by the IFN-stimulated CD11c + DCs is a TRAIL-specific phenomenon. In contrast to the CD11c + DCs, IFN-stimulated IL-3Rα + pre-DCs did not demonstrate significant cytotoxic activity against the same TRAIL-sensitive targets. We elected to positively select for IL-3Rα + pre-DCs by sorting with an anti–IL-3R mAb, as this appears not to alter the functional capacity of these cells 10 . We cannot rule out the possibility, however, that IL-3R triggering by the anti–IL-3R mAb did, in part, enhance cell viability. We routinely observed >90% cell viability of the IL-3Rα + pre-DCs for prolonged periods (up to 20 h) when cultured in the presence of human serum. This is significantly longer than previous reports, where IL-3Rα + pre-DCs cultured in the presence of FBS underwent rapid cell death 9 . Thus, the lack of IL-3Rα + pre-DC cytotoxicity in this study was not due to poor viability. It is interesting that these two blood DCs respond differently with regard to induced TRAIL expression and TRAIL-mediated death. Previous reports have demonstrated that these two DC subsets differ with regard to phagocytic capacity, T cell stimulation capacity, and cell surface phenotype before and after stimulation ( 9 10 ; Fanger, N.A., unpublished observations). The differential expression of TRAIL after IFN-γ stimulation suggests further that these two DC subsets have different roles in directing antitumor responses via TRAIL. It is possible, however, that the IL-3Rα + pre-DCs are able to express TRAIL upon further maturation/differentiation or with stimuli not examined in this study. It has been shown that when IL-3Rα + pre-DCs are cultured with IL-3 ± GM-CSF and CD40L, a phenotypically and functionally different cell results by selective proliferation and death 9 . Ongoing studies will determine whether these more mature/differentiated IL-3Rα + pre-DCs can express TRAIL after IFN activation or other stimuli. The melanoma cell line WM 164 and normal primary cells were resistant to both TRAIL-mediated apoptosis and TRAIL-expressing DCs, even though they express one and both of the apoptosis-inducing TRAIL receptors (TRAILR1 and -R2), respectively ( 29 ; Griffith, T.S., unpublished observation). The mechanism(s) that regulate sensitivity and resistance to TRAIL-induced apoptosis remain unclear. It was initially hypothesized that the expression of the non–death-inducing, or “decoy,” TRAIL receptors (TRAILR3 and -R4) were responsible for resistance to TRAIL 38 39 40 . However, we have examined the expression of the four TRAIL receptors at both the mRNA and protein level in a variety of human tumor cell lines and found there to be no correlation between decoy receptor expression and TRAIL sensitivity/resistance 24 29 . Likewise, additional studies have failed to clearly show a link between decoy receptor expression and resistance 41 42 . Thus, it is unlikely that the decoy receptor hypothesis is the sole explanation for resistance to TRAIL-induced apoptosis. One component of the cell death machinery that appears to play a role in determining sensitivity and resistance to TRAIL is FLICE (Fas-associated death domain–like IL-1β–converting enzyme)-inhibitory protein (FLIP). It is believed that FLIP prevents the binding of caspase-8 to the death domain of cross-linked death receptor, inhibiting any downstream apoptotic signaling events. Intracellular levels of FLIP are high in the TRAIL-resistant melanoma WM 164 and in the normal cells used in this study ( 24 ; Griffith, T.S., unpublished observations). High FLIP levels have also been shown to correlate with resistance to Fas-mediated apoptosis in naive peripheral T cells 43 44 . Although FLIP may have a protective function in these cells, it is likely to be one of several intracellular proteins that cooperate with other proteins (both intracellular and at the cell surface) to regulate sensitivity to TRAIL-induced death. The tumoricidal activity reported here suggests that CD11c + DCs may be one of several cells responsible for the active killing and removal of spontaneously arising tumors in the body. To date, NK cells, Mφ, T cells, and now DCs have been shown to express TRAIL under certain conditions, providing tumoricidal capability to a variety of effector cells that patrol the body 27 34 35 . Whereas all these cell types may employ TRAIL to induce cell death outright, DCs may use TRAIL to generate apoptotic bodies for subsequent uptake, processing, and presentation of target cell antigens to CD8 + T cells, resulting in a stimulatory (i.e., CTL) or tolerogenic response. Indeed, several reports have demonstrated that DCs engulf apoptotic bodies and present antigen derived from these cell fragments in an MHC class I–restricted fashion, resulting in CTL activity 13 14 . T cell tolerance is important in preventing the induction of autoimmunity. It can occur in the thymus, resulting in the deletion of self-reactive thymocytes before they are released into the periphery 45 . Peripheral tolerance can also occur when specialized, tissue-specific antigens are encountered 17 46 47 . In this situation, a DC presenting self-antigen may also be expressing TRAIL, or some other apoptosis-inducing molecule, which would kill the antigen-specific T cell once it came into contact with the DC. Recent studies have demonstrated that DCs can kill CD4 + T cells through the expression of FasL 48 49 . Thus, the expression of TRAIL and/or FasL on DCs may prove to be a mechanism for deleting T cells in vivo. The data presented here also suggest that DC TRAIL expression may contribute to other physiologic and pathologic situations, such as HIV infection. DCs are one of the first cells to encounter antigen at areas of inflammation in mucous membranes 50 , which are the major sites of initiation of HIV infection. After the interaction between DCs and the virus at these sites, the DCs migrate to the draining lymph nodes, where they stimulate CD4 + T cells. In the process, the T cells become infected, leading to the replication and spread of the virus 12 51 . The viral infection may also induce the production of various cytokines, such as IFN-γ and -α, activating an antiviral immune response. It could be hypothesized from our results that IFN production would stimulate the DCs to express TRAIL, which could potentially kill any activated T cells in the area. | Study | biomedical | en | 0.999997 |
10523614 | The generation of C3 −/− , C4 −/− , CR2 −/− , and CD19 −/− mice has been described previously 1 18 20 25 . C57BL/6 and 129Sv mice were purchased from the Institute for Laboratory Animals (Veterinary Hospital, Zurich, Switzerland). C57BL/6 and (C57BL/6 × 129Sv)F1 were used as controls. Experiments were done in a conventional mouse house facility, and mice were used at 6–12 wk of age. VSV Indiana (VSV-IND; Mudd-Summers isolate) and VSV New Jersey (VSV-NJ; Pringle isolate) were originally received from Dr. D. Kolakovsky (University of Geneva, Switzerland) and were grown on BHK21 cells. Lymphocytic choriomeningitis virus (LCMV)-WE was originally obtained from Dr. F. Lehmann Grube (Heinrich Pette Institute, Hamburg, Germany) and was propagated on L929 fibroblast cells. Poliovirus stock solutions of serotype II were obtained from the Swiss Serum and Vaccine Institute (Bern, Switzerland). Inactivated poliovirus vaccine containing all three major serotypes (Salk) was purchased from BERNA, Switzerland. Recombinant baculoviruses expressing the glycoprotein of VSV (VSV G) and the nucleoprotein of LCMV (LCMV NP) were gifts from Dr. D.H.L. Bishop (NERC Institute of Virology, Oxford, UK). They were derived from nuclear polyhedrosis virus and were grown at 28°C in Spodoptera frugiperda cells in spinner cultures 32 . VSV titers in different organs were analyzed by a plaque-forming assay. 1:10 serial dilutions of organ homogenates were incubated on a vero cell monolayer in 24-well plates for 1 h at 37°C in an atmosphere with 5% CO 2 . Overlay with methylcellulose, incubation, and staining of plaques was similarly done as described for the neutralization assay. Serum of immunized mice was prediluted 40-fold in MEM containing 2% FCS. Serial twofold dilutions were mixed with equal volumes of VSV (500 pfu/ml) and incubated for 90 min at 37°C in an atmosphere with 5% CO 2 . 100 μl of the serum–virus mixture was transferred onto vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. The monolayers were overlaid with 100 μl DMEM containing 1% methylcellulose and incubated for 24 h at 37°C. The overlay was flicked off, and the monolayer was fixed and stained with 0.5% crystal violet. The highest dilution of serum that reduced the number of plaques by 50% was taken as titer. To determine IgG titers, undiluted serum was pretreated with an equal volume of 0.1 mM β-ME in saline. Poliovirus neutralization assays were performed similarly, but samples were prediluted 1:20. We used an ELISA with the following steps: (a) coating with baculovirus-derived LCMV NP (1 μg/ml); (b) blocking with 2% BSA (Fluka AG) in PBS; (c) addition of 10-fold–prediluted sera, titrated 1:3 over 12 dilution steps; (d) detection with IgM- or IgG-specific horseradish peroxidase–labeled goat anti–mouse antibodies (0.5 μg/ml; Southern Biotechnology Associates, Inc.); and (e) addition of substrate ABTS (2.2′-azino-bis-[3-ethylbenzthiazoline-6-sulfonate]; Boehringer Mannheim) and H 2 O 2 (Fluka AG). Plates were coated overnight at 4°C; all other incubations were done for 60–90 min at room temperature (RT). Between incubations, plates were washed three times with PBS containing 0.05% Tween-20. OD was measured at 405 nm in an ELISA reader, and antibody titers were determined as the serum dilutions yielding an absorption of twice background levels. Antibody-forming cell (AFC) frequencies were determined as described 33 . In brief, 25 square-well polystyrene plates were coated with purified VSV-IND (≈10 11 pfu/ml). On the next day, plates were blocked with 2% BSA in PBS for 2 h. Titrated amounts of single-cell suspensions were added in 2% MEM and incubated for 5 h at 37°C. After washing with PBS–Tween, goat anti–mouse IgM or IgG antibody (2 μl/ml; EY Labs.) was added, and plates were incubated for 2 h at 37°C. After washing with PBS–Tween, alkaline phosphatase–labeled donkey anti–goat antibody (1 μg/ml; Jackson ImmunoResearch Labs, Inc.) was added, and plates were incubated overnight at RT. The next day, plates were washed, and the substrate solution (5-bromo-4-chloro-3-indolyl phosphate at 1 μg/ml in 0.6% agarose) was added to develop blue color spots. Mice were treated intraperitoneally on days 3 and 1 before infection with 1 mg of anti-CD4 mAb YTS191.1 34 . This treatment completely abrogates the switch from IgM to IgG and depletes CD4 + T helper cells to below detection level by FACS™ analysis (not shown). Freshly removed organs were immersed in HBSS and snap frozen in liquid nitrogen. 5-μm-thick tissue sections were cut in a cryostat, placed on siliconized glass slides, air dried, fixed with acetone for 10 min, and stored at −70°C. For staining of cell differentiation markers, rehydrated sections were incubated with rat mAbs against marginal zone macrophages (ERTR-9; reference 35 ) and against marginal zone metallophils (MOMA-1; Biomedicals). Primary rat antibodies were revealed by sequential incubation with goat antibodies to rat Igs (Caltag Labs.) and alkaline phosphatase–labeled donkey antibodies to goat Igs (Jackson ImmunoResearch Labs., Inc.). Dilutions of secondary antibodies were made in TBS containing 5% normal mouse serum. Incubations were done at RT for 30 min; TBS was used for all washing steps. Alkaline phosphatase was visualized using naphthol AS-BI (6-bromo-2-hydroxy-3-naphtholic acid-2-methoxy anilide) phosphate and new fuchsin as substrate. Endogenous alkaline phosphatase was blocked by levamisole. Color reactions were performed at RT for 15 min with reagents from Sigma Chemical Co. Sections were counterstained with hemalum, and coverslips were mounted with glycerol and gelatin. Staining for VSV antigen was done as described 36 . Primary neutralizing antibody responses against VSV or recombinant VSV G protein and ELISA binding antibodies against LCMV NP were assayed in C3 −/− and control mice. The early (day 2–6) IgM response to VSV was completely TI-1; thereafter, VSV induced a rapid and strong TD neutralizing IgG response starting around day 6–7 after infection, reaching a plateau level after 3 wk 31 . VSV G on the membranes of cells infected with a recombinant vaccinia virus expressing VSV G (Vacc VSV G) and baculovirus-expressing VSV G protein have been shown to be TI-2 antigens 27 . In contrast, IgM and IgG antibodies to LCMV NP (an internal viral antigen) are strictly dependent on Th cells. After infection with the two replicating viruses expressing the VSV G as TI antigen , a comparable antibody response was observed in wild-type and C3 −/− mice; the initial TI IgM response was, surprisingly, somewhat delayed in C3 −/− mice, whereas the TD switch to IgG was comparable in C3 −/− and control mice. In contrast, after immunization with nonreplicating viral antigens , a reduction in the TD IgG antibody responses by a factor of ∼8 against inactivated VSV and a factor of ∼60–100 against VSV G protein was observed. It has been shown previously that for immunization with nonreplicating antigens, higher amounts of antigen had to be used to induce an antibody response comparable to that after infection with 2 × 10 6 pfu live VSV 37 . In this study, we used 2 × 10 8 pfu equivalents of UV-inactivated VSV and 10 μg baculovirus-derived VSV G protein . This comparison was based on virus particles counted by electron microscopy and on one virus particle expressing ∼1,300 VSV G proteins in its envelope 29 . With a high initial antigen dose, the early IgM response could be restored in C3 −/− mice . Interestingly, the capacity of C3 −/− mice to mount TD IgM and IgG antibodies against LCMV NP after infection with 200 pfu LCMV i.v. was similar to that of controls . Immunization with UV-inactivated LCMV subcutaneously (300 μl of purified LCMV in CFA corresponding to ∼20 μg of protein) also revealed an ∼10-fold reduction of the IgG titer . The reduced IgG titers after immunization with nonreplicating viral antigens confirm earlier results with model TD antigens (bacteriophage ΦX174, sheep red blood cells) in C3 −/− and CR2 −/− mice 1 18 19 . In contrast, during infections with live virus, where efficient T cell help and/or antigen persistence is provided , switching to IgG is here shown to be largely complement independent. The influence of antigen dose and antigen persistence in vivo on the antibody response was analyzed in C3 −/− and control mice that were immunized repetitively every other day with 2 × 10 8 pfu UV-inactivated VSV . Repetitive injections of high doses of nonreplicating VSV particles induced comparable IgG titers in C3 −/− and control mice. This indicates, first, that B cells of C3 −/− mice are comparably functional in vivo and second, that the influence of complement on the specific immune response probably reflects the importance of antigen dose and persistence. The reduced IgG titers in C3 −/− mice may be a consequence of an impaired interaction of antigen-bound complement cleavage products with CD21/CD35. To test this hypothesis, the immune response of CR2 −/− mice to viral antigens was analyzed . The blocked switch to IgG in CR2 −/− mice after immunization with 2 × 10 8 pfu UV-inactivated VSV confirms the importance of the CD21–CD21L interaction in the enhancement of B cell stimulation and/or antigen trapping on FDCs in response to nonreplicating antigen. In contrast, the IgG response after infection with live viruses in C3 −/− and CR2 −/− (not shown) was within normal ranges. Recovery of mice from primary or secondary VSV infection depends mainly on the neutralizing antibody response and on IFN-α 31 38 39 . Most of the C3 −/− mice infected with 2 × 10 6 pfu died within 8–10 d after infection despite a quite normal antibody response. VSV infects neuronal tissue and causes paralysis and death after infection of the spinal cord and the brain. A titration of the infectious dose of VSV revealed an increased susceptibility to VSV by at least a factor of 100–1,000 ( Table ) in complement-deficient mice. Whereas six of nine C3 −/− mice infected intravenously with 10 5 pfu died, only one of six control animals died after infection with 10 7 pfu VSV. High VSV titers were found in the brains of paralyzed mice ( Table ), implying infection of the central nervous system by VSV as cause of disease and death. In contrast, no increased susceptibility to VSV was observed after infection of CR2 −/− mice (not shown). A number of factors may have contributed to this drastic increase in susceptibility to disease (as a comparison, the LD 50 of antibody-deficient μMT mice is ∼10 3 pfu VSV; reference 40 ) may be explained as follows: First, the observed delay in the neutralizing IgM antibody response may allow VSV to reach its target tissue, the brain, before a sufficiently high neutralizing antibody titer is mounted. Second, complement components may directly—or indirectly via antibodies—inactivate VSV and reduce peripheral infection. Third, opsonization with complement could lead to a more efficient recruiting of the virus to secondary lymphoid organs and therefore an enhanced clearance of virus from the circulation. These possibilities are analyzed in the following sections. The observation that the early (day 2–6) TI IgM responses against VSV and Vacc VSV G were reduced whereas the TD IgM response after infection with LCMV was normal was unexpected, as earlier studies with nonreplicating model antigens had suggested that mainly TD antibody responses were enhanced by complement-mediated stimulation of the B cell coreceptor complex (CR2–CD19–TAPA-1) 5 6 7 8 10 . Analysis of B cell responses in mice depleted of CD4 + T cells revealed that the IgM response was reduced in C3 −/− by a factor of 250 compared with control mice on day 6 after infection with 2 × 10 6 pfu VSV i.v. , i.e., in the absence of C3, the normally TI antibody response was almost completely blocked. Neutralizing IgG (distinguished from IgM by reduction with 0.1 M β-ME, an unequivocal means of destroying IgM; reference 41 ) on day 8 after immunization was at least two titer steps lower than total Ig, indicating that the antibodies measured until day 8 largely represented neutralizing IgM. The efficiency of the CD4 + T cell depletion protocol was verified by FACS™ analysis of the CD4 + T cell counts in the blood (not shown). In addition, no switch to IgG was observed in mice treated with anti-CD4 + antibodies, indicating that depletion of T cell help in vivo was efficient . To assess the pathway of complement activation and the mechanisms by which complement contributes to Th cell–independent B cell activation, we analyzed the early neutralizing IgM response to VSV in CD4 + T cell–depleted C4 −/− , CR2 −/− , and CD19 −/− mice and compared it to the antibody response in equally treated control mice . Similar to C3 −/− mice, the early neutralizing IgM antibody response to VSV was delayed in C4 −/− mice and depended largely on T cell help. This indicated that early during VSV infection, the complement cascade was probably activated via the classical pathway. In contrast, mice deficient in the CR1 and -2 or the coreceptor component CD19 mounted early neutralizing IgM titers that were independent of T cell help. Therefore, the effect of complement on the early antibody response could not be explained by the lack of stimulation via the B cell coreceptor complex. In an earlier study, complement inactivation using cobra venom factor (CVF) could not reveal an influence of complement on the IgM responses against VSV 42 . However, although inactivation using CVF may be efficient for serum complement components, it was shown to be rather inefficient for locally produced complement components in secondary lymphoid organs 43 . To analyze whether the impaired TI antibody response in C-deficient mice was unique to VSV or was a general phenomenon, we studied antibody responses of C3 −/− and control mice after immunization with Vacc VSV G or poliomyelitis virus . IgM responses to both of these TI-2 antigens were also largely dependent on T cell help in C3 −/− mice. Because the TI activation of B cells was not dependent on CR1 or -2, the effect may be mediated by CR3 (CD11b, CD18) or CR4 (CD11c, CD18) 44 . These receptors are mainly expressed on macrophages. Immunohistological analysis after immunization with VSV antigen showed a high concentration of VSV antigen accumulated in the splenic marginal zone in control (C57BL/6 × 129Sv)F1 mice . In contrast, this local concentration was virtually absent in C3 −/− mice . The staining of the marginal zone macrophages with MOMA-1 and ERTR-9 (specific for marginal zone macrophages; not shown) confirmed that C3 −/− mice possessed a normal marginal zone structure. Interestingly, VSV titers in the spleen 6 h after infection with 2 × 10 8 pfu VSV were comparable in C3 −/− and control mice ( Table ). This difference may be explained by the fact that immunohistochemically, only high concentrations of antigen can be stained. In contrast, under conditions where antigen is distributed more diffusely in the spleen, as in C3 −/− mice, no intensive immunohistological signal can be detected. Thus, complement mainly influenced the specific targeting of the antigen to marginal zone macrophages but less influenced the general uptake of virus in the spleen. The only slightly impaired antibody response in C3 −/− mice was in contrast to the massively increased mortality after infection with VSV. We therefore tested the protective capacity of a panel of seven monoclonal IgM and five monoclonal IgG VSV-specific neutralizing antibodies after transfer into C3 −/− or control mice 45 . The results obtained with the antibodies with the highest and the lowest in vitro neutralization titers are shown in Table . Viral titers in the spleens of C3 −/− and control mice 6 h after infection with 2 × 10 8 pfu VSV were similar, ∼6 log 10 , and comparable titers were also obtained in peripheral organs (liver and kidney; data not shown). Injection of neutralizing IgM or IgG antibodies 20–30 min before VSV infection reduced viral organ titers in control mice by 100–1,000-fold but only reduced titers in C3 −/− mice ∼10-fold ( Table ). The augmentation of the neutralizing capacity of mAbs by complement was confirmed with the administration of pooled, complement-inactivated polyclonal IgM of day 4 VSV immune mice 20–30 min before infection with 2 × 10 8 pfu VSV ( Table ). In contrast, the neutralization of noncytopathic LCMV with IgM or IgG mAbs seems to be largely independent of complement in vivo ( Table ). These results differ from earlier in vitro observations that suggested a direct role of complement in protection against LCMV 46 . In summary, the VSV-neutralizing antibodies are more efficient in vivo in the presence of an intact complement system. Complement has been shown to be involved in targeting antigen to CD21 and CD35 on FDCs in GCs, where the survival of GC B cells is dependent on the expression of CRs 23 24 47 . To assess antiviral B cell memory, C3 −/− , C4 −/− , CR2 −/− , or CD19 −/− mice were infected with 2 × 10 6 pfu VSV, and antibody titers were followed up to 150 d. C3 −/− , C4 −/− , and CR2 −/− mice maintained IgG antibody titers comparably to control mice . In contrast, CD19 −/− mice lost memory antibody titers within 90 d , confirming earlier results 33 . On day 120 after the initial infection, the number of AFCs was assessed in the bone marrow and spleen . C3 −/− and control mice had similar numbers of AFCs in the bone marrow and spleen, a finding that correlated with the observed antibody titers. The AFCs in C4 −/− and CR2 −/− mice in the bone marrow were reduced by 80–90% and in the spleen by ∼50% compared with the number of AFCs present in control mice. The AFCs in CD19 −/− mice were reduced by >99.9% in the bone marrow and the spleen, confirming earlier results analyzing B cell memory in CD19 −/− mice 33 . The reduced numbers of AFCs in C4 −/− and CR2 −/− mice by a factor of 5 or 2 in spleen or bone marrow, respectively, were still sufficient to maintain long-term antibody titers after immunization with VSV. However, in CD19 −/− mice, where AFCs are reduced by a factor of 100–1,000, neutralizing antibody titers could not be maintained. B cell memory was also assessed in C3 −/− mice 150 d after infection with Vacc VSV G, VSV G protein, and LCMV. Long-term antibody titers after these different immunization protocols were comparably maintained in C3 −/− and control mice (not shown). Complement may be involved in a viral infection in various ways 3 , and its importance for host protection is documented here by the increased susceptibility of C3 −/− mice to a model infection with the cytopathic virus VSV. VSV activates the complement cascade via the classical pathway, i.e., antibodies initially activate the C1 convertase or, as described for some retroviruses, the classical pathway is directly activated by virus-infected cells independent of antibodies 48 . Natural IgM antibodies to VSV are present in the serum of antigen-inexperienced mice and can bind to virus and activate the complement cascade 49 50 . Complement may then directly augment the efficiency of antibodies to neutralize VSV. Early in vitro studies had shown that the active cleavage product C3b bound to VSV and was incorporated into its surface and, thereby, may have prevented infection of target cells 50 . Also, a role of complement has been demonstrated in in vitro studies with various viruses including LCMV and HIV 4 51 52 , but in extension of earlier in vitro experiments 51 , in this study we could not detect a direct influence of complement on the severity of LCMV infection in vivo. A surprising effect of complement was found here on viruses that elicit an antibody response independently of T cell help. Several viruses have been shown to be TI antigens, e.g., influenza, polio, rabies virions, and others 53 . After infection with a cytopathic virus such as VSV, early defense mechanisms are crucial to prevent infection of neuronal tissue. Complement does not seem to directly influence early distribution of VSV, as C3 −/− and control mice have comparable titers of VSV in the spleen and in peripheral organs 6 h after infection. Nevertheless, complement-coated virus is targeted more efficiently to CR-expressing cells. This effect has been extensively analyzed and documented for CD21 and CD35 expressed on FDCs 23 47 , suggesting an influence on antigen persistence in GCs that may have an impact on long-term B cell memory. After infection with VSV, LCMV, and different recombinant antigens, we did not observe any impairment in B cell memory in mice deficient in complement components or CRs, although AFCs in C4 −/− and CR2 −/− mice were reduced by a factor of 2–5. The binding of antigen IgG complexes on FDC seems to be sufficient to maintain long-term B cell memory after a viral infection 47 . The observations that C3 −/− , C4 −/− , and CR2 −/− mice maintained B cell memory, whereas CD19 −/− mice had a drastic reduction in IgG antibody titers and AFCs 90–150 d after infection remains unexplained. So far, it was assumed that CD19 signaled solely after binding of C3d to CR2, as no specific ligand for CD19 is known 5 33 . However, the different phenotype of CD19 −/− and CR2 −/− mice is best explained by an intrinsic role of CD19 in BCR signaling. Alternatively, there might be a ligand for CD19 that is independent of the CR. In a different set of experiments, B cell memory to another infectious virus, human herpes simplex type 1, that replicates only to a very limited extent in mice, particularly when injected subcutaneously (a condition probably more comparable to immunization with UV-inactivated VSV), was found to be classical pathway and CD21 dependent 53a . Thus, in addition to antigen dose and replication capacity, the antigenic structure and the route of infection might be important determinants of whether and to what extent complement is required for humoral responses. Early on during a viral infection, a sufficient antigen concentration on CR3– and -4–expressing macrophages in the splenic marginal zone seems to be crucial to elicit an early, TI B cell response. Marginal zone macrophages have been shown to be important for the induction of TI-2 antibody responses to nonreplicating model antigens 54 . This mechanism causes a very efficient early IgM response necessary for survival of the infection. Any delay in the early neutralizing antibody response may allow the virus to reach neuronal tissue before an efficient neutralizing antibody response is mounted. Our results suggested that lowering the threshold of B cell activation by binding to the B cell coreceptor (CR2, CD19, TAPA-1) does not seem to play a major role after infection with live viral antigens. However, signaling via the B cell coreceptor enhanced the IgG response when nonreplicating antigens were used. This difference may be explained by nonspecific inflammatory reactions and cytokine secretion plus prolonged antigen persistence during infections with live virus that may compensate for the lack of costimulation via CR2/CD19 55 . In conclusion, during a viral infection, complement is an important link to adaptive immunity: it helps to recruit antigen to marginal zone macrophages and stimulate B cells of the marginal zone and thereby enhances specific TI antibody responses. Thus, TI IgM antibody responses are complement dependent. | Study | biomedical | en | 0.999996 |
10523615 | Recombinant GM-CSF was provided by Dr. D. Bron (Institut Bordet, Brussels, Belgium). IFN-γ was obtained from Genzyme. sCD40L trimeric protein was provided by Drs. C. Maliszewski and R. Armitage (Immunex Corp., Seattle, WA). Staphylococcus aureus Cowan I strain (SAC) was used at 0.01% (wt/vol) (Pansorbin; Calbiochem-Behring) and LPS at 10 μg/ml (Sigma Chemical Co.). Anti-CD47 mAbs B6H12 (mouse IgG1) and BRIC126 (mouse IgG2b) were purchased from Serotec. Isotype-matched negative control mAb (mouse IgG1) was prepared in our laboratory. Anti-CD18 mAb (mouse IgG1; American Type Culture Collection) was used at 10 μg/ml. Neutralizing anti–IL-10 mAb (clone 19 F1.1; American Type Culture Collection) and neutralizing anti–TGF-β (polyclonal chicken Ig) were used at 10 and 30 μg/ml, respectively. TSP was purchased from GIBCO BRL, and EDTA and EGTA were from Sigma Chemical Co. The 4N1K peptide (KRFYVVMWKK) was obtained from Genosys and corresponds to the COOH-terminal domain of TSP 16 ; 4NGG (KRFYGGMWKK) was a gift from W. Frazier (Washington University, St. Louis, MO). PBMCs were isolated by density gradient centrifugation of heparinized blood from healthy volunteers (total n = 30) using Lymphoprep (Nycomed). Enriched monocytes were prepared by cold aggregation as described 9 , followed by one cycle of rosetting with S-(2-aminoethyl) isothiouronium bromide (Aldrich)–treated SRBCs to deplete residual T and NK cells. Monocyte purity was shown to be >95% by flow cytometry (FACScan™; Becton Dickinson) using PE-conjugated anti-CD14 mAb (Ancell). Monocytes were cultured at 10 6 /ml in 96-well round-bottomed Falcon plates (Becton Dickinson). Cultures were performed in quadruplicate in complete serum-free HB101 medium (Irvine Scientific) containing Ca 2+ (600 μM) and Mg 2+ (490 μM) and supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, 100 IU penicillin, and 100 μg/ml streptomycin. IL-12p70 release was assessed by a two-site sandwich ELISA using clone 20C2 as the capture mAb and clone 4D6 as the second mAb as described 9 . Both mAbs were provided by Dr. M. Gately (Hoffmann-LaRoche, Nutley, NJ). The sensitivity of the assay was 6 pg/ml. TNF-α was measured using a sandwich ELISA as described previously 19 , and IL-10 was determined by a sandwich solid-phase RIA using anti–IL-10 mAb (clone 9D7) as the capture mAb and 125 I-labeled anti–IL-10 (clone 12G8) as the detecting probe. IL-1β, IL-6, GM-CSF, and TGF-β ELISA kits were purchased from R&D Systems. All of the measurements were performed in duplicate with a coefficient variation of <10%. The paired t test was used to determine statistical significance of the data. * P < 0.05; ** P < 0.01; *** P < 0.005. We examined the effect of soluble CD47 mAb on IL-12 release by purified monocytes costimulated by IFN-γ and T cell–dependent (sCD40L and GM-CSF) or –independent (SAC) signals. As depicted in Fig. 1 A, CD47 mAb significantly suppressed IL-12 secretion in response to either stimuli, whereas isotype-matched IgG1 mouse mAb had no effect. In agreement with Marth and Kelsall 3 , anti-CD18 mAb, used as a cell-binding irrelevant mAb, did not suppress IL-12 production (data not shown). We noted an inhibition of IL-12 release even when monocytes were cultured with SAC alone, indicating that CD47 mAb did not simply impair the enhancing effect of IFN-γ on IL-12 production. Our unpublished observations revealed a similar suppressive effect by CD47 mAb after LPS and IFN-γ costimulation. The blockade of IL-12 production in response to SAC and IFN-γ was rather selective, since the release of other monocyte products (TNF-α, IL-1, IL-6, and GM-CSF) remained largely unaffected . Note a threefold increase in IL-6 secretion, which did not reach statistical significance. Cell viability, as determined by trypan blue dye exclusion, was unchanged. Also, the inhibition of IL-12 was probably not mediated via an increase of autocrine antiinflammatory cytokines (IL-10 and TGF-β), known to efficiently downregulate bacteria-induced monokine release 1 . As shown in Fig. 1 C, IL-10 and TGF-β levels remained unaffected by CD47 mAb treatment. Moreover, neutralizing anti–IL-10 or anti–TGF-β antibodies added alone or simultaneously did not overcome CD47 mAb–mediated inhibition of IL-12 production by SAC- and IFN-γ–stimulated monocytes . We next evaluated the effect of TSP, the natural ligand of CD47 16 , on monokine release. As shown in Table , TSP significantly reduced IL-12 production by SAC- and IFN-γ–stimulated monocytes, whereas it slightly enhanced TNF-α release. Note that monocytes coexpressed two other TSP receptors (CD36 and αvβ3 ). Since the site of interaction between CD47 and TSP is limited to the cell-binding domain (CBD) of TSP, we examined the role of a synthetic peptide (4N1K) encoding this particular domain 16 . We found that 4N1K, but not control mutant peptide 4NNG, dose-dependently inhibited IL-12 release in response to SAC and IFN-γ without affecting TNF-α production . This specific inhibition of IL-12 release after CD47 engagement was reminiscent of recent studies reporting that FcγR cross-linking selectively downregulated IL-12 secretion in monocytes via an increase in extracellular calcium influx 2 . We analyzed whether this mechanism might be involved in the CD47 mAb–mediated IL-12 suppression. First, F(ab′) 2 fragments of CD47 mAb dose-dependently inhibited IL-12p70 release, demonstrating that the inhibitory effect was not Fc mediated . Furthermore, monovalent Fab fragments still suppressed IL-12 secretion , suggesting that CD47 mAb either prevented monocyte/monocyte interactions and the delivery of a positive signal, or directly delivered to the monocytes a negative signal for IL-12 production. Second, IL-12 downregulation was observed in monocyte cultures supplemented with a fivefold excess of the Ca 2+ and Mg 2+ chelating agents EDTA or EGTA, demonstrating that the suppressive effect was independent of extracellular Ca 2+ influx . Moreover, addition of EDTA or EGTA in the absence of mAb did not result in inhibition of TNF-α or IL-12 release. Since TSP signaling of focal adhesion in endothelial cells requires activation of phosphoinositide 3-kinase (PI 3-K ), we explored the involvement of this pathway in CD47 mAb–mediated suppression of IL-12 release by using two PI 3-K inhibitors, wortmannin and Ly294002. We found that both PI 3-K inhibitors restored IL-12 release in a dose-dependent manner with no effect on TNF-α secretion (data not shown). However, neither protein kinase C nor protein tyrosine kinase inhibitors (e.g., herbimycin) prevented CD47 mAb–mediated inhibition of IL-12 release (not shown). Taken collectively, our results suggest that engagement of CD47 on monocytes by mAb, its natural ligand TSP, and 4N1K peptide specifically suppressed IL-12 secretion through an Fc and Ca 2+ –independent but PI 3-K–dependent pathway. The critical role of IL-12–mediated protection against mycobacterial infection was recently confirmed in IL-12 receptor–deficient patients 21 . Nevertheless, uncontrolled production of IL-12 is pathogenic in some organ-specific autoimmune diseases 1 and endotoxic shock 22 , underscoring the requirement of potent negative regulatory feedback mechanisms. The present findings demonstrate that engagement of CD47 by its natural ligand, TSP, or by CD47 mAb, represents a novel pathway to selectively downregulate IL-12 production. Inhibition of IL-12 release by CD47 ligation occurred in activated monocytes through a PI 3-K–dependent pathway. Indeed, two specific inhibitors of PI 3-K overcame CD47-mediated IL-12 suppression with no effect on TNF-α production. Although these data are not a definite proof of the involvement of PI 3-K, they are supported by the observations that TSP-mediated functions such as cell spreading or focal adhesion disassembly required activation of PI 3-K 20 . Besides inhibitory cytokines, including IL-4, IL-10, and TGF-β, which suppress the production of a large number of proinflammatory products (e.g., TNF-α, IL-1, IL-6, and IL-12) by activated monocytes, several monocyte surface receptors reportedly downregulate IL-12 production in a somewhat selective manner. Histoplasma capsulatum and measles virus have been shown to decrease IL-12 release by signaling through CD11b and CD46, respectively 3 4 . IL-12 downmodulation that resulted from scavenger receptor and FcγR ligation is due to extracellular Ca 2+ influx 2 and increase in IL-10 production in the case of FcγRI 23 . In the present study, the CD47 mAb inhibition was Fc and Ca 2+ independent since intact, divalent, and monovalent fragments of CD47 mAb suppressed IL-12 regardless of the presence of divalent cation chelating agents. CD47 antigen, also named integrin-associated protein (IAP), regulates the function and the binding of Vn to αvβ3, its associated integrin 13 . However, Vn does not bind CD47, and the natural ligand of CD47 is reported to be TSP 16 . TSP interacts with CD47 through its CBD, but binds other surface receptors such as CD36 or αvβ3 via distinct domains 17 24 . Since TSP reportedly activates latent TGF-β in vitro and in vivo 25 and ligation of CD36 by CD36 mAb downregulates IL-12 and TNF-α production and increases IL-10 26 , we used 4N1K peptide encoding the CBD of TSP to demonstrate that TSP-mediated inhibition of IL-12 resulted from its direct effect on CD47 antigen. In addition, CD47 ligation did not decrease TNF-α nor increase IL-10 production. Note that TSP uses its properdin-like domain to activate TGF-β 17 and that TGF-β production was not upregulated after CD47 signaling . IL-12 downmodulation, in the absence of TNF-α inhibition, was also observed in IFN regulatory factor (IRF-1 27 ) and IFN consensus sequence binding protein (ICSBP)-deficient mice 28 . These two transcription factors belong to the IRF family and bind IL-12p40 promoter as well as other IFN-stimulated genes. As a direct consequence of the primary defect in IL-12 production, the deficient mice displayed an impaired Th1 response and IFN-γ–dependent host resistance to intracellular pathogens. Similarly, our unpublished observations indicate that CD47 signaling strongly impaired IL-12–induced proliferative and IFN-γ responses of activated T cells. The role of CD47 antigen, which is expressed by all leukocytes, has been demonstrated in the transendothelial migration of neutrophils, lymphocytes, and more recently, monocytes 15 . Our present findings reveal that CD47 ligation by TSP modulates the function of monocytes. Therefore, we propose that engagement of CD47 is a novel and unexplored pathway to selectively downregulate IL-12 production. Inhibition of the IL-12 response is crucial for the treatment of organ-specific autoimmune diseases 1 . For instance, administration of anti–IL-12 mAb completely abrogated established colitis of trinitrobenzene sulfonic acid (TNBS)-treated mice. IL-12 neutralization was also revealed to be an effective treatment of autoimmune encephalitis and insulin-dependent diabetes. Finally, in addition to its ability to downregulate IL-12 response through its CBD, TSP is known to display several important antiinflammatory activities via distinct domains. TSP facilitates elimination of apoptotic cells 29 , activates TGF-β in vivo and in vitro 25 , potently inhibits angiogenesis 17 , and suppresses HIV infectivity through its binding to HIVg120 24 . Moreover, absence of TSP causes pneumonia 30 . Therefore, since TSP is transiently expressed at high concentration in damaged and inflamed tissue, its selective suppression of the IL-12 response through interaction with the CD47 molecule may be relevant in limiting the duration and the intensity of the inflammatory response. The importance of such a process awaits confirmation in CD47- and TSP-deficient mice. | Study | biomedical | en | 0.999995 |
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