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10209051 | Stat4-deficient DO11.10 TCR-transgenic mice have been described previously ( 12 , 19 ). 2C TCR transgenic mice ( 20 ) were obtained from Dr. T. Hansen (Washington University, St. Louis, MO). Recombinant human IL-2, IL-4, IL-12, and KJ1-26 ( 21 ) were used as previously described ( 19 ). Recombinant murine IL-18 (Research Diagnostics Inc.) was used at 50 ng/ml. Anti–IL-12 (TOSH) ( 22 ) and anti– IFN-γ (H22) (from Dr. R.D. Schreiber, Washington University, St. Louis, MO) were used at 10 μg/ml. Anti-CD3 (2C11) (from Dr. A. Shaw, Washington University, St. Louis, MO) was coated at 10 μg/ml for primary stimulations and 1 μg/ml for secondary stimulation, and anti-CD28 (PV1) (from Dr. Carl June, Naval Medical Research Institute, Bethesda, MD) was used at 1 μg/ml. All other staining reagents were purchased from PharMingen . Sorted CD4 + DO11.10 T cells (2 × 10 5 /ml) were activated with 0.3 μM OVA peptide (OVA), IL-2, IL-12, and irradiated BALB/c splenocytes as previously described ( 3 ). In other experiments, DO11.10 splenocytes (3 × 10 6 /ml) were activated with OVA, IL-2, IL-12 (Th1), or IL-4 (Th2) as indicated in the figure legends. CD8 + T cells were sorted from spleen and lymph node cells of 2C mice and activated (2 × 10 5 /ml) using irradiated BALB/c splenocytes (1.5 × 10 6 /ml). CD4 + and CD8 + T cells were sorted from spleen and lymph node cells of Stat4-deficient or wild-type mice, and stimulated (4 × 10 5 /ml) with irradiated C57BL/6 splenocytes (4 × 10 6 /ml), IL-2, and the indicated cytokines and antibodies. IFN-γ was measured by ELISA as previously described ( 3 ). Intracellular cytokine staining was performed as described elsewhere ( 18 , 23 ). T cells were stimulated overnight with OVA and either irradiated APCs or plate-bound anti-CD3, and Brefeldin A (10 μg/ml; Epicenter Technologies) was added for the final 4 h. Cells were harvested, washed, and stained for CD4, CD8, and KJ1-26 as indicated in the figure legends. After washing, cells were fixed, washed, permeabilized, and stained for IFN-γ. Previous analyses of Stat4-deficient mice reported five- to sixfold reduced IFN-γ production based in part on polyclonal cellular activation of unseparated splenocytes ( 12 , 13 ). To examine the requirement for Stat4 in antigen-specific CD4 + T cells, we used DO11.10 TCR-transgenic mice crossed to either wild-type or Stat4-deficient backgrounds. Splenocytes from un-immunized mice were primed in vitro and induced toward Th1 and Th2 phenotypes ( 4 ) . As expected, wild-type DO11.10 T cells primed in the presence of IL-12 generated high levels of IFN-γ upon secondary stimulation. In contrast, Stat4-deficient DO11.10 T cells primed with IL-12 generated nearly 100-fold less IFN-γ, confirming that Stat4 has a significant role in CD4 + T cells for IFN-γ production. After in vitro priming, clonotype-positive (KJ1-26 + ) T cells from wild type DO11.10 transgenic mice are predominantly CD4 + . However, in Stat4-deficient mice, as much as 25% of the KJ1-26 + T cells are CD4 − and CD8 − (not shown) after in vitro priming. Double-negative T cells have been reported to exhibit differences in Th1/Th2 regulation, with impaired Th2 development ( 24 , 25 ). Thus, we wished to assess production of IFN-γ in CD4 + and CD4 − T cells using intracellular cytokine staining . Wild-type DO11.10 T cells produced abundant intracellular IFN-γ production, whereas Stat4-deficient DO11.10 T cells showed a significantly lower percentage of IFN-γ–producing cells with lower mean fluorescence intensities relative to wild-type T cells . Of the Stat4-deficient DO11.10 T cells, 6% of CD4 + cells produced IFN-γ, whereas 13% of CD4 − negative cells produced IFN-γ, implying that in KJ1-26 + T cells, CD4 + cells are more Stat4 dependent for IFN-γ production than are CD4 − cells. These and other results suggest that IFN-γ production may be regulated differently in various T cell lineages (16, 26, and Carter, L.L., unpublished observations). Therefore, we wished to compare CD4 + and CD8 + T cells from TCR-transgenic mice for their dependence on IL-12 for driving IFN-γ production . CD8 + or CD4 + T cells were sorted from 2C TCR-transgenic mice or DO11.10 mice, respectively, and primed with antigen in the presence of IL-12 or anti–IL-12 antibody for 6 d, restimulated, and assessed for IFN-γ production. CD4 + DO11.10 T cells produced high IFN-γ when primed with IL-12, but virtually undetectable IFN-γ when primed with anti–IL-12 antibody . In contrast, 2C CD8 + T cells produced high levels of IFN-γ even when primed in the presence of anti–IL-12 antibody, with IFN-γ production being reduced only twofold relative to cells primed with IL-12. Thus, CD8 + T cells show significant IL-12–independent IFN-γ production, whereas CD4 + T cells do not. In the mouse, Stat4 is uniquely activated by IL-12 ( 11 , 27 , 28 ). Therefore, IL-12–independent IFN-γ production by CD8 + T cells suggests either that Stat4 activation is IL-12 independent or that IFN-γ production is Stat4 independent. To distinguish these possibilities, we analyzed purified CD4 + and CD8 + T cells from Stat4-deficient and wild-type BALB/c mice. T cells were primed in the presence of IL-12 with either allogeneic stimulators or plate-bound anti-CD3 and anti-CD28 . When primed and reactivated with allogeneic stimulators , Stat4-deficient CD4 + T cells produced very little IFN-γ. In comparison, Stat4-deficient CD8 + T cells produced significantly more IFN-γ, although the level observed was reduced three- to fourfold relative to the wild-type CD8 + control. When T cells were reactivated with anti-CD3 , Stat4-deficient CD4 + T cells remained poor IFN-γ producers, whereas Stat4-deficient CD8 + T cells produced IFN-γ at levels similar to wild-type CD8 + controls . When T cells were primed with anti-CD3/anti-CD28 and IL-12, and reactivated with anti-CD3, Stat4-deficient CD4 + T cells again produced very low levels of IFN-γ, whereas Stat4-deficient CD8 + T cells produced high levels of IFN-γ . We extended these results with intracellular cytokine staining . Purified CD4 + and CD8 + T cells from Stat4-deficient and wild-type mice were primed in the presence of IL-12 using either APCs or anti-CD3/anti-CD28, and reactivated with APCs or anti-CD3 . CD4 + T cells again showed a strict requirement for Stat4 in IFN-γ production with both forms of activation. In contrast, Stat4-deficient CD8 + T cells produced abundant IFN-γ with either form of activation. With anti-CD3 treatment, equivalent percentages of Stat4-deficient and wild-type CD8 + T cells produced IFN-γ, whereas with activation by APCs, IFN-γ + Stat4-deficient CD8 + T cells were reduced twofold. Thus, in contrast to CD4 + T cells, CD8 + T cells show significant Stat4-independent IFN-γ production, which is most apparent with direct TCR-mediated cellular activation. Since APCs can produce IL-12 and IL-18 ( 4 , 29 , 30 ), T cell activation using APCs could engage both the TCR and the IL-12/IL-18 pathway for IFN-γ production. Therefore, we asked if these pathways were differentially Stat4 dependent in CD4 + and CD8 + T cells . Purified CD4 + and CD8 + T cells from Stat4-deficient and wild-type mice were primed with IL-12 and allogeneic APCs and reactivated on day 6 with either anti-CD3 or IL-12/ IL-18. In response to anti-CD3, wild-type CD4 + , but not Stat4-deficient CD4 + , T cells produced IFN-γ. As above, both wild-type and Stat4-deficient CD8 + T cells produced IFN-γ. However, in response to IL-12/IL-18 treatment, both CD4 + and CD8 + Stat4-deficient T cells failed to produce IFN-γ. Thus, the IL-12/IL-18 pathway for IFN-γ production is strictly Stat4 dependent in both CD4 + and CD8 + T cells. In contrast, the TCR-induced pathway for IFN-γ production is Stat4 dependent only in CD4 + , and not CD8 + , T cells. Previous observations have suggested the existence of both IL-12–dependent and –independent pathways for IFN-γ production ( 12 , 13 , 16 , 31 – 33 ). However, since few of these studies analyzed purified cell types, the effects of Stat4 in specific lineages were potentially obscured. Furthermore, recent studies have demonstrated that IFN-γ gene transcription can be activated by two distinct signaling pathways, one by TCR signaling and other by IL-12 and IL-18 ( 18 ), and these pathways were not individually examined in the previous studies. Therefore, the aim of this study was to analyze differences between CD4 + and CD8 + T cells in their regulation of these two pathways for IFN-γ production. In this paper, we make several new observations. First, we show that the IL-12/IL-18 pathway for induction of IFN-γ operates in CD8 + as well as CD4 + T cells. Second, we formally demonstrate that the IL-12/IL-18 pathway is strictly Stat4 dependent in both CD4 + and CD8 + T cells. Third, we have identified an unexpected difference between CD4 + and CD8 + T cells in TCR signaling. Specifically, CD4 + T cells produce IFN-γ in a completely Stat4-dependent manner, whereas CD8 + T cells are Stat4 independent for TCR-induced IFN-γ production. Two pathways are now recognized for IFN-γ induction ( 17 , 18 ), one via TCR-signaling and another through IL-12 and IL-18 that acts independently of antigen stimulation ( 17 ). The TCR- and IL-12/IL-18– induced pathways were shown to be pharmacologically distinct and to induce different transcription factors ( 18 ). In this study, we show that IL-12/IL-18 induction of IFN-γ operates in CD8 + as well as CD4 + T cells . The existence of antigen-independent IFN-γ production by previously activated T cells from both CD4 and CD8 lineage has significant implications for immune regulation. By stimulating production of cytokines in an antigen-independent manner, this pathway allows antigen-specific T cells to operate like innate immune cells. The Stat4-dependence of the IL-12/ IL-18–induced pathway in both CD4 + and CD8 + lineages suggest a common IFN-γ regulatory mechanism. In contrast to IL-12/IL-18– induced IFN-γ, TCR-induced signaling revealed a striking difference in the requirement for Stat4 between CD4 + and CD8 + T cells. Unseparated Stat4-deficient splenocytes displayed a partial reduction in IFN-γ production in previous studies ( 12 , 13 ), whereas pure populations of CD4 + T cells show a much more stringent requirement for Stat4 . In contrast, Stat4-deficient CD8 + T cells generated abundant IFN-γ particularly when activated through the TCR. When CD8 + T cells were activated using APCs, a partial loss of IFN-γ production was observed in Stat4-deficient CD8 + T cells relative to wild-type controls , suggesting that activation with APCs engages both TCR (Stat4-independent) and IL-12/IL-18 (Stat4-dependent) pathways. Activation of CD8 + T cells using anti-CD3 restricts activation to the TCR (Stat4-independent) pathway, resulting in equivalent levels of IFN-γ production by Stat4-deficient and wild-type CD8 + T cells. Distinct regulation of IFN-γ gene activation between CD4 + and CD8 + T cells has previously been suggested ( 26 ). A Stat4-independent mechanism for IFN-γ production development has recently been described ( 34 ), but as it operated only in the absence of Stat6 and in CD4 + T cells, it is distinct from the pathway described here. Differences in TCR signaling between CD4 + and CD8 + lineages could reside at several levels. First, CD4 + and CD8 + T cells may differ in expression of signaling components downstream of the TCR. For example, certain mitogen-activated protein (MAP) kinases implicated in IFN-γ induction ( 35 , 36 ) could be differentially expressed or activated in CD4 + versus CD8 + T cells, being Stat4 dependent only in CD4 + T cells. Second, chromatin accessibility of the IFN-γ gene may differ between primary CD4 + and CD8 + lineages. In this model, the IFN-γ gene would be accessible to TCR-induced factors independently of Stat4 in CD8 + T cells, but not in CD4 + T cells. However, IFN-γ chromatin structure in CD4 + versus CD8 + T cells has not yet been compared. Finally, coreceptor signaling could account for the present observations. CD8 may provide a signal that bypasses a Stat4 requirement in IFN-γ production, or conversely CD4 may provide a signal imposing such a requirement. Indeed, differences between coreceptor association with src family kinase Lck have been reported ( 37 – 40 ), and lack of CD4 expression impairs Th2 responses ( 24 , 25 ). In summary, the study presented here makes the first distinction between CD4 + and CD8 + T cells for the role of Stat4 in regulation of IFN-γ expression. Given the importance of IFN-γ in responses to pathogens and in autoimmune processes, it will be important to determine the basis of these lineage-specific differences in the Stat4-requirement for IFN-γ gene regulation. | Study | biomedical | en | 0.999998 |
10224276 | Recombinant human MCSF was the gift of Genetics Institute, Cambridge, MA. Recombinant human IFN-γ was the gift of Genentech , South San Francisco, CA. Recombinant human CD40 ligand (CD40L) homotrimer was the gift of W. Fanslow, Immunex Corp. , Seattle, WA. The IDO inhibitor 1-methyl- d , l -tryptophan ( 16 ) was purchased from Aldrich Chemical Co. 6-nitro-tryptophan ( 17 ) was synthesized by D. Boykin, Georgia State University, Atlanta, GA, using a modification of the method of Moriya et al. ( 18 ). Polyclonal antiserum against human IFN-γ was obtained from Biosource International . All other reagents were obtained from Sigma Chemical Co. unless otherwise specified. Human peripheral blood monocytes and lymphocytes were isolated from healthy volunteer donors by leukocytapheresis and counterflow centrifugal elutriation, following appropriate informed consent under a protocol approved by our Institutional Review Board. Monocytes (>95% purity by cell surface markers) were cultured in 96-well plates as previously described ( 4 ) using RPMI 1640 with 10% newborn calf serum (Hyclone) plus MCSF (200 U/ml). T cell activation studies in cocultures were performed as previously described ( 4 ), using the above medium supplemented with an additional 5% FCS. In brief, Møs (5 × 10 4 cells/well) were allowed to differentiate for 4–6 d in MCSF, and then autologous lymphocytes (2 × 10 5 cells/well) were added along with mitogen. The mitogens used in this study were anti-CD3 mAb (100 ng/ml, clone OKT3; American Type Culture Collection) and staphylococcal enterotoxin B (5 μg/ml; Sigma Chemical Co. ). Both gave equivalent results; the data shown are from anti-CD3 unless otherwise specified. T cell proliferation was assessed by standard thymidine incorporation assay as described ( 3 ). When T cell activation was studied without Møs, fresh autologous monocytes were added (1:4) as nonsuppressive accessory cells. Conditioned medium from cocultures of T cells and Møs was prepared by harvesting supernatant 48 h after T cell addition. Conditioned medium was then used to support a second round of T cell activation. Mitogen and other additives were prepared in tryptophan-free buffers. A chemically defined, serum-free medium ( 19 ) selectively deficient in tryptophan was prepared using tryptophan-free RPMI 1640 (Select-amine kit; GIBCO BRL ) supplemented with insulin (10 μg/ml), iron-saturated transferrin (5 μg/ml), and BSA (1 mg/ml ultra-pure grade; measured concentration of free tryptophan <5 nM). Preliminary validation experiments confirmed that T cell proliferation in this medium was undetectable but was comparable to serum-based medium when tryptophan was added. To study T cells in the absence of Møs, T cells were activated using anti-CD3 mAb adsorbed onto plastic tissue culture wells (0.5 μg/cm 2 in bicarbonate buffer, pH 9) plus soluble anti-CD28 mAb (1 μg/ml; PharMingen ). The tryptophan-degrading activity of Møs reflects a multifactored combination of IDO expression, tryptophan transport into the cells, and intracellular conditions that posttranslationally affect enzyme activity ( 20 ). Therefore, when tryptophan depletion was the outcome of interest, we measured the rate of disappearance of tryptophan from culture supernatants over time. Tryptophan was assayed using the method of Bloxam and Warren ( 21 ). Proteins were precipitated with 10% TCA and free tryptophan assayed after conversion to norharman using formaldehyde and FeCl 3 . The reaction product was measured spectrofluorometrically (excitation 360 nm, emission 460 nm) and compared against a standard preparation of tryptophan. Validation studies showed this assay to be linear in the range of 0.1–100 μM, with an estimated threshold sensitivity of 0.05 μM. Where it was desirable to show that tryptophan depletion in cultures was due to IDO activity, culture supernatants were assayed by HPLC for the presence of kynurenine. IDO catalyzes the oxidation of tryptophan to N -formylkynurenine, which in Møs is rapidly converted into kynurenine ( 22 ) and then to other downstream metabolites ( 7 ). With the exception of tryptophan oxygenase, which is found only in hepatocytes, IDO is the only enzyme capable of degrading tryptophan along the kynurenine pathway ( 8 ). Thus, the appearance of kynurenine in cultures was unambiguous evidence of functional IDO activity. However, because kynurenine can be converted into other downstream metabolites, this assay was not quantitative. Where quantitative data were required, the tryptophan depletion assay described above was used. HPLC assays were performed by the Medical College of Georgia Molecular Biology Core Facility. Samples were prepared by extracting 150 μl culture supernatant with 1 ml methanol. Precipitated proteins were removed by centrifugation and the supernatant dried under vacuum. Samples were resuspended in 100 μl initial mobile phase (deionized water) and an aliquot injected onto a C-18 column (Phenomenex Luna C-18; 250 × 4.6 mm; 5 μm). Samples were eluted with a linear gradient of acetonitrile in water (0–80% over 20 min), and absorbance was measured at 254 nm. Standards for tryptophan, kynurenine, and 1-methyl-tryptophan were run with each assay to establish retention times. In preliminary validation studies, the identity and purity of each peak was confirmed by mass spectroscopy. Total protein synthesis was measured as incorporation of tritiated leucine (4 μCi/ml) over 24 h. TCA-insoluble proteins were precipitated and washed three times in 5% TCA, and the precipitate was analyzed by liquid scintillation counting. Amino acid concentrations in culture supernatants were measured by HPLC in our clinical Neonatal Nutrition Laboratory. Møs were harvested with EDTA and total RNA prepared. Sample RNA (1 μg) was reverse transcribed with avian myeloblastosis virus (AMV)-RT, and a 182-bp fragment amplified with the following primers: forward, bp 237–254 of the published sequence ( 23 ); reverse, bp 402–418, spanning exons 3–4. Product formation was assessed by agarose gel electrophoresis and ethidium bromide staining. PCR product was isolated from the gel and reamplified with internal primers to confirm specificity. Two-color FACS ® analysis was performed using directly conjugated mAbs as previously described ( 24 ). T lymphocytes were identified by gating on CD3-positive cells, and expression of CD69, CD25, and CD71 was measured in the second color. Experiments for all figures were replicated at least three times, and representative data are shown. Data points were measured in triplicate and the mean reported. Error bars show standard deviation. Where SD was <10%, error bars have been omitted for clarity. Comparisons of multiple groups within a single experiment were by ANOVA. Supernatants were harvested from cocultures of Møs and mitogen-activated T cells after 48 h. Fresh lymphocytes were suspended in conditioned medium and activated with additional mitogen. Fig. 1 shows that conditioned medium completely failed to support T cell proliferation (<1% of the proliferation in fresh medium). However, the addition of tryptophan to conditioned medium fully restored its ability to support T cell proliferation, indicating that tryptophan was the only component that had been depleted. Consistent with this finding, amino acid analysis of conditioned media showed that all other essential amino acids were present, and only tryptophan was undetectable (data not shown). Titration of reagent tryptophan into conditioned medium gave a half-maximal concentration for T cell proliferation of 0.5–1 μM , compared with a measured concentration of tryptophan in coculture-conditioned medium of <50 nM (the detection limit of our assay). Control-conditioned media from Møs alone, from cocultures of Møs + T cells without mitogen, or from T cells activated with fresh monocytes instead of Møs all supported T cell proliferation comparably to fresh medium (90–140% of control; n = 3–4/group). The kinetics of tryptophan elimination were measured by coincubating Møs and T cells with mitogen for 24 h to allow upregulation of the tryptophan depletion pathway and then adding fresh tryptophan and following its disappearance. As shown in Fig. 3 A, tryptophan was eliminated by first-order kinetics with a half-life of 2–3 h. The initial rate of elimination when tryptophan was not limiting was up to 20,000 pmol/ 10 6 cells/h. This far exceeded the consumption attributable to cellular metabolism , as Møs without activated T cells depleted tryptophan at a rate of 300 ± 130 pmol/10 6 cells/h (cumulative measurement obtained over 7 d; data not shown). This implied that the majority of tryptophan depletion by activated Møs was due to an inducible system, which we suspected was IDO. Consistent with this finding, abundant IDO mRNA was detectable by RT-PCR in Møs after activation, whereas before activation, IDO message was undetectable . To confirm the presence of IDO activity, culture supernatants were assayed for kynurenine. As shown in Fig. 3 C, depletion of tryptophan was accompanied by a corresponding increase in kynurenine production, confirming the presence of functional IDO activity. We next asked whether pharmacologic inhibition of IDO could prevent suppression of T cells in cocultures. The compound 1-methyl-tryptophan has been reported to be a potent competitive inhibitor of IDO activity when tested in vitro using purified enzyme ( 16 , 17 ). To determine whether this agent could inhibit IDO activity in intact Møs, we added 1-methyl-tryptophan to activated Mø cultures. As shown in Fig. 4 A, the presence of 1-methyl-tryptophan markedly reduced the degradation of tryptophan by Møs, and this was accompanied by a corresponding inhibition of kynurenine production , confirming that the target of the inhibitor was IDO. Functionally, the addition of 1-methyl-tryptophan to cocultures abrogated the ability of Møs to suppress T cell proliferation in a dose-dependent manner . Although this finding was consistent with the proposed role for IDO in Mø-mediated suppression, it might in theory indicate an unanticipated immunostimulatory role for 1-methyl-tryptophan itself. To exclude this possibility, we synthesized a second analogue of tryptophan, 6-nitro-tryptophan, which has also been reported to inhibit purified IDO enzyme in vitro ( 17 ). As shown in Fig. 4 C, 6-nitro-tryptophan also prevented Mø- mediated suppression in a dose-dependent fashion . Finally, we tested the effects of supplemental tryptophan on suppression. As shown in Fig. 4 D, high levels of tryptophan did prevent suppression of T cells, provided that the number of Møs in cocultures was kept low. At our usual concentrations of Mø, it proved impossible to supplement with sufficient tryptophan to overcome its rapid degradation. Thus, by the use of two pharmacologic inhibitors of IDO and by tryptophan supplementation, the mechanism of T cell suppression in our system appeared to be depletion of tryptophan by IDO. Møs did not degrade tryptophan simply as a result of contact with T cells. Rather, there was an obligate requirement that the T cells attempt to activate . In light of the existing studies implicating IFN-γ as an inducer of IDO ( 25 – 27 ), we suspected that IFN-γ from activating T cells might be the signal for IDO induction. Consistent with this idea, low but detectable levels of IFN-γ were present in cocultures within 4–6 h of T cell activation, coincident with the time that tryptophan degradation began . Neutralizing antibodies against IFN-γ reduced the induction of tryptophan-degrading activity and reduced suppression of T cells by Møs , supporting a role for IFN-γ in the signaling pathway. However, the dose–response relationship using recombinant IFN-γ revealed that relatively high concentrations of IFN-γ were required for full induction of tryptophan-degrading activity . We therefore asked whether there was an additional signal that might act in concert with IFN-γ. CD40L is upregulated early in T cell activation and is known to act synergistically with IFN-γ to activate other Mø functions ( 28 ). Fig. 5 D shows that CD40L exerted marked synergy with IFN-γ, shifting the dose–response curve for IFN-γ one to two orders of magnitude so that significant tryptophan depletion began at IFN-γ concentrations of <1 U/ml. We have previously shown that T cells activated in coculture with MCSF-derived Møs initially enter the cell cycle but arrest before the first G1/S transition ( 4 ). We therefore asked whether a comparable phenomenon occurred when T cells were activated in the absence of tryptophan. Purified T cells (without monocytes or Møs) were cultured in tryptophan-free medium using immobilized anti-CD3 plus anti-CD28 mAb as activating stimuli. In this system, T cells stimulated in the presence of tryptophan activated normally, whereas T cells stimulated without tryptophan arrested before entry into the first S phase, as shown by the complete absence of DNA synthesis . This arrest was not due to an absence of protein synthesis, as T cells without tryptophan successfully upregulated CD69, CD25 (high-affinity IL-2 receptor), and CD71 (transferrin receptor) and secreted IL-2 and IFN-γ , all of which require new protein synthesis ( 29 ). Total protein synthesis, measured as incorporation of radiolabeled leucine during the first 24 h of activation, continued at a rate 40– 55% of controls ( n = 3; see Materials and Methods), despite the absence of exogenous tryptophan. Nonetheless, no entry into S phase occurred. Thus, T cells activated in the absence of exogenous tryptophan arrested in a fashion similar to that which we had previously observed in coculture. The upregulation of early G1 markers suggested that some portion of G1 was tryptophan independent. To test this hypothesis, T cells were activated for various times in the absence of tryptophan, and then tryptophan was added and the time to entry into S phase determined. Control cells, cultured with tryptophan throughout, reproducibly entered S phase 28–32 h after initial TCR engagement (times are reported as 4-h ranges to reflect the limit of precision of the assay). In contrast, T cells that had been preactivated under tryptophan-free conditions required only 12–16 h to enter S phase after tryptophan was added , indicating that significant progression through G1 had occurred in the absence of tryptophan. The tryptophan-sensitive arrest point was stable, with T cells surviving >72 h in the absence of tryptophan with no loss of viability. When tryptophan was added to arrested cells, the time of entry into S phase was consistently 12–16 h, regardless of whether cells had been preactivated for 36, 48, or 72 h without tryptophan. This suggested that the arrest occurred at a specific point in G1 and that this position in the cell cycle was maintained until tryptophan was restored. From the preceding experiments, we estimated that the tryptophan-independent portion of G1 was ∼14 h (calculated as the difference between the average time to S phase for resting T cells versus the time to S phase for preactivated cells). To test this estimate, we deprived T cells of tryptophan during the initial 14 h of activation, then added tryptophan just before the putative arrest point. As shown in Fig. 9 B, cultures deprived of tryptophan for the first 14 h entered S phase identically to T cells supplied with tryptophan throughout, supporting the hypothesis that the initial portion of G1 was independent of tryptophan. In additional experiments (not shown), delaying the addition of tryptophan beyond 14 h introduced a corresponding delay in entry into S phase, supporting the proposed localization of the arrest point close to hour 14. Resting (G0) T cells require TCR signaling in order to enter G1, but subsequent progression through the cell cycle rapidly becomes TCR independent (for a review see reference 29 ). In our system, commitment to TCR- independent cell division was first detectable ∼6 h after TCR engagement, and most cells were committed by hour 12. As shown in Fig. 10 , this commitment occurred identically regardless of whether tryptophan was present or absent during the relevant time period. As long as the cells were not allowed to arrest (i.e., tryptophan was supplied before the tryptophan-sensitive checkpoint), commitment to cell division proceeded normally. In contrast to the experiments shown in Fig. 10 , however, once T cells entered the arrested state, simply restoring tryptophan was no longer sufficient to allow cell cycle progression. T cells were activated for 48 h in tryptophan-deficient medium using immobilized anti-CD3/CD28. The arrested cells were then removed from contact with anti-CD3, washed free of anti-CD28, and transferred to medium containing normal levels of tryptophan. As shown in Fig. 11 , despite their previous 48-h exposure to anti-CD3, the arrested T cells still required additional TCR signaling plus the presence of tryptophan to exit the arrested state. Even costimulation via CD28 was not sufficient to promote cell cycle progression in the absence of TCR engagement. In this study, we show that tryptophan catabolism via IDO is the mechanism by which MCSF-derived Møs suppress T cell proliferation in vitro. We have recently tested this hypothesis of IDO-mediated T cell suppression in vivo using the model of allogeneic pregnancy. This model was chosen because it has long been recognized as paradoxical that the maternal immune system tolerates a genetically foreign fetus throughout gestation ( 30 ). IDO is known to be expressed in human placenta and has been reported to be localized to the zone of contact between fetal-derived tissues and the maternal immune system ( 31 ). Using 1-methyl-tryptophan as a pharmacologic inhibitor of IDO, we have demonstrated that IDO is a required component of the mechanism by which the allogeneic fetus protects itself from rejection by the maternal immune system and that inhibition of IDO breaks maternal tolerance to the allogeneic fetus ( 15 ). In the same report, we also showed that pharmacologic inhibition of IDO enhances the activation of autoreactive T cells. Thus, by two measures—breaking tolerance and enhancing autoreactivity— these data support a role for IDO in regulating T cell responses in vivo. IDO has previously been viewed primarily as a host defense mechanism, inhibiting proliferation of intracellular pathogens ( 6 , 9 – 13 ) or cancer cell lines ( 14 ) by depriving them of tryptophan (for a review see reference 8 ). In these settings, the proposed role of IDO has been to eliminate the cell's own stores of tryptophan. To our knowledge, no role for IDO in regulating the proliferation of adjacent cells has been suggested. However, both direct and indirect evidence indicates that IDO is widely expressed throughout the immune system ( 32 , 33 ) and, specifically, that it is localized to a subset of cells with a Mø or dendritic cell morphology ( 33 – 35 ). These IDO-expressing cells are found at several putative sites of immune tolerance or privilege, including thymus, mucosa of the gut, epididymis, placenta, and the anterior chamber of the eye ( 32 , 33 , 36 , 37 ). This pattern of widespread expression throughout the immune system is difficult to reconcile with a simple mechanism of host defense. We hypothesize that IDO expression by APCs functions to suppress undesirable T cell activation and thus helps maintain peripheral tolerance. Two models might be proposed by which IDO could suppress T cells in vivo: it might catalyze the production of a suppressive metabolite of tryptophan, or it could deplete local tryptophan below some threshold level required for T cell activation. In repeated experiments, we have been unable to detect any evidence of an immunosuppressive metabolite in coculture supernatants ( 4 ). Furthermore, our experiments with isolated T cells imply a specific checkpoint in early T cell activation that is sensitive to low concentrations of tryptophan. For these reasons, we favor the tryptophan depletion hypothesis. Implicit in this hypothesis is the assumption that cells expressing IDO in vivo could create a local microenvironment in which tryptophan is low, despite the availability of ample tryptophan elsewhere. In this regard, it is well established that delivery of a substrate into local microenvironments is sharply limited by the rate of diffusion ( K d ) through the interstitial space ( 38 , 39 ). In the face of even normal metabolic demands, substrate concentrations rapidly fall to undetectable levels within a few cell diameters of the source of delivery ( 39 ). Because the rate of tryptophan consumption by IDO-expressing Møs is orders of magnitude greater than normal metabolic demands, it is plausible that such Møs could create local conditions of very low tryptophan concentrations. Although this hypothesis is now speculative with regard to tryptophan, the phenomenon is well documented with regard to, for example, the local hypoxic state created within muscle tissue during exercise. Because tryptophan degradation by IDO is much greater than consumption by metabolic demands , it is likely that IDO constitutes the major route of tryptophan depletion by activated Møs. However, IDO could act in combination with other pathways. Møs have a high rate of protein synthesis, and the incorporation of free tryptophan into proteins could contribute to local tryptophan depletion. Indeed, the tRNA synthetase for tryptophan (the WRS gene) is unique among tRNA synthetases in that it is massively induced in Mø lineage cell lines (but not lymphoid lines) by the same signals that induce IDO ( 40 ). It has been proposed that this induction allows Møs to compete preferentially for tryptophan when the concentration of substrate is low. Likewise, any pathway that transported tryptophan into Møs, whether for protein synthesis, degradation by IDO, or incorporation into other biosynthetic pathways, would also serve to deplete local tryptophan. Thus, IDO could act in concert with other catabolic pathways to render Møs an effective local “sink” for tryptophan. The proposed tryptophan depletion model gains support from the apparent existence of a cell cycle arrest point sensitive to tryptophan concentration. Although the absence of any essential nutrient is, by definition, incompatible with long-term proliferation, the arrest point we describe appears more specific than simple protein starvation. First, although protein synthesis is reduced in the absence of exogenous tryptophan, it still occurs at a significant rate, presumably reflecting a combination of endogenous tryptophan stores and recycling of tryptophan from catabolism of endogenous and exogenous proteins ( 41 ). Yet despite ongoing protein synthesis, cell cycle progression is not simply delayed but rather is completely arrested. Second, the arrest induced by tryptophan deprivation occurs at a reproducible point in the cell cycle and remains stable once entered, suggesting a regulated process. Taken together, these attributes suggest a specific, tryptophan-sensitive cell cycle arrest point. It has been noted by several groups that deprivation of certain amino acids—tryptophan in particular—exerts an inhibitory effect on cell cycle progression that cannot be explained by the effect on protein synthesis ( 42 – 45 ). For that reason, it has been suggested that levels of these amino acids may function as specific checkpoints regulating cell cycle progression. However, the biologic significance of such amino acid–specific checkpoints and the mechanism by which the levels of amino acids might be manipulated in order to regulate T cell activation has remained obscure. We now propose a system in which regulation of local tryptophan concentration functions as a means of communication between APCs and T cells, with APCs regulating the tryptophan level via IDO and T cells responding with either activation or arrest, depending on the level they detect. As a strategy to inhibit T cell activation, arresting progression through the cell cycle is not unique to tryptophan metabolism. The immunosuppressive drugs mycophenolate, rapamycin, and leflunomide all induce a mid-G1 arrest in activating T cells, and this is believed to account in whole or part for their immunosuppressant action ( 46 – 48 ). Recent evidence suggests that T cells require one or more rounds of cell division to acquire a variety of effector functions ( 49 – 51 ), so inhibiting proliferation may also inhibit functional activity. In our system, it is currently unknown how T cells sense the level of tryptophan and trigger cell cycle arrest. Tryptophan-sensing systems in bacteria have been well described ( 52 ), but comparable systems in eukaryotes have not yet been identified. However, mammalian genes such as tryptophan oxygenase are known to be regulated by changes in tryptophan levels ( 53 ), so such sensing systems can be inferred to exist. The requirement for a second signal from the TCR in order to exit the arrested state is an important finding in light of our proposed biologic model. Under this model, T cells that attempt to activate while in contact with an IDO-expressing APC are inhibited by the local absence of tryptophan. In theory, however, once such T cells were committed to cell division, they could migrate elsewhere and complete the activation process under tryptophan-sufficient conditions. The data presented in Fig. 11 show that once T cells have arrested, simply regaining tryptophan is no longer sufficient to allow continued activation. Despite the fact that T cells would normally have become independent of TCR signaling before the tryptophan-sensitive checkpoint , once they enter the arrested state they apparently reverse this commitment and reimpose upon themselves a requirement for a second round of TCR signaling. From a biologic standpoint, this would mean that a T cell arrested by an IDO-expressing APC would be obliged to find a second, nonsuppressive APC presenting the same antigen in order to exit the arrested state. What would be the fate of an arrested T cell if no such supportive APC could be found? In vitro, we find that arrested cells undergo progressive apoptosis after several days if not rescued by TCR engagement ( 4 ). Whether this means that they would likewise die in vivo, enter some form of anergy, or return to a resting state remains to be determined. However, the arrested state we describe differs from classical anergy ( 54 ) in several interesting respects. First, the cells retain their responsiveness to TCR engagement . Second, costimulation via CD28 is not sufficient to rescue cells once they arrest. And third, arrested cells die if not rescued within a relatively brief window of time. Taken together, these attributes suggest that T cells arrested by tryptophan deprivation are not immediately deleted from the repertoire but that they must find a permissive APC and complete the activation process if they are to survive. In conclusion, our hypothesis regarding the biologic role of IDO-expressing APCs is that they are involved in maintaining peripheral tolerance to self antigens. Our in vitro model has focused on MCSF-derived Møs as one example of immunosuppressive APCs, but dendritic cells or other APCs that possess inducible IDO could likewise be immunosuppressive. We speculate that tryptophan catabolism may constitute a previously unsuspected mechanism contributing to the regulation of peripheral T cell activation. | Other | biomedical | en | 0.999998 |
10224277 | Porcine aortic endothelial cells were isolated as described previously ( 27 ) by gentle mechanical scraping of the intima of the descending part of porcine aorta. Harvests of endothelial cells were plated at a density of 10 6 cells per 100-mm plastic dish. The cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 in air. The “basal culture medium” consisted of medium 199 with Earle's salt, supplemented with 100 IU/ml penicillin G, 100 μg/ml streptomycin, and 20% (vol/vol) newborn calf serum (NCS). 1 The medium was renewed every other day. After 4 d, when the cells had grown to confluence, they were trypsinized in PBS (composed of [mM]: 137 NaCl, 2.7 KCl, 1.5 KH 2 PO 4 , and 8.0 Na 2 HPO 4 , at pH 7.4, supplemented with 0.05% [wt/vol] trypsin and 0.02% [wt/vol] EDTA). Endothelial cells were seeded at a density of 7 × 10 4 cells/cm 2 on either 24-mm round polycarbonate filters (pore size 0.4 μm) or 20-mm round glass coverslips for determination of albumin flux and immunostaining, respectively, and were cultured in basal culture medium (for compositions, see above). Experiments were performed with confluent monolayers, 4 d after seeding. The purity of these cultures was >99% endothelial cells as determined by uptake of DiI-ac-LDL, contrasted with <1% cells positive for α−smooth muscle actin. The permeability of the endothelial cell monolayer was studied in a two-compartment system separated by a filter membrane ( 24 , 28 ). Both compartments contained as basal medium modified Tyrode's solution (composition in mM: 150 NaCl, 2.7 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 1.0 CaCl 2 , and 30.0 N -2-hydroxyethylpiperazine- N ′-2-ethanesulfonic acid; pH 7.4, 37°C) supplemented with 2% (vol/vol) NCS. There was no hydrostatic pressure gradient between the two compartments. The “luminal” compartment containing the monolayer had a volume of 2.5 ml, and the “abluminal” had a volume of 6.5 ml. The fluid in the abluminal compartment was constantly stirred. Trypan blue–labeled albumin (60 μM) was added to the luminal compartment. The appearance of the labeled albumin in the abluminal compartment was continuously monitored by pumping the liquid through a spectrophotometer (Specord 10; Carl Zeiss ). Increases of the concentration of labeled albumin were detected with a time delay of <15 s. The concentration of labeled albumin in the luminal compartment was determined every 10 min of incubation. It did not change significantly in the time frame of the experiments. The albumin flux ( F , expressed as mol/[s × cm 2 ]) across the monolayer with the surface ( S ) was determined from the rise of albumin concentration (d[A] 2 ) during the time interval (d t ) in the abluminal compartment (volume V ): \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{F}}=(d[A]_{2})/d{\mathit{t}}{\times}{\mathit{V}})/{\mathit{S}}.\end{equation*}\end{document} To facilitate the comparison of data obtained in this study with those of other studies, the permeability coefficient ( P , expressed as cm/s) of the combined system of monolayer and filter support was calculated from F according to Fick's law of diffusion as follows: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathit{P}}={\mathit{F}}/([A]_{1}-[A]_{2})\end{equation*}\end{document} where [A] 1 and [A] 2 denote tracer concentrations in the luminal and abluminal compartments, respectively. Because the driving force ([A] 1 − [A] 2 ) remained virtually unchanged in the course of the described experiments, the relative changes in F correspond to similar changes in the permeability coefficient. The basal medium used in incubations was modified Tyrode's solution (see above). Macromolecule permeability of the endothelial monolayer, transferred to the incubation chamber, was determined after an initial equilibration period of 20 min. The basal albumin permeability of each monolayer filter system was then determined for another 20 min of incubation. Agents were added as indicated, and the response of the albumin permeability was recorded for an additional 80 min. In a set of experiments, endothelial monolayers were preincubated in basal medium (for composition, see above) supplemented with thrombin-activated factor XIII A (1 U/ml) at 37°C in a cell culture incubator for 2, 4, and 6 h. The endothelial monolayers were then transferred to the incubation chamber, and albumin permeability of these pretreated monolayers was determined after an initial equilibration period of 20 min. Hearts from 250-g male Wistar rats were mounted immediately after isolation on a Langendorff perfusion system in a temperature-controlled chamber (37°C), as described previously ( 29 ). During normoxic perfusion, the chamber was flushed with humidified air, and during anoxic perfusion, with a 95% N 2 (vol/vol)/5% CO 2 (vol/vol) mixture. Under normoxic conditions, the hearts were perfused at a constant flow of 10 ml/ min with an oxygenated saline medium (composition in mM: 140.0 NaCl, 24.0 NaHCO 3 , 2.7 KCl, 0.4 KH 2 PO 4 , 1 MgSO 4 , 1.8 CaCl 2 , 5 glucose, pH 7.4; gassed with 95% O 2 [vol/vol]/5% CO 2 [vol/ vol]). For low-flow ischemia, this normoxic period was followed by 40 min anoxic perfusion at 0.5 ml/min (composition of the perfusion medium as above; pH 7.4; gassed with 95% N 2 [vol/vol]/5% CO 2 [vol/vol]). After low-flow ischemia, hearts were again resupplied with oxygen by returning to the initial perfusion conditions. Factor XIII A was added to the perfusion medium 5 min before the onset of low-flow ischemia. It remained in the perfusion medium during the entire period of low-flow ischemia and reperfusion. Activation of the plasma factor XIII and factor XIII A was performed by incubations of known amounts of factor XIII in the presence of sepharose-coupled thrombin at 37°C in Tris buffer (200 mM, pH 7.4) for 20 min. The activated factor XIII was then separated from thrombin-sepharose by centrifugation. The contamination with thrombin of these supernatants was below detection limits. Factor XIII activity was determined by using the assay described by Fickenscher et al. ( 30 ) without thrombin in the assay. Factor XIII A was inactivated using the alkylating agent iodoacetamide as described by Curtis et al. ( 31 ). To inactivate factor XIII, aliquots of the thrombin-activated factor XIII A containing ∼12 μM (corresponding to 1 mg protein/ml) were incubated in the presence of 24 μM iodoacetamide at 37°C for 10 min. 48 μM glutathione was then added to react with the residual amounts of iodoacetamide, and incubations were continued for 5 min at room temperature. After this procedure, the activity of factor XIII A was below detection limits. Aliquots of the inactivated factor XIII A (∼10 μg protein equivalent to 1 U factor XIII A) were added to the cells. The final concentrations of iodoacetamide and glutathione were 0.24 and 0.48 μM, respectively. At those concentrations, neither substance affected basal permeability of the endothelial monolayers. Confluent endothelial monolayers were washed three times with PBS, then fixed with 5% paraformaldehyde for 10 min at 20°C, and washed again three times with PBS. The cells were covered with 100 μl polyclonal rabbit anti-factor XIII A or anti-factor XIII B antibodies (diluted 1:200 in PBS), and incubated for 6 h at 37°C. The coverslips were then washed three times with PBS, covered with 100 μl of mouse anti–rabbit IgG coupled to FITC (diluted 1:100 in PBS), and incubated for 6 h at 37°C. The coverslips were finally embedded in a 40% glycerol/PBS solution (pH 8.5) on glass slides. Cell monolayers were visualized using an inverse fluorescence microscope (model IX 70; Olympus ). After permeability experiments, confluent endothelial monolayers on filter membranes were washed three times with PBS, and fixed with 5% paraformaldehyde for 10 min at 20°C as described for immunofluorescence microscopy. The cells were covered with 100 μl polyclonal rabbit anti-factor XIII A or anti-factor XIII B antibodies (diluted 1:200 in PBS), and incubated overnight at room temperature. The filters were then washed three times with PBS, covered by 100 μl of donkey anti– rabbit IgG coupled to peroxidase (diluted 1:150 in PBS), and incubated at room temperature for 1 h. The filters were washed twice with PBS and twice with Tris-HCl (10 mM, pH 7.4) and then incubated with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide as substrates for the peroxidase reaction in the presence of nickel ammonium sulfite for 45 min. The filters were then washed again three times with Tris-HCl and exposed to a 1% solution of OsO 4 at 4°C for 1 h. After washing twice with Tris-HCl and twice with maleate buffer (pH 5.2), the specimens were incubated in a 1% uranyl acetate solution in maleate buffer in the dark at room temperature for 1 h. Subsequently, the specimens were washed again three times with maleate buffer, dehydrated in 70% ethanol, and transferred to 2,2′-dimethoxypropan, followed by embedding in spurr resin. Polymerization of the embedded specimens was performed at 60–70°C overnight. Ultrathin cross-sections of the monolayers were cut, stained with lead citrate, and viewed with a transmission electron microscope (model EM 902; Carl Zeiss ). Data are given as means ± SD of n = 6 experiments using independent cell preparations. Statistical analysis of data was performed according to Student's unpaired t test. Probability ( P ) values <0.05 were considered significant. Donkey anti–rabbit IgG coupled to peroxidase was from Amersham Buchler ; Falcon plastic tissue culture dishes were from Becton Dickinson ; polyclonal anti-factor XIII A antibody, and polyclonal anti-factor XIII B antibody DADE were from Behring Diagnostics; glutathione was from Boehringer Mannheim ; plasma factor XIII and isolated factor XIII B subunit purified from Fibrogammin HS™, factor XIII A subunit (recombinant human factor XIII expressed in yeast and purified to homogeneity [impurities <100 ppm]), and human thrombin were from Centeon Pharma GmbH; Transwell ® polycarbonate filter inserts (24-mm diameter, 0.4-μm pore size) were from Costar; NCS), medium 199, penicillin-streptomycin, and trypsin-EDTA were from GIBCO Life Technologies; DAB (ISOPAC™) and DiI-ac-LDL (acetylated low-density lipoprotein labeled with 1,1′-dioctadecyl-1-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate) were from Paesel & Lorei; spurr resin was from Serva; anti–rabbit IgG coupled to peroxidase or FITC, and iodoacetamide were from Sigma . All other chemicals were of the best available quality, usually analytical grade. It was tested initially whether the activity of factor XIII added to endothelial monolayers is changed throughout the time course of a permeability experiment. The following additions to the luminal compartment of the incubation chambers were made: thrombin-activated or nonactivated plasma factor XIII, and thrombin-activated or nonactivated factor XIII A subunit. As shown in Fig. 1 , the measured activities remained stable during the entire experimental period. Macromolecule permeability of endothelial monolayers was continuously monitored by determining the flux of albumin across the monolayers. Under control conditions, mean permeability was 5.9 ± 0.6 × 10 −6 cm/s . It remained constant during the entire period of observation. Addition of the thrombin-activated plasma factor XIII (1 U/ml) caused a rapid decrease of albumin permeability, which was reduced by 30% after 20 min. In contrast to the activated plasma factor XIII, addition of the nonactivated plasma factor XIII had no effect on permeability. Exposure of endothelial monolayers to the thrombin- activated factor XIII A subunit (10 μg/ml, equivalent to ∼1 U/ml) also led to a rapid reduction of permeability, by 34% within 20 min . The nonactivated factor XIII A (10 μg/ml) as well as additions of the iodoacetamide-inactivated factor XIII A (10 μg/ml) had no effect on permeability. Likewise, the isolated factor XIII B subunit (10 μg/ml) did not affect the albumin permeability of the endothelial monolayers . In a set of experiments, it was tested whether the activated factor XIII A can affect albumin permeability of endothelial monolayers for a prolonged period of time. For that reason, endothelial monolayers were preincubated with thrombin-activated factor XIII A for 2, 4, and 6 h. Albumin permeability was then determined. As shown in Table I , the reduction of albumin permeability induced by the activated factor XIII A persists for 6 h. The activated factor XIII A reduced albumin permeability with increasing activity , with half-maximal effect at 0.9 U/ml. In contrast, the nonactivated factor XIII A as well as factor XIII B had no significant effect on albumin permeability when applied in the same range of protein concentration. For immunostaining, a polyclonal rabbit anti-factor XIII A antibody was used which recognizes the activated as well as the nonactivated factor XIII A ( 32 ). Immunostaining of endothelial monolayers incubated for 20 min in the presence of thrombin-activated factor XIII A (1 U/ml) revealed factor XIII A–positive staining along the interface of adjacent endothelial cells . In monolayers that were exposed to nonactivated factor XIII A at equivalent protein concentration (10 μg protein/ml), immunostaining for factor XIII A remained absent . As control, endothelial monolayers that had not been incubated in the presence of factor XIII A were exposed to either the first anti-factor XIII A and second antibody or the second antibody alone . No specific staining was observed with these protocols. In a second set of experiments, endothelial monolayers were incubated in the presence of factor XIII B (10 μg protein/ml) which had been preexposed or not to thrombin. For immunohistochemistry, a specific polyclonal antibody raised against factor XIII B ( 32 ) was used, which we confirmed to stain isolated factor XIII B (not shown). No specific staining for factor XIII B was detected in the monolayers . To analyze the localization of factor XIII A in cross-sections of endothelial monolayers in greater detail, these were incubated for 20 min in the presence or absence of thrombin-activated or nonactivated factor XIII A. The endothelial monolayers were then processed for transmission electron microscopy. When activated factor XIII A had been applied, factor XIII A immunoreactivity was identified by the accumulation of an electron-dense DAB reaction product at the intercellular cleft and of the basal endothelial surface along the margin of the cells . In contrast, no DAB reaction product was observed in intercellular clefts of control monolayers or in endothelial monolayers exposed to nonactivated factor XIII A . As shown in previous studies from our laboratory ( 28 , 33 ), metabolic inhibition (MI) of mitochondrial and glycolytic energy production causes a rapid rise in macromolecule permeability. In the present study, it was tested whether the activated factor XIII A can attenuate the hyperpermeability in energy-depleted endothelial monolayers. Addition of 1 mM KCN (inhibitor of mitochondrial respiration) plus 1 mM 2-deoxy- d -glucose (2-DG, inhibitor of glycolytic ATP production) caused an increase in permeability by 23% within 10 min . Exposure of endothelial monolayers to 1 U/ml of activated factor XIII A led to a 30% reduction of permeability. In the presence of activated factor XIII A, addition of the metabolic inhibitors no longer caused an increase in permeability. The level of permeability remained even as low as that obtained by addition of the activated factor XIII A before MI. In immunomicroscopy, the staining of factor XIII A at cell–cell interfaces was enhanced when the monolayers were exposed to metabolic inhibitors . As can be seen by comparison of immunostaining and phase–contrast images of the same section, the enlarged zones of factor XIII A–positive staining correspond to gaps opening between adjacent cells. To analyze whether the activated factor XIII A can also affect endothelial barrier function in the coronary system, the isolated perfused heart was used and changes of myocardial water content were determined. Under control conditions, the myocardial water content of the normoxic perfused rat heart was, on average, 430 ml/100 g dry wt over a period of 160 min of observation . To provoke an increase in vascular permeability, hearts were exposed to a 40-min period of low-flow ischemia followed by a period of 60 min of normoxic reperfusion. Ischemia-reperfusion experiments were performed with addition of either the nonactivated or the activated factor XIII A 5 min before onset of anoxic low-flow perfusion. With the nonactivated factor XIII A, the water content of reperfused hearts rose to 530 ml/100 g dry wt. In the presence of the activated factor XIII A (5 U/ml), myocardial water content remained as it was before reperfusion. The central question of this study was whether factor XIII can directly influence endothelial barrier function. In the model of cultured endothelial monolayers, we found that activated factor XIII not only lowers the basal permeability for macromolecules but also prevents the increase in permeability provoked by an inhibition of endothelial energy production. In the isolated whole heart, activated factor XIII was able to prevent edema formation caused by ischemia-reperfusion. The endothelial effects of factor XIII are exerted only by the activated form of the A subunit. Confluent monolayers of cultured porcine aortic cells were used as a model ( 24 , 28 , 33 ). To characterize the barrier of these monolayers towards macromolecules, the passage of albumin across the monolayers was studied. Changes in macromolecule permeability in this model are attributed to changes in paracellular permeability ( 25 ). The basal level of permeability in this model is not the lowest possible, and can therefore be used to investigate factors improving endothelial barrier function without prior stimulation ( 23 , 34 ). The nonactivated plasma factor XIII did not affect permeability of the monolayers. However, when activated by exposure to sepharose-coupled thrombin, plasma factor XIII markedly lowered the permeability. To analyze which part of the heterodimeric complex is responsible for this effect, a recombinant A subunit and a purified B subunit of factor XIII were applied in the permeability experiments. The A subunit was equipotent to plasma factor XIII when activated by exposure to thrombin. The lowering effect on permeability of the factor XIII A was dependent on its enzymatic activity. If factor XIII A was inactivated by the alkylating agent iodoacetamide, it no longer reduced permeability. The B subunit had no effect. The results thus show that the activated A subunit of factor XIII represents the active principle of the permeability-lowering effect. Active factor XIII is a transglutaminase capable of cross-linking various types of proteins ( 2 ) and is entrapped in the stable protein meshwork formed. With immunomicroscopy, we found factor XIII deposited at the endothelial monolayer under exactly those conditions where factor XIII reduced monolayer permeability, i.e., when the activated A subunit was present. Immunoreactivity of factor XIII A was localized under these circumstances along the interfaces of adjacent endothelial cells. Electron microscopy revealed that it was concentrated in the narrow gaps between adjacent cells and at the basal endothelial surface between the cells and the filter support. Mass deposition of factor XIII A was not found at any other site within the endothelial monolayers. The B subunit did not form depositions on the monolayer when applied. There are a variety of proteins like fibronectin and vitronectin residing in the intercellular clefts and the subendothelial matrix which are involved in cell-to-cell and cell-to-matrix adhesion of endothelial cells and which represent substrates for factor XIII cross-linking reactions ( 12 , 14 ). Interestingly, the small intercellular clefts represent the principle paracellular pathway for passage of macromolecules in these monolayers. Therefore, the microscopic observations suggest that active factor XIII A reduces monolayer permeability because it reacts with extracellular matrix proteins at these strategic sites of the endothelial barrier. In doing so it may itself become entrapped, as in fibrin clots. We showed previously, using the same experimental model, that energy depletion of endothelial cells causes a rapid rise in monolayer permeability ( 28 , 33 ). This rise in permeability is associated with a widening of intercellular gaps. We find now that in the presence of active factor XIII A, the rise in permeability is abolished even though the energy-depleted cells in the monolayer remain retracted from each other. The latter observation indicates that factor XIII does not prevent the immediate structural consequences of energy loss within endothelial monolayers. The explanation for the protective effect of factor XIII seems to lie in another finding, that the intercellular gaps contain massive depositions of factor XIII immunoreactivity. This finding is consistent with the above hypothesis that factor XIII reduces monolayer permeability by cross-linking of proteins at the paracellular passageways. To study whether the activated factor XIII A can affect endothelial barrier function in an intact coronary system, saline-perfused rat hearts were used as a model. Low-flow ischemia and subsequent reperfusion caused a marked increase in myocardial water content, as also reported by others ( 26 ). When the perfusion medium was supplemented with the activated factor XIII A before onset of low-flow ischemia, an increase in myocardial water content did not occur. These data show that the activated factor XIII A can prevent development of hyperpermeability in this perfused heart model. This study has revealed a new function of factor XIII, i.e., stabilization of endothelial barrier function. It shows that this function is due to a direct effect on the endothelial monolayer. The observations in microscopy indicate that factor XIII can reduce the permeability through an endothelial monolayer by interactions with proteins of the extracellular matrix between cells. As the permeability-lowering effect is restricted to the active form of factor XIII, which acts as an enzymatic cross-linker of proteins, this effect seems to be due to narrowing of the sieving meshwork in the paracellular transendothelial passageways. The experiments on energy-depleted monolayers and ischemic-reperfused hearts indicate that the active factor XIII can be used to prevent edema formation caused by endothelial metabolic disturbances. | Study | biomedical | en | 0.999997 |
10224278 | Grf40 cDNA fragments were isolated by a yeast two-hybrid screen of a human PHA-PBL cDNA library ( Clontech ). The bait plasmid was constructed by insertion of a cDNA fragment encoding the full-length human AMSH protein (our unpublished protocol) in pAS2-1 ( Clontech ). The bait plasmid was transformed into the yeast strain CG1945 ( Clontech ), followed by transformation with the human PHA-PBL cDNA library. The transformed strains were selected on dropout plates (Trp − , Leu − , His − ) with 5 mM 3-aminotriazole. Positive colonies were subsequently tested for the expression of lacZ. One clone was determined to contain a homologous sequence to Grb2 ( 7 ). To obtain the full-length cDNA, the 800-bp fragment of the above clone was used as a probe for screening a λgt11 oligo(dT)-primed cDNA library of PHA-PBL. The sequences of 10 clones were identical, and two of them contained an open reading frame coding for 330 amino acids. This sequence contains an in-frame stop codon at 114 bp upstream of the first methionine codon, and the sequence around the first methionine (nucleotides 193–195) matches the favorable Kozak consensus sequence. A full-length cDNA encoding Grf40 was thus isolated. Grf40 cDNA was generated by PCR using the above full-length cDNA clone as a template and subcloned into Myc-Tag-pcDNA3.1(+) to generate the plasmid sequence (EQKLISEEDL). pMycGrf40-dSH3N, pMycGrf40-dSH2, pMycGrf40-dSH3C, and pMycGrf40-dSH3NC are Myc-tagged Grf40 mutants deleted of the SH3 domain of the NH 2 terminus (amino acid position Met 1 –Pro 56 ), deleted of the SH2 domain (amino acid position Lys 57 –Thr 149 ), of the SH3 domain of the COOH terminus (amino acid position Ala 278 –Arg 330 ), and of the SH3 domains of the NH 2 and COOH termini (amino acid positions Met 1 –Pro 56 and Ala 278 –Arg 330 ), respectively. pMycGrb2 and pMycGrb2-dSH2 are expression plasmids for the Myc-tagged wild-type Grb2 and Grb2 mutant deleted of the SH2 domain (amino acid position Trp 60 –Glu 152 ), respectively. SLP-76 cDNA was generated by PCR and subcloned into pFLAG-CMV-2 ( Eastman Kodak Co. ) to generate the plasmid pFlagSLP, possessing an NH 2 -terminal Flag epitope tag (DYKDDDDK). pFlagSLP-157-533, pFlagSLP-217-533, pFlagSLP-241-533, and pFlagSLP-281-533 are expression plasmids for Flag-tagged SLP-76 mutants deleted of amino acid positions Met 1 –Leu 156 , Met 1 –His 216 , Met 1 –Lys 240 , and Met 1 –Pro 280 , respectively. pCX-SLP76 is an expression plasmid for the wild-type SLP-76 cloned into the pCXN2 vector ( 19 ). Luciferase reporter constructs were as follows: pNFATLuc was constructed by insertion of three tandem copies of the NF-AT binding region (−286 to −249 of human IL-2) linked to the human IL-2 promoter (−64 to +47) into the pGL3-basic vector ( Promega ) ( 20 ); pIL2Luc was constructed by insertion of the human IL-2 promoter (−541 to +57) into the pGL3-basic vector ( 21 ). pENL is a β-galactosidase expression plasmid ( 22 ). All constructs were sequenced for verification with a DNA sequencer (model 377; Applied Biosystems, Inc.). Cell lines used were human T cell lines, Jurkat and MOLT-4; human B cell lines, Daudi, Raji, and Ramos; a human monocytic cell line, THP-1; a human eosinophilic cell line, Eol-3; a human GM-CSF–responsive cell line, TF-1; a human lung fibroblastic cell line, WI-26; a human epithelial cell line, HeLa; and an SV40-transformed monkey kidney cell line, COS7. TF-1 was maintained in RPMI 1640 medium supplemented with 10% FCS and recombinant GM-CSF. WI-26 and COS7 were maintained in DME supplemented with 5% FCS. Other cell lines were maintained in RPMI 1640 supplemented with 10% FCS. The following Abs were used in this study: anti-CD3ε mAb OKT3 (American Type Culture Collection); antiphosphotyrosine mAb 4G10, anti-Myc polyclonal Ab and anti-LAT Ab (Upstate Biotechnology); antiphosphotyrosine mAb PY-20 (ICN Biomedicals); anti-Myc mAb (9E10) and anti-Grb2 Ab (sc-255) ( Santa Cruz Biotechnology ); anti-Flag mAb (M2; Eastman Kodak Co. ). Anti-Grf40 rabbit antiserum was prepared by immunization with a peptide (Val 174 –Pro 194 ) of human Grf40. Anti–SLP-76 rabbit antiserum was prepared by immunization with a peptide (Gly 302 –Glu 321 ) of human SLP-76. Anti-B19 human parvovirus mAb (Par1) was used as a control mAb. Immunoprecipitation and immunoblotting were carried out as described previously ( 23 ). In brief, cells were lysed with a cell extraction buffer (1% NP-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM Na 3 VO 4 , 2 mM PMSF, and 20 μg/ml aprotinin), and immunoprecipitated with the indicated Abs or antisera. The immunoprecipitates were separated by SDS-PAGE and then transferred to polyvinylidene difluoride filters ( Millipore ). After incubation in PBS containing 2% BSA and 0.1% Tween 20, the filters were probed with the indicated Abs and visualized using the ECL detection system ( Amersham Pharmacia Biotech ). Northern blot analyses were performed as described previously ( 24 ). In brief, a Multiple Tissue Northern blot containing poly(A) + RNA preparations derived from various human tissues was purchased ( Clontech ). They were probed with radiolabeled cDNA fragments of Grf40 and β-actin. Signals were analyzed with a Bio-Image Analyzer, BAS 1500 (Fuji Film and Photo, Inc.). COS7 cells were electroporated with the indicated plasmids in OPTI-MEM I ( GIBCO BRL ) at a density of 6 × 10 6 cells/700 μl/cuvette with a gene pulser (Bio-Rad Laboratories) set at 1,000 V and 200 μF, and then subjected to a Western blot assay 48 h after the transfection. For luciferase assays, Jurkat cells were electroporated with 2.5 μg of pENL and the indicated dose of pIL2Luc or pNFATLuc, along with expression plasmids for SLP-76, and Grf40 or Grb2 in OPTI-MEM I at a density of 5 × 10 6 cells/400 μl at 200 V and 950 μF. The cells were cultured at 37°C for 24 h, and then stimulated for 8 h with 10 μg/ml OKT3 plus 50 ng/ml PMA or with 10 μg/ml OKT3 alone. The cells were then lysed in 300 μl of PicaGene ReporterLysis Buffer (Toyo Ink) and assayed for luciferase and β-galactosidase activities as described previously ( 25 ). We previously reported a signal transducing adaptor molecule, STAM, which is associated with Janus kinase (Jak)2 and Jak3 and is involved in signal transduction mediated by IL-2 and GM-CSF ( 26 ). We have also recently cloned a cDNA clone encoding a novel molecule, named AMSH, which binds to STAM (our unpublished results). To address the functional significance of AMSH, we attempted to identify molecules associated with AMSH using the yeast two-hybrid assay system. One full-length cDNA clone was isolated from a human PHA-PBL cDNA library. The cDNA clone encodes a molecule homologous to Grb2, named Grf40 (for Grb2 family member of 40 kD). The nucleotide sequence of the Grf40 gene has been deposited with GenBank, and is available from EMBL/GenBank/DDBJ under accession no. AF042380 . The deduced amino acid sequence of Grf40 consists of 330 amino acid residues. The schematic structure of Grf40 was compared with Grb2 ( 7 ) and Grap, another Grb2 family member ( 27 , 28 ). The NH 2 - and COOH-terminal SH3 domains and an intermediate SH2 domain of Grf40 are highly homologous to those of Grb2 and Grap, while a unique insert region (amino acid position Arg 156 –Arg 277 ) containing proline/ glutamine-rich sequences was seen in Grf40 but not Grb2 and Grap . These results indicate that Grf40 is a new member of the Grb2 family. Various human cell lines were examined for expression of Grf40 by immunoblotting with anti-Grf40 Ab. Two T cell lines, MOLT-4 and Jurkat, were strongly positive for expression of the 40-kD Grf40, and two B cell lines, Daudi and Raji, were weakly positive, but the other cell lines, including a B cell line (Ramos), myeloid cell lines (THP-1, TF-1, and Eol-3), and the nonhematopoietic cell lines (HeLa and WI-26) were all negative for this expression . In contrast to Grf40, appreciable expression of Grb2 was seen in all the cell lines . Northern blot analyses on various human cell lines and tissues showed two Grf40-specific transcripts at 3.5 and 1.5 kb in Jurkat, MOLT-4, three myeloid cell lines (KU812, K562, and M-TAT) and PHA-PBL (data not shown), and in human immunotissues such as thymus, spleen, small intestine, and PBL, whereas marginal levels of the transcripts were detected in other tissues, including prostate, testis, ovary, colon, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas . These results suggest that Grf40, unlike Grb2, is predominantly expressed in immunotissues and hematopoietic cells, particularly T cells. Since Grb2 has been shown to bind to SLP-76 and LAT, which are 76- and 36/38-kD tyrosine-phosphorylated proteins essential for TCR-mediated signaling, respectively ( 10 , 11 , 18 ), we asked ourselves whether or not Grf40 is also associated with SLP-76 and LAT. We detected 76- and 36/38-kD tyrosine-phosphorylated proteins that coimmunoprecipitated with Grf40 in Jurkat cells after stimulation by TCR cross-linking with OKT3 . We then confirmed that the 76- and 36/38-kD tyrosine-phosphorylated proteins were SLP-76 and LAT, respectively, by stimulating Jurkat cells with OKT3. Their lysates were immunoprecipitated with anti-Grf40 Ab, and the immunoprecipitates were then immunoblotted with anti-LAT, anti–SLP-76, or anti-Grf40 Ab. Grf40 precipitated SLP-76 irrespective of TCR stimulation, but precipitated LAT only after TCR stimulation . These results indicate the association of Grf40 with SLP-76 and LAT in Jurkat cells. To determine the association site of Grf40 for SLP-76, we carried out further coimmunoprecipitation assays between the various deletion mutants of Grf40 and SLP-76. COS7 cells were transiently transfected with Myc-tagged wild-type Grf40 and four Grf40 mutants deleted of the NH 2 -terminal SH3 domain (Grf40-dSH3N), the COOH-terminal SH3 domain (Grf40-dSH3C), both the NH 2 - and COOH-terminal SH3 domains (Grf40-dSH3NC), or the SH2 domain (Grf40-dSH2). The transfected COS7 cells were immunoprecipitated with anti–SLP-76 Ab or anti-Myc mAb, and then immunoblotted with anti-Myc mAb or anti–SLP-76 Ab. Wild-type Grf40 and the Grf40-dSH2 and Grf40-dSH3N mutants were coimmunoprecipitated with SLP-76, but the Grf40-dSH3C and Grf40-dSH3NC mutants were not tested . Conversely, SLP-76 was coimmunoprecipitated with Grf40-dSH2, Grf40-dSH3N, and wild-type Grf40, but not with Grf40-dSH3C and Grf40-dSH3NC mutants (data not shown). These results indicate that the COOH-terminal SH3 domain of Grf40 is an association site for SLP-76. We next determined the association site of SLP-76 for Grf40 by using various SLP-76 mutants. Flag-tagged wild-type and four mutants of SLP-76 were introduced into COS7 cells together with Myc-tagged Grf40, and then immunoprecipitated and immunoblotted with anti-Flag and anti-Myc Abs. Myc-tagged Grf40 was coimmunoprecipitated with the SLP-76 mutants consisting of and containing the amino acid position Glu 217 –Pro 533 , but not with the SLP-76 mutant consisting of the amino acid position Pro 241 –Pro 533 . These results indicate that the Grf40 binding site is located in the amino acid position Glu 217 –Lys 240 of SLP-76 . This Grf40 binding site of SLP-76 almost overlaps the amino acid position Asn 224 –Asp 244 , which has been shown to be the Grb2 binding site ( 15 ). On the other hand, the binding site of Grb2 for LAT has been shown to be the SH2 domain of Grb2, which is thought to bind to the phosphorylated tyrosine residue ( 18 ). Together with this notion, we showed that LAT is tyrosine phosphorylated and subsequently coimmunoprecipitated with Grf40 after TCR stimulation, suggesting that the SH2 domain of Grf40 is possibly the binding site for LAT. Since the COOH-terminal SH3 domain of Grb2 has been shown to be the binding site for SLP-76 ( 10 , 11 ), we examined the competitive binding ability between Grf40 and Grb2 to SLP-76. COS7 cells were transiently transfected with 2.5-μg plasmids of Myc-tagged Grf40 and Myc-tagged Grb2 in association with different doses (0–1.0 μg) of Flag-tagged SLP-76 plasmid. Their lysates were immunoprecipitated with anti-Flag mAb and then immunoblotted with anti-Myc polyclonal Ab. Coimmunoprecipitation of Myc-tagged Grf40 with SLP-76 gradually decreased upon reducing the SLP-76 plasmid dose to 0.05 μg, whereas the Myc-tagged Grb2 coimmunoprecipitation with SLP-76 was detectable only at a 1.0-μg dose of SLP-76 plasmid . Expression levels of the plasmids introduced were quantified by immunoblotting, confirming that there was no significant difference in the amounts between Myc-tagged Grf40 and Myc-tagged Grb2 . These results suggest the possibility that Grf40 associated much stronger with SLP-76 than did Grb2. To confirm this further, COS7 cells were transiently transfected with low doses of SLP-76 plasmid (0.2 μg) and Myc-Grf40 plasmid (2.5 μg) together with various doses (0–10 μg) of Myc-tagged Grb2 plasmid. Their lysates were immunoprecipitated with anti-Flag mAb and then immunoblotted with anti-Myc polyclonal Ab. Even when up to 10-μg plasmid doses of Myc-tagged Grb2 were cotransfected, coimmunoprecipitation of Myc-tagged Grf40 with SLP-76 was still unchanged . Furthermore, although the increased expression of Myc-tagged Grb2 was dependent on its plasmid dose, which was considerably higher than that of Grf40 at a 10-μg plasmid dose of Myc-tagged Grb2, Myc-tagged Grb2 was not coimmunoprecipitated with SLP-76 . These results indicate that Grf40 competes with Grb2 in its binding to SLP-76, and the binding affinity of Grf40 to SLP-76 is apparently higher than that of Grb2. Since the SLP-76 mutant deleted of the Grb2 binding site, which overlaps the Grf40 binding site, failed to increase IL-2 promoter activity upon TCR stimulation ( 15 , 16 ), it is possible that not only Grb2 but also Grf40 plays a critical role in the SLP-76–dependent increase in IL-2 promoter activity. To address the functional significance of Grf40 in TCR-mediated signaling, we performed luciferase assays with reporter genes containing the IL-2 promoter and the NF-AT binding domain. Overexpression of wild-type Grf40 did not lead to either basal or TCR-mediated activation of the IL-2 promoter and NF-AT (data not shown). Since Grf40 interacts with SLP-76 in Jurkat cells, and overexpression of SLP-76 is known to augment TCR-mediated stimulation of the IL-2 promoter and NF-AT activity ( 15 , 16 ), we used Jurkat cells transiently transfected with SLP-76 to examine the effect of Grf40 in TCR-mediated signaling. Transfections of wild-type Grf40 and Grf40-dSH3N mutant into Jurkat cells overexpressing SLP-76 led to significant increases in IL-2 promoter activity upon stimulation with OKT3 plus PMA, whereas transfection of Grf40-dSH2 mutant induced a marked inhibition of IL-2 promoter activity compared with transfection of an empty vector . These results indicate that Grf40-dSH2 mutant has a dominant-negative effect in TCR stimulation, suggesting that the SH2 domain of Grf40 interacts with an essential molecule for TCR-mediated signaling, which is possibly LAT ( 18 ). Similar results were obtained in NF-AT luciferase assays with Jurkat cells stimulated with OKT3 . Furthermore, the Grf40 mutants (Grf40-dSH3C and Grf40-dSH3NC) deleted of the COOH-terminal SH3 domain, which is the binding site for SLP-76, also lost their ability to increase IL-2 promoter activity , suggesting that a Grf40–SLP-76 complex formation is required for SLP-76–dependent TCR stimulation. These results indicate that Grf40 is involved in signaling the stimulation of the IL-2 promoter and NF-AT activities mediated by OKT3 and PMA. Since Grb2 has also been considered to be involved in the modulation of TCR-mediated signal transduction ( 9 ), we compared the functional significance of Grf40 and Grb2 in TCR-mediated stimulation of IL-2 promoter activity. Jurkat cells overexpressing SLP-76 were transfected with the wild-types and SH2 deletion mutants of Grf40 and Grb2, in association with an IL-2 promoter–driven luciferase construct. They were stimulated with OKT3 plus PMA, and assayed for luciferase activity. An appreciable enhancement of IL-2 promoter activity was seen with wild-type Grf40, but scarcely with wild-type Grb2, whereas Grf40-dSH2 mutant showed a marked dominant-negative effect in the IL-2 luciferase assay compared with Grb2-dSH2 mutant . The plasmid dose dependency in the IL-2 luciferase assay was compared between the Grf40-dSH2 and Grb2-dSH2 mutants. The suppressive effects on the luciferase activities were significantly stronger in Grf40-dSH2 mutant than Grb2-dSH2 mutant at various plasmid doses . These results indicate that Grf40 is involved in TCR-mediated signaling more effectively than Grb2. This conclusion is in accordance with the observation of greater binding affinity of Grf40 to SLP-76 compared with Grb2. This study showed a critical involvement of Grf40 in the SLP-76–dependent signaling mediated by the TCR. One might consider the possibility that Grf40 mutants exert their effects in TCR-mediated signaling by altering expression levels of SLP-76. However, this possibility is negligible because the expression of pCX-SLP76 was confirmed to be unaffected by transient expression of Grf40, Grb2, and their mutants in COS7 cells (data not shown). Hence, the interaction of Grf40 with SLP-76 and LAT is thought to be critical for TCR-mediated signaling. The NH 2 -terminal SH3 domain of Grb2 is known to be a binding site for Sos, a Ras guanine nucleotide exchange factor, which has been considered to be involved in Ras activation upon TCR stimulation ( 9 ). We confirmed the association of Grb2 with Sos in Jurkat cells; however, a complex formation between Grf40 and Sos was undetectable in these cells (data not shown). Therefore, we suspect that Grf40 does not direct the Ras activation signaling mediated by the TCR. However, Grf40 contributes to TCR-mediated activation of the IL-2 promoter and NF-AT more effectively than Grb2, suggesting the critical involvement of Grf40 in TCR-mediated signaling. In this context, it is of interest that SLP-76 associated with Grf40 also binds to Vav, a Rac/Rho guanine nucleotide exchange factor, and that the interaction between SLP-76 and Vav has been shown to participate in IL-2 gene activation upon TCR stimulation ( 17 ). These observations provide a model pathway in which activated ZAP-70 tyrosine kinase after TCR ligation phosphorylates LAT ( 18 ), which then binds to the preformed Grf40–SLP-76 complex and recruits it to ZAP-70 ( 29 ), which further phosphorylates SLP-76 to be associated with Vav, leading to the downstream signaling of the TCR. Northern blot and immunoblot analyses revealed that Grf40 is expressed predominantly in immunotissues and hematopoietic cells, particularly T cells, in contrast to Grb2. Such restricted distribution of Grf40 may reflect the more efficient involvement of Grf40 in TCR-mediated signaling compared with Grb2. Although Grap has also been shown to be specific for hematopoietic and lymphocytic cells ( 28 ), the functional significance of Grap is still unknown. The genome sequence of GRB2L has been registered in GenBank/EMBL/DDBJ , which contains the entire sequence of Grf40. Since GRB2L has been mapped to human chromosome 22q12, Grf40 is thought to have the same chromosomal location. Furthermore, cDNA clones identical to Grf40 were reported as Grap2 ( 30 ), and human Gads ( 31 ) and their mouse homologues, named Mona ( 32 ) and mouse Gads ( 33 ), were also reported after the submission of this paper. Although the report regarding human Gads showed similar results as our study, they did not show any comparative study between Gads and Grb2. We here show evidence suggesting that Grf40 plays a more critical role in the TCR-mediated signaling than Grb2. | Study | biomedical | en | 0.999997 |
10224279 | PS-ODNs used in this study were synthesized by Oligo Etc. Sequences used were as previously described ( 23 ): c-myc antisense, AACGTTGAGGGGCAT, located in exon 2 of initiation site of translation; sense c-myc , ATGCCCCTCAACGTT; non-sense, AGTGGCGGAGACTCT; and scrambled, AAGCATACGGGGTGT containing a GGGG motif ( 32 ). The oligonucleotides were dissolved in 30 mM Hepes (pH 7.0). Purified mAbs to human CD8 (G10-1, IgG2a), CD16 (FC-2, IgG2b), CD20 (1F5, IgG2a), and HLA-DR (HB10a, IgG2a) were produced in our lab and used to purify human primary CD4 + T cells as previously described ( 14 ). Goat anti–mouse IgG conjugated to magnetic microbeads was purchased from Miltenyi Biotec. mAbs to human CD3 (64.1, IgG2a) and CD28 (9.3, IgG2a) were used to activate CD4 + T cells as previously described ( 14 ). Phospho–c-Myc (Thr58/Ser62) polyclonal antibody was purchased from New England Biolabs . Anti–human c-Myc mAb (9E10, IgG1), rabbit polyclonal antibody specific to NH 2 -terminal region 1–262 amino acids of c-Myc (N-262), and rabbit polyclonal anti-ERK1 (c-16) antiserum were obtained from Santa Cruz Biotechnology . PE-conjugated anti–HIV-1 p24 protein mAb was purchased from Coulter Corp. TUNEL (TdT-mediated dUTP nick-end labeling) detection kits were obtained from Boehringer Mannheim . Enriched preparations of human CD4 + T cells were isolated from peripheral blood samples from healthy, HIV-seronegative donors as follows: PBLs were obtained by centrifugation over Ficoll-Hypaque, and then E-rosette–positive (Er + ) cells were isolated as previously described ( 33 ). CD4 + T cells were obtained by negative selection of Er + cells depleting CD8 + , CD16 + , CD20 + , and HLA-DR + cells with mAb-coated beads. The purity of isolated CD4 + cells was >97% as monitored by flow cytometry. Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin, 10 mg/ml streptomycin, 1 mM pyruvate, and nonessential amino acids. HIV-1 strain Lai was prepared as previously described ( 14 ). Cells were infected with HIV-1 at a multiplicity of infection of 0.01 per cell. DNA was extracted from HIV-1– and heat-inactivated HIV-1–infected cells as previously described ( 14 ). PCR was performed as described ( 14 ) with some modifications including: 50 ng of DNA/sample for amplification of β-globin, 100 ng for LTR/LTR products, 250 ng for LTR/ gag products, and 750 ng for LTR/circle products. PCR mixtures contained 1 μM of each primer, 200 μM each of the four deoxynucleoside triphosphates, 1.5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 0.2 U Tag DNA polymerase ( GIBCO BRL ). The final volume was 50 μl. The reaction was subjected to 32 cycles (30 cycles for β-globin) of denaturation for 45 s at 94°C, annealing for 1 min at 60°C, and elongation for 2 min at 70°C. PCR products were subjected to 2% agarose gel containing 0.01 μg/ml ethidium bromide and were visualized by UV light. Primers used in this study have been described ( 14 ). After various treatments, 5 × 10 6 primary CD4 + T cells were lysed in 500 μl lysis buffer (2% NP-40, 0.5% sodium deoxycholate, 0.2% SDS, 25 mM Tris-HCl, 50 mM NaCl, 1 mM PMSF, 1 mM Na 3 VO 4 , 10 μM E-64 [trans-epoxysuccinylt- l -leucylamido (4-guanidino)-butane], 1 μg/ml pepstatin, 10 μg/ml leupeptin, and 0.1% aprotinin). After incubation on ice for 30 min the cells were sonicated. The cell lysates (equivalent to 10 6 primary CD4 + T cells) were mixed with 2× SDS loading buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 83 mM dithiothreitol, and 0.02% bromophenol blue), incubated at 100°C for 5 min, electrophoresed by 8% SDS-polyacrylamide gel, and then transferred to nitrocellulose membranes (Schreicher & Schuell). The membranes were blocked with 5% nonfat milk-TBST (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20) at 4°C overnight, followed by incubation with primary antibodies (in 5% BSA-TBST) at 4°C overnight. After washing, membranes were incubated with horseradish peroxidase–conjugated second antibodies at room temperature for 1 h. Bands on the blotted membranes were detected by incubation with enhanced chemiluminescence reagent (ECL) ( Amersham ) for 1 min and exposure to Kodak X-Omat film ( Eastman-Kodak Co. ). Apoptotic cell death followed by HIV-1 infection was detected by TUNEL according to the manufacturer's protocol ( Boehringer Mannheim ). After TUNEL staining, cells were resuspended in 100 μl PBS containing 1% BSA; PE-conjugated anti–HIV-1 p24 mAb was added and incubated at 4°C for 20 min. The cells were washed with cold PBS, suspended in 1% paraformaldehyde ( Sigma Chemical Co. ), and kept at 4°C in the dark until flow cytometry analysis by means of FACScan ® ( Becton Dickinson ). Cell cycle stages were determined by measuring DNA content with propidium iodide as previously described ( 34 ). Cell proliferation was estimated by [ 3 H]thymidine incorporation: 10 5 primary CD4 + T cells in the presence or absence of oligodeoxynucleotides were stimulated with CD3 (10 μg/ml) and CD28 (20 μg/ml) mAbs in triplicate in 96-well plates. The cells were incubated at 37°C in a 5% CO 2 incubator for 3 d. Each well was pulsed for 16 h with 0.5 μCi [ 3 H]thymidine, and then the incorporation of [ 3 H]thymidine radioactivity was monitored by a beta counter. In our previous study we found that HIV-1 nuclear import required a CSA-sensitive pathway, and that both TCR and CD28 ligation are essential for this process ( 14 ). Similarly, the expression of c-Myc in primary CD4 + T cells required costimulation with CD3 and CD28 mAbs ; neither CD3 nor CD28 ligation alone induced c-Myc expression. Time course experiments showed that c-Myc expression increased by 4 h, peaked at 24 h after costimulation, and was sustained for 48 h. Moreover, CSA inhibited c-Myc expression . Because of this correlation, we tested whether c-Myc might be a key regulator of HIV-1 DNA nuclear import in primary T cells. Since no c-Myc–specific inhibitor is yet available, we used a c-myc antisense PS-ODN to inhibit c-Myc function. By competitively inhibiting HIV-1 reverse transcriptase binding to the virus genome–cellular primer complex, PS-ODNs have an inhibitory effect on the initiation of HIV-1 reverse transcription in a sequence-independent manner ( 35 , 36 ). However, sequence-independent PS-ODNs do not exhibit any anti–HIV-1 activity once initiation of virus reverse transcription has begun ( 35 – 38 ). To avoid nonspecific anti-HIV activity of sequence-independent PS-ODNs, we first infected activated CD4 + T cells with HIV-1 for 24 h and then administrated graded doses of c-myc antisense, sense, or non-sense PS-ODNs to the infected cells. As shown in Fig. 1 B, initiation of reverse transcription (LTR/LTR product) and full-length viral DNA synthesis (LTR/gag product) were not affected by the c-myc antisense PS-ODN. However, nuclear import of HIV-1 DNA (LTR circles) was blocked by c-myc antisense PS-ODN even at doses as low as 1 μM. Doses below to 0.2 μM were less efficient at inhibiting LTR circle formation (data not shown). Neither c-myc sense nor non-sense PS-ODN had any effect on viral DNA nucleus translocation up to 8 μM . Consequently, HIV-1–infected cells treated with c-myc antisense PS-ODN did not produce p24 gag protein or undergo apoptosis . Under conditions in which HIV-1 had already entered the nucleus (e.g., at 48 h), c-myc antisense PS-ODN did block viral p24 expression (data not shown). Lack of an effect by c-myc antisense PS-ODN on full-length viral DNA synthesis was not simply because the oligonucleotides were added too late to the cultures (after 24 h infection), as full-length viral DNA was not detectable until at least 40 h after HIV infection in activated CD4 + T cells (reference 14 and data not shown). Thus, c-myc antisense PS-ODN apparently selectively acts on the stage of HIV-1 DNA nuclear import. We next studied whether c-myc antisense PS-ODN specifically inhibited full-length c-Myc protein expression. Using mAb 9E10 specific to the COOH-terminal end of c-Myc ( 39 ), we consistently observed that in the presence of c-myc antisense, sense, or non-sense PS-ODNs, the two major forms of c-Myc proteins, p64 and p67, remained relatively unchanged . However, c-myc antisense PS-ODN selectively induced the accumulation of 46- and 50-kD proteins, whose expression levels were higher than that of the full-length c-Myc. Neither the c-myc sense nor non-sense PS-ODN induced accumulation of these two proteins . These data are consistent with previous studies showing that expression of c-Myc short (c-MycS) proteins in some tumor cell lines arised from two translational initiation sites downstream of the full-length c-Myc start codon ( 40 – 45 ). These downstream-initiated c-MycS proteins lack most of the NH 2 -terminal transactivation domain; they are produced through a leaky scanning mechanism, since optimization of the traditional initiation codon for full-length c-Myc results in less synthesis of the c-MycS proteins ( 45 ). Because the c-myc antisense oligonucleotide we used corresponds to the initiation site of full-length c-Myc mRNA, and the two smaller proteins we detected are about the same size as c-MycS isoforms, it seemed likely that the 46- and 50-kD proteins are produced through the same mechanism leading to deletion of the NH 2 -terminal region. This possibility was substantiated by the fact that antibodies specific to either NH 2 -terminal phosphorylated Thr58/Ser62 or the whole NH 2 terminus region of c-Myc failed to recognize 46- and 50-kD proteins . However, both antibodies were able to recognize p64 and p67 full-length c-Myc, which did not change expression in cells treated with different PS-ODNs . The same result was obtained in a CD4 + lymphoid cell line, CEM (data not shown). Thus, the c-myc antisense oligonucleotides, but not control PS-ODNs, selectively induce NH 2 -terminally truncated c-Myc proteins that are known to act as dominant negative inhibitors by competitively suppressing full-length c-Myc functions ( 45 – 48 ). Blockage of HIV-1 DNA nuclear import by c-myc antisense PS-ODN most likely is mediated by these NH 2 -terminally truncated c-Myc proteins. Finally, we tested whether c-myc antisense PS-ODN could inhibit the entry of cell cycle and proliferation induced in primary CD4 + T cells after TCR and CD28 ligation. Treating CD4 + T cells with 6 μM of c-myc antisense oligonucleotide, which efficiently blocked HIV-1 LTR circle formation, could not inhibit cell cycle progression . Similarly, c-myc antisense, sense, and non-sense PS-ODN had no effects on CD4 + T cell proliferation induced by CD3 plus CD28 mAbs . These data are consistent with previous findings that NH 2 -terminally truncated c-Myc proteins do not interfere with cell growth ( 45 , 49 ). The study presented here reveals a novel function of c-Myc for regulation of HIV-1 nuclear import. Blocking of HIV-1 DNA nuclear import by c-myc antisense PS-ODN appeared to be mediated through the presence of 46- and 50-kD NH 2 -terminally truncated c-Myc proteins, which do not affect cell cycle progression or cell proliferation . Our data imply that the mechanism by which c-Myc controls HIV-1 DNA nuclear import is distinct from those controlling cell cycle progression. However, precisely where and how c-Myc is required for HIV-1 DNA nuclear import in proliferating CD4 + T cells remains to be discovered. NH 2 -terminal–defective c-Myc proteins are able to heterodimerize with Max, translocate to nucleus, repress gene expression, stimulate cellular proliferation, and induce cell apoptosis ( 49 ). However, c-MycS proteins are not able to activate gene transcription ( 49 ). It is probable that c-Myc regulates HIV-1 DNA nuclear import through its transactivation activity by regulation downstream gene expression. The ability of HIV-1 to infect nondividing cells, such as monocytes, terminally differentiated macrophages, mucosal dendritic cells, or γ-irradiated cells, is believed to be a unique feature since oncoretroviruses only can establish infection when the cells undergo mitosis ( 50 – 57 ). The ability of HIV-1 to infect nondividing cells is presumably related to the fact that its PIC can be recognized by the cell nuclear import machinery ( 58 – 61 ) and actively transported through nucleopores ( 62 ). Moreover, a cellular serine/threonine protein kinase, mitogen-activated protein kinase (MAPK), can associate with HIV-1 PIC to facilitate nuclear targeting of viral DNA ( 63 – 66 ). It is unclear whether HIV-1 DNA nuclear import in proliferating CD4 T cells is regulated through the identical pathway seen in nondividing cells. A reasonable possibility is that c-Myc affects the expression of genes encoding cellular proteins involved in nuclear transport. Further elucidation of the role of c-Myc in regulation of expression of cellular nuclear importing molecules might help us to understand how c-Myc regulates HIV-1 DNA nuclear import. | Study | biomedical | en | 0.999997 |
10224280 | A cDNA encoding the P230 form of BCR/ ABL was generated by a PCR strategy. A 5′ primer containing the AatII site at nucleotide 3184 in the BCR cDNA open reading frame and the adjacent e19/a2 BCR/ABL junction sequence and a 3′ primer containing the KpnI site at nucleotide 744 of the human c- ABL type Ia cDNA were used to generate a 307-bp PCR product from AatII to KpnI, spanning the P230 BCR/ABL junction. The product was subcloned and completely sequenced to verify the correct reading frame and lack of other mutations, then used to generate a complete P230 cDNA. The P190, P210, and P230 BCR/ABL cDNAs were introduced as EcoRI fragments into the unique EcoRI site of the murine stem cell virus (MSCV)- based retroviral vector, MSCVneoEB ( 34 ). Immunoprecipitation of Abl proteins from transfected 293 cells or transduced Ba/F3 cells and immune complex kinase assay was as described previously ( 19 , 35 ), except that a glutathione S -transferase (GST)-c-Crk 120–225 fusion protein was used as an exogenous substrate. To normalize for the amount of Abl proteins, cells were labeled for 12 h with [ 35 S] l -methionine before harvesting. The amount of 32 P incorporation was quantitated by PhosphorImager analysis (STORM 850; Molecular Dynamics), whereas 35 S incorporation was quantitated by digital camera and analysis by NIH Image v1.59 software. Relative protein levels, which were within 15% of one another, were corrected for methionine content and used to normalize 32 P incorporation to calculate relative kinase activity. All DNAs were purified by two rounds of buoyant density centrifugation in CsCl. 10 μg of retroviral vector DNA and 5 μg of MCV-ecopac, an ecotropic single-genome packaging construct ( 36 ), were transfected per 6-cm dish of 293T cl. 17 cells ( 37 ), as described ( 36 ). Medium was changed at 24 h, and virus supernatant was harvested at 48 h after transfection. Supernatant was passed through a 0.45-μ filter, aliquoted, and frozen at −80°C. An aliquot was thawed, the virus titer for neomycin resistance was determined by transduction of NIH 3T3 cells, and a screen for the presence of replication-competent helper virus was carried out with a lacZ -3T3 indicator cell line. All viruses had titers of 3.5–4.0 × 10 6 neomycin-resistant CFU/ml, passed the correct proviral structure by Southern blot analysis, and were free of detectable helper virus activity. 32D cl3 cells and Ba/F3 cells were grown, transduced, and selected for G418 resistance and IL-3 independence as described previously ( 19 ). At 96 h after transduction, viable cells were counted and plated at 4 × 10 4 cells in 1.0 ml RPMI medium without IL-3 and neomycin in triplicate wells of a 24-well plate. Cells were incubated at 37°C, and the viable cell count was determined daily for 3 d. All animal studies were approved by the Animal Use and Care Committees of the Center for Blood Research and Harvard Medical School. Balb/c mice (The Jackson Laboratory or Taconic Farms) from 6 to 12 wk of age were used in all experiments. In some experiments, male donor mice were primed by intravenous injection with 5-fluorouracil (5-FU; 200 mg/kg) 4 d before harvest. Male donors were killed by CO 2 asphyxiation, femur and tibia were collected, and bone marrow was harvested by flushing with syringe and 26-gauge needle. Cells were counted and plated without removal of erythrocytes at 2 × 10 7 cells per 10-cm plate in prestimulation medium ( 38 ) of DME, 15% (vol/vol) inactivated FCS, 5% (vol/vol) WEHI-3B conditioned medium, penicillin/ streptomycin, 1.0 μg/ml ciprofloxacin, 200 μM l -glutamine, 6 ng/ml recombinant murine IL-3 ( Genzyme ), 10 ng/ml recombinant murine IL-6 ( Genzyme ), and 50–100 ng/ml recombinant murine stem cell factor (SCF; PeproTech ). With non–5-FU–treated marrow, 10 ng/ml recombinant murine IL-7 ( Genzyme ) was also included. After prestimulation for 24 h at 37°C, viable cells were counted and transduced with retroviral stocks in the same medium containing 50% retroviral supernatant, 10 mM Hepes, pH 7.4, and 2 μg/ml polybrene. To increase transduction efficiency ( 39 ), virus and cells were cosedimented at 1,000 g for 90 min in a Sorvall RT-6000 centrifuge. Medium was changed after a 2–4-h adsorption period. At 48 h, a second round of transduction and cosedimentation was performed, and the cells were collected 2 h later, washed once in HBSS, and counted. Recipient female mice were prepared by two doses of 450-cGy gamma irradiation separated by 3 h. Transduced marrow cells were transplanted by injection of 0.2–0.5 × 10 6 cells (5-FU–treated marrow) or 1.0 × 10 6 cells (non–5-FU–treated marrow) in 0.4–0.5 ml HBSS into the lateral tail vein. After transplant, recipients were housed in microisolator cages supplied with acidified (pH 2.0) water. After transplant, recipient mice were evaluated daily for signs of morbidity, weight loss, failure to thrive, and splenomegaly. Premorbid animals were killed by CO 2 asphyxiation, peripheral blood was obtained from the retroorbital venous plexus, and hematopoietic tissues were removed. Depending on the individual animal, hematopoietic tissues and cells were used for several applications, including histopathology, in vitro culture, FACS ® analysis, secondary transplantation, genomic DNA preparation, protein lysate preparation, or lineage analysis (see below). The clinical features and histopathology of BCR/ ABL -induced CML-like disease, B lymphoid leukemia, and macrophage tumors were very similar to those observed previously ( 26 ). Peripheral blood samples generally served as a nearly pure source of neutrophils and their precursors; on occasion, neutrophils were purified further by positive selection for Ly-6G (Gr-1) antigen expression by immunomagnetic beads (MicroMACS ® ; Miltenyi Biotec). Macrophages were isolated from peritoneal washes, bone marrow, liver, or spleen by adherence and often subsequent culture on bacterial petri dishes in the presence of L929-conditioned medium as a source of CSF-1. Erythroid cells were purified from spleen by sedimentation through Ficoll-hypaque or by positive selection with TER119 mAb and immunomagnetic beads. B lymphoid cells were purified from pooled lymph nodes or spleen by positive selection with anti-B220 mAb, and T cells were purified by isolation of thymocytes or by positive selection of spleen or peripheral blood with anti–Thy-1.2 mAb. In all cases, the purity of the population was assessed by Wright-Giemsa staining of a cytospin specimen, and only those samples with at least 80% purity as judged from cell morphology were used for Southern blot analysis. Genomic DNA was prepared from each population, digested with BglII, and hybridized with a radioactive probe from the proviral neomycin resistance gene to determine the number of distinct proviral integrations in each sample. Subsequently, the blots were stripped and reprobed with a radioactive probe from the human c- ABL gene that detects a common 2.2-kb fragment from all proviruses, allowing determination of the total proviral content of each hematopoietic lineage. To control for differences in DNA loading, the intensity of hybridization of a fragment from the endogenous mouse c- abl gene was used as an internal control. Intensity of hybridization was determined by PhosphorImager analysis (Molecular Dynamics), and the ratio of intensity of hybridization of the BCR/ABL fragment to murine c- abl fragment was compared with that of genomic DNA standards containing a single proviral copy, with the results expressed as proviral copy number per diploid genome. A proviral copy number of 0.2 or less is consistent with the absence of provirus from a particular hematopoietic lineage. For adoptive transfer of the CML-like syndrome, 1–2 × 10 6 bone marrow and/or splenocytes from primary animals were injected intravenously into sublethally irradiated (450 cGy) female Balb/c recipient mice. For isolation of day 12 spleen colonies, pairs of lethally irradiated (900 cGy) Balb/c mice were injected intravenously with 3 × 10 3 , 1 × 10 4 , and 3 × 10 4 nucleated marrow cells from primary animals. Recipients were killed 12 d later, spleens were isolated, and macroscopic colonies were dissected out with the aid of a stereoscopic microscope. For those colonies not visibly red in appearance, cytospin preparations were examined by Wright-Giemsa staining to ensure the mixed myeloid origin of the colony. Genomic DNA was then prepared and analyzed as described above. Protein lysates were prepared from peripheral blood myeloid cells by resuspension of 10 7 cells in 75 μl RIPA buffer with immediate addition of an equal volume of 95°C 2× sample buffer and boiling for 10 min. Amounts of protein were standardized by SDS-PAGE and Coomassie blue staining. From other hematopoietic tissues and cultures, cell lysates in RIPA were quantitated by Bio-Rad protein assay. SDS-PAGE and Western blotting with anti-Abl (8E9; PharMingen ) and antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.) antibodies were performed as described ( 19 ). Previous studies indicated that P190 Bcr/Abl had intrinsically higher tyrosine kinase activity, measured as autophosphorylation or phosphorylation of an exogenous substrate, than did P210, while both were higher than c-Abl ( 18 , 19 ). In a direct comparison after immunoprecipitation from transfected 293 cells, we again confirmed this and found a significantly lower kinase activity for P230 Bcr/Abl . Corrected for levels of Abl protein, P190 had 7-fold increased phosphorylation of a GST-Crk substrate relative to c-Abl, whereas P210 and P230 had 5.4- and 3.7-fold increased activity, respectively, with similar results for autophosphorylation. There was no difference in the kinase activity of c-Abl and the oncogenic SH3-deleted c-Abl protein, as observed previously ( 40 ). Similar results were obtained when the Bcr/Abl proteins were immunoprecipitated from stably transformed Ba/F3 cells (see below; data not shown). To compare the transforming ability of the three BCR/ABL oncogenes in cultured hematopoietic cells, we introduced them by retroviral transduction into IL-3–dependent myeloid (32D cl3) and lymphoid (Ba/F3) cell lines. P210 BCR/ABL has been previously demonstrated to transform each cell line to become independent of IL-3 for survival and growth ( 41 , 42 ) by a mechanism that does not involve autocrine production of growth factors. To avoid any differences due to prolonged culture in the presence of BCR/ABL , populations of transduced cells were selected in neomycin and immediately deprived of IL-3, with measurement of cell survival and proliferation within 96 h of transduction. With both cell types, populations of cells that were independent of IL-3 for survival and growth were selected with equal efficiency after transduction with all three forms of BCR/ABL , but not with the parental MSCVneoEB virus, demonstrating that P190, P210, and P230 are all capable of transforming myeloid and lymphoid cells to cytokine independence . There was no difference in the growth rate of 32D cells transduced with any of the three forms of BCR/ABL in the absence of IL-3 or of vector-transduced cells in the presence of IL-3 . In contrast, BCR/ABL -transduced Ba/F3 cells in the absence of IL-3 proliferated more slowly than vector-transduced cells in the presence of IL-3, with P190-transduced cells showing the highest proliferation rate, followed by P210 and then P230 . The level of expression of the three Bcr/Abl proteins and the spectrum of tyrosine phosphorylated proteins were similar within each of the two cell types (data not shown). These results suggest that the three forms of BCR/ABL induce an identical proliferative response in myeloid factor-dependent cells, but deliver a submaximal growth stimulus in lymphoid cells that parallels their intrinsic tyrosine kinase activity. To compare directly the ability of the three forms of BCR/ABL to induce a myeloproliferative syndrome in mice, we used the bone marrow transduction/transplantation model system ( 26 ), with several modifications to the original protocol designed principally to increase transduction efficiency (see Materials and Methods). With the modified protocol, we observed that all mice transplanted with bone marrow transduced with each of the three forms of BCR/ABL developed a fatal CML-like myeloproliferative syndrome within 4 wk after transplantation when marrow from 5-FU–treated donors was used . The disease was characterized principally by massive expansion of maturing myeloid cells in bone marrow, spleen, liver, and peripheral blood. The peripheral blood leukocyte count at death was 2–4 × 10 5 /μl, composed predominantly of mature neutrophils, along with metamyelocytes, myelocytes, and promyelocytes, indicative of a shift towards less differentiated myeloid cells. The spleen and liver were greatly enlarged and disrupted by large numbers of maturing myeloid cells from the neutrophil lineage, along with significant erythropoiesis and increased megakaryocytes. Nearly all mice with the CML-like syndrome also had small to medium-sized collections of macrophages centered on portal areas in the liver. In addition, all animals had focal consolidation of the lungs with maturing myeloid cells and extensive intraparenchymal hemorrhage, which may have been the ultimate cause of death of these animals. Bone marrow showed increased cellularity with maturation and an increased myeloid to erythroid ratio, while lymph nodes and thymus were normal. In most cases, the CML-like syndrome could be efficiently transferred to secondary recipients by injection of bone marrow (data not shown), but as previously reported ( 43 ), for some primary animals the disease was not readily transplanted. There was no significant difference in the histopathology, average survival, peripheral blood leukocyte count, or spleen weight of the disease induced by the three forms of BCR/ABL . Because the spleen is the main reservoir of myeloid cells in these diseased mice, this implies that mice transplanted with P190-, P210-, and P230-transduced marrow have identical total myeloid cell burdens at death, indicating that the proliferative stimulus induced in myeloid cells by the three BCR/ ABL oncogenes is very similar. Although human P190 + CMLs may be characterized by monocytosis ( 13 ), the percentage of monocytes in peripheral blood of mice with CML-like disease was <10% and did not differ significantly between BCR/ABL genotypes (data not shown). Mice with the CML-like syndrome were analyzed for the presence of Bcr/Abl protein by Western blot and for the BCR/ABL provirus by Southern blot of genomic DNA. Bcr/Abl protein was detected in peripheral blood leukocytes and in peritoneal macrophages , confirming the expression of the BCR/ABL provirus in two distinct myeloid lineages. To identify the BCR/ABL provirus, genomic DNA from total spleen, liver, peripheral blood, or bone marrow was digested with XbaI (which cuts once in each proviral LTR) or BglII (which cuts once in proviral DNA sequences 3′ of the BCR/ABL cDNA insert) and probed with a radioactive fragment from the neomycin resistance gene. The XbaI digest indicated that these tissues contained the BCR/ABL provirus at about one proviral copy per cell , confirming that the increased myeloid cells in these animals were a primary part of the malignant process and not a secondary or reactive phenomenon ( 30 , 44 ). The BglII digest, which yields a distinct hybridizing fragment for each unique proviral integration site, demonstrated that multiple independent clones contributed to the myeloid cell expansion in these mice , with 4–12 clones (average 9) observed in recipients of P190-, P210-, and P230-transduced marrow. Interestingly, the relative abundance of some clones differed between DNA samples from the same animal, suggesting a variable contribution of individual clones to myelopoiesis in different tissues. The presence of polyclonal disease suggests that the presence of BCR/ABL alone is sufficient to induce the CML-like syndrome. To determine which hematopoietic lineages were involved in the malignant process, we performed lineage analysis by purifying different hematopoietic cell populations and determining the BCR/ABL proviral status by Southern blot. With all three oncogenes, mice with the CML-like syndrome carried the BCR/ABL provirus at or above single-copy levels in neutrophils, macrophages, erythroid cells, and splenic B lymphoid cells, and in some cases peripheral node lymphocytes and thymocytes . Most of the independent proviral clones present were found in each lineage. These data indicate transduction of a bone marrow target cell capable of differentiation via multiple myeloid and lymphoid pathways, suggesting that the BCR/ABL target cell for the CML-like syndrome is a multipotential progenitor cell. To further define the nature of this target cell, we performed secondary transplant experiments to isolate day 12 spleen colonies derived from selected primary mice with the CML-like syndrome. Such colonies are clonal, of mixed myeloid origin, have limited self-renewal potential, and are derived from a cell (CFU-S 12 ) with characteristics of an early multipotential progenitor with some stem cell–like properties ( 45 ). It was previously shown ( 26 ) that an animal with the CML-like syndrome carried the retroviral provirus in the majority of secondary day 12 spleen colonies, suggesting that the target cell for the CML-like syndrome was an early progenitor that could generate CFU-S 12 . In this study, we also detected the BCR/ABL provirus in the majority of day 12 spleen colonies in secondary transplants from mice with the CML-like syndrome . Collectively, in 10 primary animals with the CML-like syndrome, 64% (28 out of 44) of day 12 spleen colonies from secondary transplants carried the BCR/ABL provirus . Interestingly, of the many distinct proviral clones present in the bone marrow of the primary animals, a small subset of clones was detected frequently or exclusively in secondary day 12 spleen colonies. For example, two minor clones were found in four out of seven colonies derived from an animal with P190-induced CML-like disease , whereas a single minor clone generated seven out of eight colonies from a mouse with P210-induced disease . We observed a similar phenomenon in secondary recipients that developed the CML-like syndrome after transplantation of larger numbers of marrow cells. Instead of developing polyclonal disease with the same spectrum of proviral clones as the primary animal, myeloid cells from secondary mice with CML-like disease typically contained only a single minor clone from the donor . In one animal, the same clone that contributed exclusively to day 12 spleen colonies was also recovered from secondary mice with CML-like disease . These findings suggest that the target cell for induction of the CML-like syndrome is heterogeneous, with all clones exhibiting multilineage repopulating ability but only some clones capable of efficient generation of CFU-S 12 and secondary disease. Because animals with the CML-like syndrome die within 4 wk of transplantation, it is not possible to assess the incidence or severity of BCR/ABL -induced hematologic malignancies of longer latency or duration in this system. Multipotential myeloid target cells for BCR/ABL are enriched for by 5-FU treatment ( 46 ), whereas the target cell for Abelson virus–induced pre-B leukemia is fairly abundant in normal marrow from Balb/c mice ( 47 ). Therefore, we reasoned that the use of bone marrow from donors that had not been pretreated with 5-FU might allow us to observe the characteristics of other leukemias induced by BCR/ABL , including lymphoid leukemia. Indeed, when marrow from non–5-FU–treated donors was used for retroviral transduction, we observed three distinct hematopoietic neoplasms with each of the three forms of BCR/ABL . We observed the CML-like syndrome in about half of transplant recipients. The survival of mice with the CML-like syndrome induced by transduction of non–5-FU marrow (21–49 d) was somewhat longer than for recipients of 5-FU–treated marrow. Otherwise, the syndrome appeared pathologically identical to that observed with 5-FU–treated marrow, with increased neutrophil counts, hepatosplenomegaly, and pulmonary hemorrhage. Analysis of proviral integration in myeloid cells showed that significantly fewer (one to three) independent proviral clones contributed to the CML-like disease in these mice, compared with recipients of transduced marrow from 5-FU–treated donors . Lineage analysis of selected mice demonstrated the same proviral clone(s) in neutrophils, macrophages, erythroid cells, and B lymphoid cells (data not shown), indicating the target cell for the CML-like disease in normal marrow, like that from 5-FU– treated marrow, had multilineage differentiation potential. The other animals developed acute leukemia of B lymphoid type, or tumors of monocyte/macrophages. Animals with B lymphoid leukemia exhibited modest splenomegaly (0.2–0.4 g) and lymphadenopathy with infiltration with lymphoblasts, and a bloody pleural effusion, containing high levels of malignant lymphoid cells, that appeared to be the cause of death. The lymphoblasts expressed high levels of Bcr/Abl protein , were negative for myeloid and T lymphoid cell surface markers, but positive for CD45R (B220), CD43, 6C3/BP-1, and CD24 (data not shown), indicating an immature B cell phenotype. The lymphoid leukemias were efficiently transplanted to secondary recipients, with animals receiving 3 × 10 6 tumor cells from lymph node or pleural effusion succumbing to an identical disease within 4–5 wk. Animals with the monocyte/macrophage tumors presented with enlargement of the liver and infiltration of periportal areas with tumors of cells with large vacuolated cytoplasm; similar tumors were often found in the spleen and mesentery, and most animals had a prominent ascites composed exclusively of macrophages. The tumor cells expressed high levels of Bcr/Abl protein and were positive for the cell surface markers Mac-1 and F4/80 but negative for lymphoid markers (data not shown). Some mice exhibited characteristics of two diseases simultaneously ; because of the distinct and very uniform features of the three malignancies, it was relatively easy to recognize these animals based upon clinical features and histopathology. The survival curve of mice transplanted with P190-transduced marrow differed significantly from that of P210- or P230-transduced recipients . Principally, this was because P190 induced B lymphoid leukemia in the majority (6/8) of recipients, which rapidly lead to the death of the host. In contrast, recipients of P210- or P230-transduced marrow that developed B lymphoid leukemia (3/9 and 5/7 mice, respectively) succumbed to these diseases much later after transplantation. The longer latency associated with P210 and P230 appeared to reflect a longer time required for establishment of the disease rather than a more aggressive leukemia, because the survival of secondary recipients transplanted with 3 × 10 6 malignant lymph node cells from either P190- or P210-induced leukemia was similar, ∼4 wk (data not shown). These data confirm earlier observations on the relative potency of P190 and P210 BCR/ABL for in vitro lymphoid transformation ( 21 ), and suggest that P190 induces lymphoid leukemia with shorter latency and perhaps greater efficiency than either P210 or P230. To further characterize the lymphoid leukemias and macrophage tumors, we analyzed genomic DNA from the primary tumors (lymph node or pleural effusion cells for the lymphoid leukemias, and liver, ascites, or purified macrophages for the macrophage tumors). In addition, we performed lineage analysis and isolated secondary day 12 spleen colonies from selected mice. In contrast to the polyclonal nature of the CML-like syndrome, we generally observed only one or two independent proviral integrations in the lymphoid leukemias and macrophage tumors . In some mice with lymphoid leukemia, different clones predominated in different anatomical sites such as lymph nodes and the pleural effusion. The presence of oligo- or monoclonal disease suggests that the presence of BCR/ABL alone is insufficient to induce these diseases, and that secondary genetic or epigenetic events are required. In contrast to the CML-like syndrome, where provirus-positive cells were observed in multiple myeloid and lymphoid lineages in addition to the neutrophils, the provirus in the B lymphoid leukemias and macrophage tumors was confined to the tumor cells, and not present in cells of other hematopoietic lineages . In animals with the macrophage disease, provirus was absent from the neutrophils, a closely related myeloid lineage. In those animals diagnosed with two diseases based on clinicopathological criteria, two different proviral integrants were usually observed, restricted to the individual tumors . In addition, provirus was uniformly absent from day 12 spleen colonies derived from bone marrow of these mice; collectively, a total of 47 colonies derived from 3 primary animals with lymphoid leukemia and 3 primary animals with macrophage disease lacked the retroviral provirus . These results suggest that the target cells for these diseases are progenitor cells whose differentiation capacity is lineage restricted and cannot give rise to CFU-S 12 . In human Ph-positive leukemia, the clear association of different forms of the BCR/ABL oncogene with distinct types of leukemia begs a biological explanation. This is most evident for P190 BCR/ABL , which is only rarely if ever observed in CML. Recently, it has been suggested that the P230 form of BCR/ABL identifies a group of patients with a distinctly benign form of CML, neutrophilic CML ( 14 ). There are two models that might explain such a phenotypic correlation with different BCR/ABL genotypes. It is possible that the three forms of BCR/ABL have different intrinsic leukemogenic activities when expressed in a hematopoietic progenitor cell that has acquired a t(9; 22) translocation. Alternatively, the three oncogenes might have identical leukemogenic properties, but their expression might be largely restricted to different hematopoietic lineages because a particular chromosome 22 breakpoint is favored in a given lineage during the formation of the Ph chromosome. For example, in this model, the BCR intron 1 breakpoint might be frequent in B lymphoid progenitor cells but uncommon in hematopoietic stem cells, explaining the rarity of P190 BCR/ABL in CML. Several studies of the cell of origin of the Ph chromosome translocation appear to support the latter model. In CML patients, the Ph chromosome is present in myeloid, erythroid, megakaryocytic, and B lymphoid cells, confirming that the t(9;22) translocation took place in a very early multipotential progenitor or stem cell ( 48 ). In Ph-positive ALL, about half of adult patients have traditional BCR intron 13 or 14 breakpoints on chromosome 22 generating the P210 form of BCR/ABL . Some of these patients have persistence of the Ph chromosome in remission and carry the Ph chromosome in myeloid cells and myeloid colonies grown in vitro, suggesting they represent cases of CML presenting in blast crisis after an unrecognized chronic phase ( 6 – 8 , 49 ). In contrast, the majority of ALL patients with the BCR intron 1 breakpoint characteristic of P190 BCR/ABL do not exhibit the additional cytogenetic abnormalities typical of CML blast crisis, lack the Ph chromosome in myeloid cells, and become Ph-negative during clinical remissions, suggesting they represent transformation of a cell type that is more limited in its differentiation potential ( 50 ). However, some patients with P190-associated ALL show persistence of the Ph chromosome in remission and in myeloid cells, suggesting a multipotential target cell ( 51 – 53 ). Detailed studies of the nature of the cell of origin in patients with neutrophilic CML have not been reported. Therefore, the available clinical and molecular studies of human Ph-positive leukemia patients do not provide a biological distinction between the above two models. The purpose of this study was to characterize the kinase activity and in vitro transformation properties of the newly described P230 Bcr/Abl protein, and to compare the in vivo leukemogenic activity of the three principal forms of Bcr/ Abl to distinguish between these alternative models. We found elevated kinase activity for P230 Bcr/Abl relative to c-Abl, although the activity of P230 was reproducibly lower than that of P210. While all three forms of BCR/ABL transformed IL-3–dependent myeloid and lymphoid cell lines to IL-3 independence, they were all less effective than IL-3 at promoting the growth of Ba/F3 lymphoid cells, with the rates of proliferation directly correlating with the intrinsic tyrosine kinase activity of Bcr/Abl. The reason for this difference is not known, but it is plausible that increased tyrosine phosphorylation of critical substrates such as STAT6 by P190 might contribute to proliferation. To test the hypothesis that the three forms of BCR/ABL have different leukemogenic properties in vivo, we used the bone marrow transduction/transplantation model system. This assay allowed us to directly determine whether P190 is able to cause a CML-like syndrome and whether P230 is less potent than P210 upon transduction of an identical spectrum of primary hematopoietic cells. When the three forms of BCR/ABL were transduced into marrow from donors treated with 5-FU, they induced an identical fatal CML-like disease in recipients transplanted with the transduced cells, with no appreciable differences in histopathology, hematologic parameters, or disease latency. These observations argue that the three forms of BCR/ABL induce an identical proliferative stimulus to myeloid cells in our experimental model. Human patients with neutrophilic CML are described as having lower peripheral blood leukocyte counts, less splenomegaly, and requiring little or no myelosuppressive therapy ( 14 ), features which imply less myeloid cell proliferation and expansion relative to traditional CML. However, our results do not support the hypothesis that P230 generates a weaker proliferative stimulus in myeloid cells than P210, and suggest that the mild clinical symptoms observed in some patients with P230 BCR/ABL are due to other variables, such as genetic differences between individuals or a selection bias ( 15 – 17 ). In recipients of transduced marrow from non–5-FU–treated donors, there is a suggestion of increased latency of P210- and P230-induced CML-like disease compared with P190, which leaves open the possibility of differences in BCR/ABL -induced CML-like disease under conditions where small numbers of proviral clones and perhaps lower Bcr/Abl expression exist. Similarly, because P190 BCR/ABL can induce a CML-like syndrome in mice which is identical to that induced by P210, it is difficult to argue that the rarity of P190 in human CML reflects different leukemogenic activity of P190 in myeloid cells. Rather, our results suggest that the restriction of P190 BCR/ABL to human acute leukemias may be due to a relative lack of BCR first intron breakpoints during the generation of the Ph chromosome in stem cells. Alternatively, patients with the P190 form of BCR/ABL may develop the Ph translocation in a stem cell but manifest only a brief chronic phase. This is supported by a recent study of human P190-positive ALL, where the Ph chromosome was detected by interphase fluorescence in situ hybridization in granulocytes of five out of five patients, suggesting multilineage involvement ( 53 ). Although there was no difference between the three BCR/ABL oncogenes in induction of a CML-like disease in recipients of transduced marrow from 5-FU–treated donors, there was a highly significant difference in survival of recipients of P190-transduced marrow compared with recipients of P210- and P230-transduced marrow when donors were not pretreated with 5-FU. Under these conditions, rather than succumbing to CML-like disease, most recipients of P190-transduced marrow developed B lymphoid leukemia. Although several recipients of P210- and P230-transduced marrow also developed B lymphoid leukemia, the disease developed significantly later with P210 and P230. These results demonstrate that P190 BCR/ABL induces lymphoid leukemia in vivo with shorter latency than P210 or P230, and are in agreement with previous studies which found P190 to be more potent than P210 at transformation of B lymphoid cells in vitro ( 21 ) and induction of lymphoid leukemia after marrow transduction ( 32 ). Our data further suggest that P230 and P210 have similar lymphoid leukemogenic activity. The mechanism of the increased lymphoid leukemogenic activity of P190 relative to P210 and P230 is not known. One possibility is that the increased intrinsic tyrosine kinase activity of P190 allows elevated tyrosine phosphorylation of a substrate critical to proliferation or transformation of lymphoid cells. One candidate for such a key substrate is STAT6, whose DNA-binding activity is activated via tyrosine phosphorylation by P190 but not P210 ( 24 ). In support of this, we have observed that bone marrow from stat6 −/− mice ( 23 ) exhibits significant resistance to transformation by P190 BCR/ABL in vitro when compared with stat6 +/+ marrow (our unpublished observations). An alternative possibility is that the presence of distinct functional motifs in the Bcr portion of P210 and P230 BCR/ABL impairs lymphoid transformation by these oncogenes. The only known domain common to P210 and P230 but lacking in P190 is a homology to Cdc24Hs/Dbl, which is a member of a class of guanine nucleotide exchange factors for small G proteins of the Rac/Cdc42 family. Our system allows us to readily test the importance of this domain in lymphoid leukemogenesis. What accounts for the very different spectrum of leukemia observed when non–5-FU–treated marrow is used? Data in this report argue that the CML-like disease, B lymphoid leukemia, and macrophage disease are the consequence of BCR/ABL transduction of distinct bone marrow target cells, and the relative abundance and/or infectability of these target cells is influenced by 5-FU treatment of donors. In mice with the CML-like syndrome, multiple myeloid and lymphoid lineages carry the same spectrum of proviral clones, demonstrating that the target cell is a progenitor with both lymphoid and myeloerythroid differentiation potential. In addition, we found the BCR/ABL provirus in the majority of secondary day 12 spleen colonies derived from primary mice with the CML-like syndrome, demonstrating that some of these target cells can generate CFU-S 12 . In contrast, the bone marrow target cells for induction of BCR/ABL -induced B lymphoid leukemia and macrophage disease have restricted in vivo differentiation potential, so that provirus-positive cells do not contribute to other hematopoietic lineages; further, the provirus is never found in day 12 spleen colonies derived by secondary transplantation of marrow from these animals. These observations suggest the BCR/ABL B lymphoid leukemia target cell is a committed B lymphoid progenitor similar or identical to the Abelson virus target cell, which is known to be an immature Thy-1 lo B220 + committed lymphoid progenitor ( 54 ) that is distinct from CFU-S 12 ( 55 ). Analysis of proviral integration patterns suggests other pathogenetic differences between BCR/ABL -induced CML-like disease and B lymphoid leukemia. The CML-like disease observed in recipients of transduced marrow from 5-FU–treated donors is polyclonal, whereas the B lymphoid leukemias are mono- or oligoclonal. The short latency of the CML-like disease together with the polyclonal nature suggest that BCR/ABL alone is capable of inducing CML-like disease, whereas the longer latency and monoclonal nature of the B lymphoid leukemias imply that expression of BCR/ABL alone may be insufficient for malignant transformation of primary B lymphocytes and that additional events are required, a concept supported by many previous observations ( 20 , 56 – 59 ). However, proof of the sufficiency of BCR/ABL for induction of the CML-like disease will require limiting dilution analysis and knowledge of the efficiency of infection of the CML target cell. Interestingly, we and others ( 29 ) have found evidence for heterogeneity among the presumptive target cells for the murine CML-like syndrome. Of the many clones contributing to the expansion of myeloid cells in several primary CML animals, only one or two relatively minor clones were observed to contribute to day 12 spleen colonies and to efficiently transfer the CML-like disease in secondary transplants. These data imply two distinct target cells for the murine CML-like syndrome, both with the capacity for multilineage differentiation in the primary animal, but with different ability for repopulation of secondary recipients. These properties are reminiscent of the hierarchy of murine hematopoietic stem cells, where a distinct population of progenitor cells enriched for CFU-S 12 has transient multilineage repopulating and self-renewal potential, and a more differentiated subset lacks secondary transplantability ( 60 ). An alternative possibility is that all the CML target cells are equivalent immediately after infection, but additional genetic or epigenetic abnormalities confer transplantability on a small subset of transduced progenitors. Distinguishing between these models will require isolation of the various target cells from whole marrow. We found small to medium-sized accumulations of macrophages in the livers of nearly every animal with the CML-like syndrome. Previous studies have shown that some animals with extensive BCR/ABL -induced macrophage tumors exhibit elevated serum GM-CSF and/or G-CSF levels, and a secondary elevation in peripheral blood neutrophils that lack the retroviral provirus and are therefore not a direct part of a malignant process ( 26 , 30 , 44 ). In our study, immunoassay of plasma from several mice with primary or secondary CML-like disease revealed normal levels of GM-CSF but increased levels of IL-3 (data not shown). The biological significance of the elevation in plasma IL-3 in these animals is not clear and is under investigation. However, in contrast to mice with primary macrophage tumors, the neutrophils in all animals with the CML-like disease contain the retroviral provirus and express Bcr/Abl protein, demonstrating that this disease represents a bona fide myeloproliferative process. The lineage analysis suggests that the macrophage collections in mice with the CML-like syndrome represent the product of differentiation of the myeloid clones, as previously suggested ( 32 ). Our original goal in expressing BCR/ABL in the murine hematopoietic system by retroviral transduction ( 26 ) was to determine whether BCR/ABL was the direct cause of CML, a point which has been established beyond reasonable doubt ( 26 – 29 ). However, our initial model system was difficult to use for comparative studies because of the low efficiency and variability of induction of the CML-like disease. In this report, the mouse bone marrow transduction/transplantation system has been improved, and we have used this system for a direct comparison of the leukemogenic properties of the three principal forms of BCR/ABL . We found no significant difference in the ability of the three oncogenes to induce a CML-like myeloproliferative syndrome, but observed that P190 BCR/ ABL had increased potency for induction of B lymphoid leukemia. This model system should be very useful for studying the molecular pathophysiology of the Ph-positive leukemias, such as testing the requirement for various functional domains of Bcr/Abl, determining signaling pathways relevant to leukemogenesis, and investigating the physiological effects of BCR/ ABL expression in primary hematopoietic cells. | Study | biomedical | en | 0.999998 |
10224281 | Transgenic mice overexpressing the IL-9 gene were generated in the FVB/N background using a construct consisting of an IL-9 genomic fragment linked to the promoter of the murine pim-1 gene, including the TATA box and the cap site, followed by two copies of the Eμ enhancer and one copy of the mMLV LTR, as described previously ( 3 ). Although this construct should be preferentially expressed in the lymphoid lineage, transgenic animals express large amounts of IL-9 message in all organs, and high levels of biologically active IL-9 (± 1 μg/ml) have been detected in the serum of transgenic but not control mice. We used females of 8–12 wk of age, except in the kinetics experiment, where we used animals of 3, 6, 12, 25, or 48 wk, or older than 18 mo. Mice were maintained in a specific pathogen–free environment. Five independent transgene-positive lines (Tg5, Tg54, Tg83, Tg25, and Tg95) were obtained. For most experiments, the results obtained with the Tg5 line are shown, as these mice give results that are representative for the other IL-9 transgenic lines. Animals carrying the IL-9 transgene and deficient in IL-5 were obtained by crossing IL-5 knockout C57BL/6 mice ( 14 ) and Tg5 or Tg54 mice. F1 mice were backcrossed with IL-5–deficient mice. 50% of the F2 mice were homozygous for the targeted IL-5 gene, and 50% of these expressed the IL-9 transgene. All of the IL-9 transgenic F2 mice expressed similar IL-9 concentrations in serum (mean of 0.2 μg/ml). Normal 8-wk-old C57BL/6 mice were used for the IL-9 injection experiment. Groups of five mice (FVB/N or C57BL/6) received recombinant murine IL-9 (1 μg/d i.p.), which was produced and purified in our laboratory as described previously ( 13 ). Control groups of mice received the same volume of buffer (PBS plus 1% mouse serum) used for the cytokine injection. The treatment was performed for 3, 7, 14, or 21 consecutive days. For the LPS injection, 10 FVB and Tg5 females were injected with 50 μg i.p. LPS (Re 595 from Salmonella minnesota ; Sigma Chemical Co. ) 3 times, at 1-wk intervals, and blood samples were collected 1 wk after the last injection. Peritoneal and pleural cells were obtained by washing the cavities with 3–5 ml of NaCl 0.9% or medium containing 20 U/ml heparin (Leo Pharmaceuticals). Heparinized blood samples were centrifuged on a Ficoll layer and incubated for 5 min in 0.15 M NH 4 Cl in order to lyse the RBCs. Spleen cells were treated similarly, to remove RBCs. Double labeling of cells was performed with biotinylated rat mAbs against Mac-1 (M1/70, rat IgG1) followed by PE-conjugated streptavidin ( Becton Dickinson ) and FITC-conjugated anti-IgM (LOMM9; provided by H. Bazin, University of Louvain) or FITC-conjugated anti–Mac-1 (Cedarlane Labs., Ltd.) plus PE-conjugated anti-CD5 ( PharMingen ). Three-color analysis was also performed with FITC-conjugated anti-IgM, PE-conjugated anti-CD5, and biotinylated anti–Mac-1 followed by RED670 ( GIBCO BRL )–conjugated streptavidin. Negative controls for double labeling were cells incubated with the respective single labeling. PE-conjugated IgG 2a isotype ( PharMingen ) was used as control of specificity of CD5 staining. After staining, cells were fixed in paraformaldehyde 1.25%, and fluorescence intensity was measured on 10 4 cells/sample on a FACScan™ apparatus ( Becton Dickinson ). An IL-9–Ig fusion protein was produced as follows. The murine IL-9 cDNA was amplified by PCR using a mutated antisense primer that introduced a BclI restriction site just before the stop codon: 5′-TCGGCTGATCAGCCTTTGCATCTCTGT-3′. The region comprising the hinge, CH2, and CH3 domains of the murine IgG3 isotype heavy chain was amplified by PCR using cDNA from the IgG 3 anti-TNP hybridoma C3110 as a template with the following primers ( 15 ): 5′-AAGACTGAGTTGATCAAGAGAATCGAGCCTAGA-3′ (sense), and 5′-AATGTCTAGATGCTGTTCTCATTTACC-3′ (antisense) containing BclI and XbaI sites for cloning. After amplification, both PCR products were digested with the appropriate restriction enzymes and cloned into the pCDNA/Amp plasmid (Invitrogen). Clones with the correct insert were transiently transfected into COS7 cells, and supernatants were collected after 3 d. For FACS ® staining, cells were incubated with 10% COS cell supernatant as the first step and with PE- or FITC-labeled polyclonal anti-IgG 3 (Southern Laboratories) as the second step. To assess the specificity of the IL-9 receptor staining, we performed the labeling in the presence or absence of an excess of free IL-9 (10 3 U). Groups of 20 animals of 8–12 wk were used for measuring antibody secretion. For antigen-specific responses, 20 females were immunized with 100 μg i.p. of KLH or TNP-Ficoll in PBS buffer, and boosted 15 d later with the same dose of antigen always without adjuvant. Sera were obtained 15 d after the first immunization and 15 d after the boost. Sera were conserved at 4°C with azide (±10 mM) before antibody measurements. Antigen-specific antibody measurements were performed as described ( 16 ). Microtiter plates (Immunoplates; Nunc, Inc.) were coated with the antigen (20 μg/ml KLH [ Calbiochem -Behring Corp.] or 10 μg/ml TNP-Ficoll) in glycine (20 mM)–buffered NaCl (30 mM), pH 9.2, and incubated overnight at room temperature. After washing in 0.15 M NaCl plus Triton 0.01%, serial dilutions of samples were added and plates were incubated for 2–3 h at 37°C. After incubation, plates were washed as before, then soaked for 7 min in saline plus NP-40 1% (Fluka AG) before further incubation. Bound Ig was detected using rabbit antibodies specific for each mouse IgG subclass and by peroxidase-conjugated goat anti–rabbit IgG, following the same washing and incubation steps. The assay was developed by adding o -phenylenediamine dihydrochloride (OPD) as a substrate. Calibration was obtained by referring to the binding of equivalent affinity anti-DNP IgG 1 , IgG 2a , IgG 2b , and IgG 3 mAbs to DNP-BSA–coated plates. Spontaneous Ig concentrations were determined as described ( 17 ). Plates were coated with goat anti–rabbit IgG followed by rabbit antibodies specific for each mouse IgG subclass or for mouse IgM (provided by J.-P. Coutelier, from our laboratory) and a rat anti–mouse IgE (LO-ME-3; provided by H. Bazin). Incubations of serial dilutions of samples or standards were followed by donkey antibodies specific for mouse IgG (or a rabbit anti– mouse IgM), both conjugated to peroxidase, and for IgE determination a biotinylated rat anti–mouse IgE (LO-ME-2) plus streptavidin-peroxidase ( Sigma Chemical Co. ) was used. Assays were performed as described above for antigen-specific ELISA. Titration of rheumatoid factor (RF) 1 was performed in serum by latex agglutination as described ( 18 ). RF titers were defined as the highest serum dilution resulting in a >10% agglutination of carboxylated polystyrene particles (0.8-μm diameter; Rhône-Poulenc) coated as described with purified mAbs obtained in our laboratory: IgG 2a or IgG 1 . All sera were decomplemented before the agglutination test by heating at 56°C for 30 min and centrifuged for 5 min at 10,000 rpm. In brief, equal volumes (25 μl) of the agglutinator and a 0.05% (wt/vol) latex suspension were incubated for 30 min in a shaking water bath at 37°C. The agglutination of IgG-coated particles in this mixture was measured by the particle-counting immunoassay (PACIA), after 100× dilution, by counting the residual nonagglutinated particles. Antibromelain-treated RBC activity was measured as described ( 19 ). Purified RBCs from FVB mice were obtained by retroorbital puncture on heparin and centrifugation on Lymphoprep layer for 20 min at 1,700 rpm. RBCs were incubated as a 50% suspension with bromelain ( Sigma Chemical Co. ) at a final concentration of 20 mg/ml in PBS for 45 min at 37°C, according to the method of Cunningham ( 20 ). RBCs were washed three times, and 25 μl of a suspension of 20 × 10 6 cells/ml was incubated with the same volume of serum dilutions for 30 min at 4°C. After washing and centrifugation, the bromelain-treated RBCs were resuspended in Hank's medium (with 3% decomplemented FCS and 0.01 M azide), incubated with rat mAb anti-IgM LO-MM9 conjugated to fluorescein (5 μg/ml), and analyzed on the FACScan™ apparatus. Results are given as the mean fluorescence intensity for each sample. Antithymocyte activity was detected by incubating 20 μl of a suspension of FVB thymocytes (25 × 10 6 cells/ml) with 50 μl of diluted serum for 30 min at 4°C in microplates. After washing, the resuspended thymocytes were treated, as described for bromelain-treated RBCs, with rat mAb anti-IgM LO-MM9 (provided by H. Bazin) conjugated to fluorescein (5 μg/ml). Results are given as the mean fluorescence intensity for each sample as measured on a FACScan™ apparatus. Peritoneal cell suspensions were adjusted to 10 7 /ml with cold Hank's medium supplemented with 3% decomplemented FCS, before addition of 1/10 volume of a 10% bromelain-treated RBC suspension, as described ( 21 ). Cells were centrifuged for 5 min at 1,600 rpm, and resuspended on a roller at 4°C for 15 min. Enrichment of rosette-forming cells was performed on a discontinuous Percoll gradient composed of 2 ml of 70% solution for the lower layer and 2 ml of 30% solution for the upper layer. Cell mixtures were gently overlaid on top of the gradient. The tubes were then centrifuged for 15 min at 2,000 rpm, and the pellets containing free RBCs and rosettes were washed; RBCs were removed by NH 4 Cl treatment followed by washes with Hank's medium, and FACS ® staining was performed as described above. Statistical analyses were performed by Mann-Whitney U statistical test for unpaired values using Instat software. The spontaneous production of Ig was analyzed in the progeny of five independent transgene-positive mice. As reported previously, IL-9 transgenic animals have >1 μg/ml of circulating IL-9 in the serum, whereas IL-9 is undetectable (<100 pg/ml) in normal mice. In individuals of all transgenic lines bred under specific pathogen–free conditions, serum concentrations of IgM, IgG 1 , IgG 2a , IgG 2b , IgG 3 , and IgE were significantly increased, as shown for a representative line (Tg5) in Fig. 1 . The most prominent were a 20- and a 9-fold enhancement in IgG 1 and IgE, respectively; IgM, IgG 2a , IgG 2b , and IgG 3 levels showed a 3–4-fold increase. A similar picture was found with the other IL-9 transgenic lines. To investigate the influence of IL-9 on antibody production against foreign antigens, wild-type control FVB and IL-9 transgenic mice were immunized with TNP-Ficoll or KLH. The specific antibody response in the serum was measured by ELISA 14 d after a primary injection or 14 d after a boost injection. The result of such an immunization against TNP-Ficoll is shown in Fig. 2 . For every IgG subclass (IgG 1 , IgG 2a , IgG 2b , and IgG 3 ), IL-9 induced a modest (two- to fivefold) but significant increase in TNP-specific antibodies ( P < 0.05 in the primary response for all IgG subclasses; P < 0.002 in the secondary response, Mann-Whitney test). The results obtained with KLH were the same as with the antigen (not shown). We next addressed the possibility that the increase of spontaneous Ig secretion could be associated with a modification in B lymphocyte populations. Total cell counts and FACS ® analysis with anti-IgM antibodies were first performed with blood cells from control and Tg5 mice. As shown in Table I , peripheral blood numbers of nucleated cells were tripled in transgenics, and B cell numbers rose from 0.6 × 10 6 /ml in control mice to 4.0 × 10 6 /ml in IL-9 transgenics, indicating a significant expansion of circulating B cells. By contrast, in the spleen, the total number of cells was only mildly increased (1.5–2-fold) in IL-9 transgenic mice, without preferential upregulation of B lymphocyte numbers. This observation indicates that splenic B lymphocytes are not specifically expanded in IL-9 transgenic mice and that their number simply reflects a global spleen enlargement. In line with these data, total cell and B lymphocyte numbers were only marginally increased in mesenteric lymph nodes of IL-9 transgenic animals. To complete our analysis, we performed washouts of the peritoneal and the pleuropericardial cavities. In these compartments, we observed a 7- and 22-fold increase, respectively, in total numbers of cells in Tg5 compared with FVB control mice. Labeling of these cells with anti-IgM antibodies revealed an expansion for the B cell population in IL-9 transgenic mice, by a factor of 15 and 56 for peritoneal and pleuropericardial cells, respectively (Table I ). These observations indicate that the absolute number of B cells in the peritoneal and pleuropericardial cavities of an IL-9 transgenic mouse is equivalent to 21% of the number of splenic B cells, whereas this ratio is only 1.5% in a normal FVB mouse. Similar observations were made with three other independent IL-9 transgenic lines (data not shown). To determine in more detail the phenotype of the expanded B cell population in the peritoneal cavity of IL-9 transgenics, FACS ® analysis was performed with anti-IgM, anti–Mac-1, and anti-CD5 antibodies. The peritoneal and pleuropericardial cavities are indeed known to be the preferential locations of B-1 lymphocytes, and these three cell surface antigens allow us to discriminate the major B lymphocyte subpopulations of the peritoneal cavity ( 22 , 23 ). B-1 lymphocytes can be distinguished from conventional B cells (B-2 lymphocytes) by the expression of an intermediate level of Mac-1, a marker expressed at higher levels by macrophages. The result of FACS ® staining of peritoneal cells is shown in Fig. 3 . This confirmed the predominant expansion of IgM + cells in IL-9 transgenic mice. Interestingly, these IgM + cells expressed an intermediate level of Mac-1, thereby corresponding to the B-1 lineage. Although in normal mice the majority of peritoneal cells consisted of macrophages that express high levels of Mac-1 and no IgM, most of the IL-9 transgenic peritoneal cells consisted of B-1 lymphocytes with expression of both surface IgM and Mac-1 . Within B-1 cells, two subpopulations are defined on the basis of CD5 expression: B-1a lymphocytes are IgM + Mac-1 + CD5 + , and B-1b lymphocytes are IgM + Mac-1 + CD5 − . A three-color labeling of peritoneal cells showed that both populations of B-1 lymphocytes were expanded, but that the major increase in the peritoneal populations concerned the B-1b cells (IgM + Mac-1 + CD5 − ) in IL-9 transgenics compared with FVB control mice . Fig. 4 shows the kinetics of accumulation of peritoneal cells in two IL-9 transgenic lines (Tg5 and Tg54). At 3 wk of age, there was already a significant difference between the number of peritoneal cells found in the peritoneal cavity of transgenic mice compared with control mice ( P < 0.008, Mann-Whitney test). This difference reached five- to sevenfold after 6–12 wk, and peritoneal numbers remained stable at this level throughout the life of the individuals. Animals of >18 mo presented a similarly enlarged cell population as younger individuals (data not shown). In aging FVB control mice, peritoneal cell numbers increased slightly compared with young individuals. In common strains, the B-1 cell population constitutes a predominant fraction of the peritoneal and pleuropericardial B cell population, but is rare in spleen and lymph nodes and absent in the peripheral blood lymphocytes of adult animals. We checked the other B cell compartments for the presence of B-1 cells by double labeling, with anti-IgM and anti–Mac-1 antibodies, of cells from different locations in IL-9 transgenic and control animals. As shown in Fig. 5 , in every location where we found an increase in B cell numbers (peritoneal and pleuropericardial cavities, and blood), IgM + Mac-1 + cells were clearly expanded. This is particularly remarkable in blood, where IgM + Mac-1 + cells were completely absent in normal mice but represented up to 4.1% of total blood cells and 15% of blood B cells in IL-9 transgenic mice. In spleen, although Mac-1 staining of IL-9 transgenic B cells was too weak to clearly distinguish a positive and negative population, increased Mac-1 staining was observed in IgM + cells (mean fluorescence intensity 41 in Tg5 vs. 25 in FVB mice), suggesting the presence of Mac-1 + B cells in Tg5 spleen. The IL-9 transgenic mice used in this study show elevated levels of this cytokine before birth (Renauld, J.-C., unpublished data). To assess whether the effect of IL-9 on B-1 lymphocytes required early and prolonged overexpression, we administered, to 8-wk-old FVB mice, daily intraperitoneal injections of mouse recombinant IL-9 (1 μg/d/mouse) for 1, 3, 7, 14, or 21 d. Total counts of peritoneal cells are given in Fig. 6 . Seven injections of IL-9 were sufficient to observe a twofold increase in peritoneal cell numbers. Seven additional injections allowed a further increase to reach a plateau after 2–3 wk of daily injections ( P < 0.008, Mann-Whitney test). Double labeling with anti-IgM and anti–Mac-1 was performed to confirm B-1 cell expansion in mice that received 21 daily injections of 1 μg of IL-9. Fig. 7 shows the preferential increase of the B-1 population in treated FVB mice as well as in another strain, C57BL/6. Total numbers of IgM + Mac-1 + cells increased by a factor of 6.8 for FVB ( P = 0.016, Mann-Whitney test) and 3.3 for C57BL/6 mice ( P = 0.05). By contrast, IgM + Mac-1 − (B-2) cells were not significantly affected by IL-9. Many reports, including the analysis of IL-5 transgenic mice, point to IL-5 as a key factor for the B-1 population ( 24 – 26 ). Although IL-5 is a potent growth factor for B-1 cells, it does not seem to be indispensable, since IL-5 knockout mice show a delay in B-1 population development and a weak reduction of peritoneal B-1 cell numbers in adults ( 14 , 27 ). To assess whether the activity of IL-9 is mediated by IL-5 or whether IL-9 can contribute to the development of B-1 cells in the absence of IL-5, we crossed IL-5–deficient mice with IL-9 transgenics (Tg5 or Tg54), and F1 mice were backcrossed with IL-5–deficient mice. Thus, 50% of the F2 hybrids have one copy of the normal IL-5 gene, and 50% of the mice express the IL-9 transgene. We performed B-1 cell numbering and determination by three-color analysis of the ratio between the B-1a (IgM + Mac-1 + CD5 + ) and B-1b (IgM + Mac-1 + CD5 − ) cells in the peritoneal washouts of individuals belonging to the four groups obtained. In F2 mice having one copy of the normal IL-5 gene, overexpression of IL-9 resulted in a significant increase in the number of the B-1 population, and this expansion concerned mainly the B-1b population, as expected from the results described above . Interestingly, IL-9 had the same effect on the number of CD5 − B-1 cells in the IL-5–deficient animals . Similar results were obtained with Tg54 hybrids (not shown). In these experiments, the IL-5 deficiency resulted in a 50% decrease in B-1 numbers, but irrespectively of IL-9 expression. Taken together, these observations demonstrate that, although IL-5 plays a role in B-1 cell development, it does not mediate the activity of IL-9 on this cell type. A simple explanation for the expansion of B-1 cells by IL-9 could be a B-1–specific expression of the IL-9 receptor. To analyze IL-9 receptor expression, peritoneal cells from normal FVB mice were stained with a fusion protein consisting of IL-9 fused to the Fc fragment of mouse IgG 3 . The binding of this protein was detected by FACS ® , using a secondary antibody specific for IgG 3 . The specificity of the binding was demonstrated by its reversion in the presence of an excess of IL-9. As shown in Fig. 9 A, a fraction (∼34%) of peritoneal IgM + Mac-1 + cells were stained with this fusion protein. The labeling with anti-CD5 antibodies showed that both B-1a and B-1b populations expressed the IL-9 receptor. By contrast, the IL-9 receptor was barely detectable at the surface of IgM + B cells from the spleen . Numerous studies have demonstrated a role for B-1 cells in the secretion of autoantibodies such as anti-RBC, antithymocyte, or RF ( 28 – 30 ). In particular, IL-5 transgenic mice showed elevated levels of anti-DNA antibodies in the serum, concomitant with the increased frequency of B-1 cells ( 25 ). The best-described specificity of the autoantibodies secreted by B-1 cells is directed against an antigen presented by RBCs treated by the proteolytic enzyme bromelain. The concentrations of autoantibodies were measured in the serum of Tg5 or FVB mice that either had or had not received three in vivo injections of LPS, since it was known that this treatment upregulates autoantibody production ( 31 ). As shown in Fig. 10 , we failed to detect any difference between IL-9 transgenic and FVB control mice concerning basal levels of anti-IgG 1 or anti-IgG 2a RFs, antithymocyte antibodies, or antibromelain-treated RBC autoantibodies. As expected, LPS injection increased the production of these antibodies but with the same intensity in IL-9 transgenic mice as in normal mice. Antibromelain-treated RBC specificity is considered to be a unique characteristic of a significant proportion of B-1 cells ( 19 ). Thus, the lack of difference in anti-RBC antibody production between IL-9 transgenic and FVB mice, even after LPS induction, suggests that IL-9–induced B-1 cells have a distinct specificity compared with the classical B-1 population. To check this hypothesis, we analyzed directly the specificity of peritoneal B cells by testing their ability to form rosettes with bromelain-treated RBCs. Peritoneal cells from FVB or Tg5 mice were incubated with bromelain-treated RBCs, and rosetting cells were separated from free peritoneocytes on a Ficoll layer. The resulting RBC-specific B cells were subsequently stained with anti–Mac-1 and anti-CD5 fluorescent antibodies. The results of two independent experiments are shown in Table III . In both FVB and IL-9 transgenic mice, enrichment in RBC-specific cells was paralleled by an increase in the proportion of CD5 + Mac-1 + cells, reflecting the high frequency of anti-RBC lymphocytes in this population. In IL-9 transgenic mice, selection of anti-RBC resulted in a significant drop in the percentage of Mac-1 + CD5 − cells, indicating that this specificity is much less frequent in this population. Thus, this experiment demonstrates that IL-9–induced B-1b cells do not exhibit the same autoimmune specificity as B-1a cells. In this paper, we report that IL-9 induces a specific expansion of B-1 lymphocytes in vivo. In normal mice, this population constitutes only 1% of the total B cells and resides mainly in the peritoneal and pleuropericardial cavities. In IL-9 transgenics, we found a 15–20-fold increase in B-1 cell numbers at these locations. In addition, although B-1 lymphocytes were absent in the blood of normal animals, they represented up to 15% of blood B cells of IL-9 transgenics, based on Mac-1 and IgM expression. Another characteristic of the peritoneal B-1 population found in IL-9 transgenics is the ratio between B-1a and B-1b cells in favor of the B-1b subset, contrasting with the more balanced situation between B-1a and B-1b subpopulations found in the FVB background. In addition, the B-1b expansion in IL-9 transgenic mice might be underestimated, since B-1 cells are supposed to lose Mac-1 in the spleen ( 22 , 23 ). Therefore, additional criteria are critically needed to study the B-1b population in spleen and lymph nodes. In most models, expansion of the B-1 population is associated with high levels of autoantibody secretion. In NZB and (NZB × NZW)F1 mice, CD5 + B cells accumulate in the peritoneal cavity and in the spleen, and produce autoantibodies against single-stranded DNA, thymocytes, or bromelain-treated mouse erythrocytes ( 28 , 32 ). In another in vivo model, increased CD19 expression also resulted in an expansion of CD5 + B cells within the peritoneum and spleen, correlating with high levels of anti-DNA antibodies and RF ( 33 ). Other models resulting from mutation in the SHP1 gene ( 34 ) or targeting of genes involved in signal transduction, such as Btk ( 35 ) or vav ( 36 , 37 ), also support the link between B-1 cell development and levels of autoantibodies. In the IL-9 transgenics, we failed to detect any increase in the production of RF, antithymocyte, or antibromelain-treated RBC antibodies. In particular, antibromelain-treated RBC specificity was mainly found in peritoneal CD5 + B-1 cells, as demonstrated in the experiments with rosette-forming cells with bromelain-treated RBCs. This is in line with a previous report indicating that the antibromelain-treated RBC specificity is restricted to CD5 + cells ( 38 ). However, we cannot formally exclude that the activity of IL-9 could be selective within the B-1b population and that expanded cells derive from a subset of B-1b cells which has properties distinct from B-1b cells from normal animals. In our model, IL-9 production induced an increase in all spontaneous Ig isotypes. The most prominent were a 20- and a 9-fold enhancement in IgG 1 and IgE, respectively; IgM, IgG 2a , IgG 2b , and IgG 3 levels showed only a 3–4-fold increase. This is again different from other models with expansion of B-1 cells, such as mice homozygous for the viable mutation motheaten me v , where all B cells are CD5 + , and IgM, IgG 3 , and IgA serum concentrations are 10–50 times greater than in control mice but IgG 1 levels remain unaffected ( 39 ). This difference might also be related to the fact that B-1b cells are more expanded than B-1a cells in IL-9 transgenic mice. Although conventional B cells did not seem to express the IL-9 receptor to the same extent as B-1 cells and were not expanded in response to IL-9, we could not rule out the possibility that they also contribute to this increase in Ig production. For example, other IL-9–responsive cells, such as mast cells, T cells, or even the B-1 cells, could mediate an indirect effect of IL-9 on conventional B cells through the production of other cytokines. In this respect, B-1 cells have been reported to be an important source of IL-10 ( 40 ). However, we failed to detect any increase in IL-10 production in IL-9 transgenic mice (our unpublished data). The observation that B-1 cell numbers were increased by IL-9 in the absence of IL-5 ruled out the possibility that the effect of IL-9 is mediated by this cytokine. IL-5 is a potent activator of B-1 cells, and mice overexpressing IL-5 are also characterized by an increase in B-1 cells. However, in contrast to IL-9 transgenics, the cellular expansion exclusively concerned the B-1a population of the spleen ( 25 ). The serum IgG 1 levels were not modified, but IgM and IgA levels were increased by eight- and fourfold, respectively, in IL-5 transgenics. Surprisingly, the IL-9 receptor appeared to be expressed at similar levels in many normal B-1a and B-1b lymphocytes, whereas the B-1b population is preferentially expanded in vivo. These data suggest that distinct homeostasis mechanisms control these subpopulations. The fact that the number of CD5 + B cells is endogenously controlled has been reported, but the mechanisms remain unclear ( 41 ). In addition, despite the high level of IL-9 receptor at the surface of B-1 cells, we failed to demonstrate in vitro proliferative responses of these cells to IL-9 (our unpublished data), suggesting that the actual role of IL-9 on B-1 cells might be related to other features, such as differentiation or survival. In this report, the increase of antigen-specific Ig concentrations in TNP-Ficoll– or KLH-immunized IL-9 transgenic mice and the specific expansion of the B-1 populations suggest that these cells could actively participate in the immune response against various proteic antigens. Future experiments will be needed to determine their actual role in more physiological responses and particularly in allergic responses, in which a role for IL-9 was recently demonstrated ( 7 ). | Study | biomedical | en | 0.999997 |
10224282 | E. coli DH5α, L. pneumophila Philadelphia 1, the M. tuberculosis strains Erdman , H37Rv , and H37Ra , M. bovis , M . bovis BCG , M. phlei , M. smegmatis , and Mycobacterium avium were cultured as described ( 5 ). M . tuberculosis Erdman extracellular and intracellular glutamine synthetase was purified as described ( 5 ) or by chromatography on Affi-Gel Blue 100–200 mesh (Bio-Rad Labs.) and size fractionation on Superdex 75 ( Pharmacia Biotech, Inc. ). Active fractions were dialyzed against 15 mM imidazole, 2.2 mM MnCl 2 , pH 7, stored at 4°C, and assessed for enzyme activity by biosynthetic and transfer assay as described ( 8 ). Proteins in the active fractions were analyzed by denaturing PAGE and stained with Coomassie brilliant blue R or silver nitrate. The NH 2 -terminal sequence of the extra- and intracellular M . tuberculosis glutamine synthetase (TEKTPDD) was presented in our earlier report ( 5 ). In that report, we showed that this NH 2 terminus of active glutamine synthetase corresponds exclusively to the DNA sequence of the gln A1 gene. Although the M . tuberculosis genome contains four genes with domains homologous with other bacterial glutamine synthetases, the gln A1 gene has the highest overall homology, and it is not known whether the other genes are transcribed or expressed ( 9 ). Protein concentrations were determined by incubation with bicinchoninic acid reagent ( Pierce Chemical Co. ). The amount and activity of extracellular glutamine synthetase released by M . tuberculosis or other microorganisms over their entire growth period (16 h–6 wk) was determined by assaying aliquots of cell-free culture supernates, taken at hourly, daily, or weekly intervals, for enzyme activity by the γ-glutamyltransferase assay ( 8 ). The theoretical possibility of leakage of cytoplasmic glutamine synthetase from dead or dying M . tuberculosis cells was assessed by monitoring the activity of the cytoplasmic marker protein lactate dehydrogenase during the 6-wk growth period, both in the culture supernate and in the cell pellet, using a commercially available diagnostic kit ( Sigma Chemical Co. ). Extracellular lactate dehydrogenase activity never exceeded 0.5% of intracellular activity. Activity and inhibition profiles of M . tuberculosis Erdman, E . coli W ( Sigma Chemical Co. ), and sheep brain glutamine synthetase ( Sigma Chemical Co. ) in the presence and absence of l -methionine- S -sulfoximine and d , l -phosphinothricin (a gift from David Eisenberg, University of California at Los Angeles, Los Angeles, CA) were determined for 1 U of each enzyme in 50 mM MgCl 2 as described, using the biologically relevant biosynthetic reaction for all assays ( 8 ). K m and K i values for the three enzymes were calculated as described ( 10 , 11 ). K i values were determined by plotting reciprocal values for the reaction velocity versus substrate concentration for concentrations of the l -glutamate substrate ranging from 2.5 to 100 mM and for concentrations of the inhibitor ranging from 2 to 200 μM in the enzyme assays. K i values, determined in triplicate assays, were expressed as K i = K m × [I] / ( K m − K m(app) ). Direct analyses of cell pellets and culture supernates for glutamine synthetase activity were performed using the transfer reaction. The recombinant M . tuberculosis glutamine synthetase, which was cloned in the E . coli /mycobacterial shuttle vector pNBV-1 as a genomic DNA fragment containing the structural gene plus extensive flanking regions ( 12 ), was expressed in M . smegmatis and exported into the extracellular milieu ( 12 ). Recombinant glutamine synthetase was purified, and its enzymatic activity and inhibition profile were determined as described above for the endogenous enzymes. Broth cultures of bacteria were inoculated at a density of 1–5 × 10 5 cells/ml and grown until stationary phase was reached (overnight–6 wk). Various amounts of l -methionine- S -sulfoximine; d , l -phosphinothricin; the standard antituberculous antibiotics amikacin, ethambutol, isoniazid, pyrazinamide, and rifampin ( Sigma Chemical Co. ); or l -glutamate/ glutamine were added at time points specified in the figure legends. Viable cells in each culture were determined by removing culture aliquots, plating washed bacteria on appropriate growth media, and enumerating colonies after standard incubation periods (overnight–2 wk). THP-1 cells, a human acute monocytic leukemia line (TIB 202; ATCC), were seeded at 10 7 cells/ml, differentiated with 100 nM PMA ( Sigma Chemical Co. ), infected with freshly grown M . tuberculosis Erdman or M . avium bacteria at a multiplicity of 1 for 90 min (thereby infecting 6–11% of the monocytes, based on a bacterial count 3 h after infection), and cultured for up to 5 d in the presence of various concentrations of l -methionine- S -sulfoximine. At various time points, THP-1 cell cultures were lysed by the addition of 0.1% SDS or by vortexing, and serial dilutions of the lysates were plated on agar medium for the enumeration of viable mycobacterial colonies ( 13 ). M . tuberculosis broth cultures in 7H9 or Sauton's medium (Difco Labs.) were inoculated at a density of 1–5 × 10 5 cells/ml and grown for 6 wk until stationary phase was reached. l -methionine- S -sulfoximine was added, either at the time of inoculation (0.2 and 2 μM final concentration) or after an initial 2-wk growth period (20 and 200 μM final concentration), to duplicate cultures. A second and third set of duplicate cultures were inoculated in parallel and treated with amikacin (0.06–1 μg/ ml) or rifampin after 2 wk of cell growth. The isolation of poly- l -glutamate/glutamine and the peptidoglycan portion of the bacteria's cell walls followed a procedure described in an earlier report ( 6 ). In brief, the procedure involves (i) treatment of disrupted cells with trypsin and chymotrypsin, (ii) delipidation of cell walls by acetone and chloroform/methanol extractions, (iii) acid hydrolysis of the cell walls, and (iv) precipitation of the poly- l -glutamate/glutamine with hydrochloric acid and redissolution in sodium hydroxide, a process that is repeated several times before the final precipitate is washed with water and lyophilized to obtain a pure white powder. The pellet after the first redissolution in sodium hydroxide contains the peptidoglycan chain. Both fractions, poly- l -glutamate/glutamine and peptidoglycan, were analyzed for their amino acid content after complete acid hydrolysis. l -Methionine- S -sul f oximine and d , l -phosphinothricin are well characterized inhibitors of prokaryotic and eukaryotic glutamine synthetases ( 10 , 11 , 14 ). Preparatory to studying their effect on growth of M . tuberculosis and other mycobacteria, we characterized their effect on purified M . tuberculosis glutamine synthetase. Additionally, we compared the sensitivity of M . tuberculosis glutamine synthetase with a representative bacterial glutamine synthetase, E . coli glutamine synthetase, and a representative mammalian glutamine synthetase, sheep brain glutamine synthetase. We have previously reported the purification and characterization of glutamine synthetase from the highly virulent Erdman strain of M . tuberculosis ( 5 ). The homogeneous enzyme is made up of 12 identical M r ∼56–58,000 subunits and active in the biosynthetic assay, with an apparent K m value for l -glutamate of 2.7 ± 0.2 mM, a specific activity of 110 μmol P i /min/mg enzyme, and a turnover number of ∼70,000 (product/min/mol enzyme). Based on its molecular mass, structure, and biochemical features, the M . tuberculosis glutamine synthetase appears very similar to other bacterial glutamine synthetases ( 15 , 16 ). To investigate the effect of glutamine synthetase inhibitors on purified M . tuberculosis , E . coli , and sheep brain glutamine synthetases, we first preincubated the enzymes in l -glutamate–free reaction buffer for various times with l -methionine- S -sulfoximine and d , l -phosphinothricin at final concentrations of 2, 20, and 200 μM. We then assayed the remaining biosynthetic activity of the enzyme over a 2-h period. The prokaryotic and eukaryotic glutamine synthetases exhibited clear differences in sensitivity to the inhibitors . In the presence of both inhibitors, the prokaryotic glutamine synthetases rapidly lost their enzymatic activity; they retained <10% of initial activity at 2 and 20 μM l -methionine- S -sulfoximine and at 2 μM d , l -phosphinothricin, and they were completely inactivated (≤0.5% initial activity) at higher concentrations of the inhibitors. In contrast, sheep brain glutamine synthetase lost activity much more slowly, retained a higher proportion of initial activity throughout the incubation, and was not completely inactivated at even the highest dose of each inhibitor. For example, at inhibitor concentrations of 2 μM, the bacterial enzymes retained only 3–10% of their initial activity, whereas the sheep brain enzyme retained ∼40% of its initial activity in the presence of l -methionine- S -sulfoximine and 60% in the presence of d , l -phosphinothricin. These results thus confirm earlier reports that eukaryotic glutamine synthetases are much more resistant to these two inhibitors than bacterial enzymes ( 10 , 11 ). Inactivation of all three glutamine synthetases was strictly dependent on the presence of ATP and magnesium ions in the incubation mix. The addition of l -glutamate to the incubation mix at a final concentration of 100 mM substantially protected the three enzymes, most likely by competing with the inhibitors for the same binding site ( 10 , 11 , 14 , 17 ). In this regard, it is noteworthy that M . tuberculosis exports large amounts of ATP into the extracellular milieu; the detectable ATP concentration in a logarithmically growing culture is ∼150–170 μM ( 5 ). However, even in the presence of l -glutamate with very prolonged incubation (30, 60, and 120 min), the bacterial enzymes showed a significant decrease in activity: to ∼40–50% of the initial activity at 2 μM, ∼20% at 20 μM, and ∼7.5% at 200 μM l -methionine- S -sulfoximine and d , l -phosphinothricin. Under the same conditions, the sheep brain enzyme retained ∼75% of its initial activity at 2 μM, ∼50% at 20 μM, and ∼25% at 200 μM l -methionine- S -sulfoximine and d , l -phosphinothricin. To further compare the effect of the inhibitors on bacterial and mammalian glutamine synthetase, we determined the K m and K i values for all three glutamine synthetases for l -glutamate concentrations between 2.5 and 100 mM (Table I ). The K m results, ranging from 2.4 to 4.0 mM, confirmed our previous findings ( 5 ) and those described for the E . coli and sheep brain enzymes ( 10 , 11 ). The K i values for M . tuberculosis and E . coli glutamine synthetases in the presence of the irreversible inhibitor l -methionine- S -sulfoximine (1.1 and 1.3 μM, respectively) were two orders of magnitude lower than the K i value for sheep brain glutamine synthetase (110.5 μM). The precision of the K i values per se was limited somewhat by the variation of ∼15% around the calculated mean value for V max in the inhibition curves. However, the data still allowed a valid comparison between the bacterial and eukaryotic enzymes and confirmed that the eukaryotic enzyme is much more tolerant to the two inhibitors than the bacterial enzymes. Having determined that M. tuberculosis glutamine synthetase is sensitive to the inhibitors l -methionine- S -sulfoximine and d , l- phosphinothricin, we examined the effect of the two inhibitors on the growth of M. tuberculosis . We first added the inhibitors at final concentrations of 20, 200, and 2,000 μM to broth cultures of M. tuberculosis Erdman strain . The inhibitors had a profound effect on the growth of the organism. l- methionine- S -sulfoximine at all three concentrations blocked cell growth almost immediately, whether added at the time the culture was inoculated or 1–3 wk later. The effect of d,l -phosphinothricin was less pronounced and not sustained; the cell density of cultures treated with this inhibitor from the time the culture was inoculated lagged the uninhibited cell cultures by 0.5–1.5 log units. As l- methionine- S -sulfoximine had such a sustained and apparently irreversible effect on cell growth, we focused our subsequent investigations on this inhibitor. To determine the minimal inhibitory concentration of l -methionine- S -sulfoximine on M . tuberculosis cultures, we incubated the bacteria for 4 wk with 0, 0.2, 2, 20, and 200 μM of the inhibitor . At the highest doses of inhibitor, 20 and 200 μM, growth of M . tuberculosis was completely inhibited. At 2 μM of inhibitor, growth was strongly inhibited, reaching its plateau at ∼2 log units below that of the control cultures. At 0.2 μM of inhibitor, growth was minimally inhibited, lagging the untreated control cultures by ∼0.1–0.2 log units. The results of the studies presented thus far suggested that the inhibitory effect of l -methionine- S -sulfoximine on M . tuberculosis growth may be due, at least in part, to the inhibitor's effect on the bacterial glutamine synthetase. Studies in which the racemic inhibitors d , l -methionine- S,R -sulfoximine or l -glutamine were added to M . tuberculosis broth cultures were consistent with this hypothesis. Of the four racemic forms of the inhibitor, only l -methionine- S -sulfoximine is active against glutamine synthetase ( 14 ). A comparison of the inhibitory effect on M . tuberculosis growth of l -methionine- S -sulfoximine and the racemate d , l -methionine- S,R -sulfoximine showed that 4–5 times higher concentrations of the racemate were required to achieve the same growth-inhibitory effect as l -methionine- S -sulfoximine at concentrations of 2, 20, and 200 μM. The addition of l -glutamine at concentrations 10–100-fold greater than l -methionine- S -sulfoximine reversed the bacteriostatic effect of the inhibitor. At inhibitor concentrations of 0.2 and 2 μM, the reversal of growth inhibition was almost complete, but at inhibitor concentrations of 20 and 200 μM, bacterial growth was only partially restored, lagging uninhibited cultures by 0.4–1.3 log units. Our finding that the glutamine synthetase inhibitor l -methionine- S -sulfoximine strongly inhibits growth of M . tuberculosis , a pathogenic mycobacterium that releases large quantities of glutamine synthetase extracellularly, suggested the possibility that bacterial sensitivity to the inhibitor is correlated with enzyme release. We first tested this hypothesis by studying the inhibitory effect of l -methionine- S -sulfoximine on the growth of several other mycobacterial strains and subsequently extended the studies to include nonmycobacterial strains as well . Consistent with this hypothesis, all pathogenic mycobacteria studied, including M . tuberculosis Erdman and H37Rv, M . bovis , and M . bovis BCG, all of which release large amounts of glutamine synthetase extracellularly, were highly sensitive to l -methionine- S -sulfoximine, whereas all nonpathogenic mycobacteria studied, including M . smegmatis and M . phlei , and the nonmycobacterial species L. pneumophila Philadelphia 1 and E . coli DH5α, none of which release glutamine synthetase extracellularly, were insensitive to the inhibitor . For the pathogenic mycobacteria, the patterns of growth inhibition were very similar to those of M . tuberculosis . M . avium , an avian mycobacterium that is an opportunistic pathogen for immunocompromised but not immunocompetent humans, and that releases small quantities of glutamine synthetase extracellularly, was intermediate between the highly pathogenic and nonpathogenic mycobacteria in its sensitivity to l -methionine- S -sulfoximine . The experiments described above did not reveal whether the growth-inhibitory effect of l -methionine- S -sulfoximine on M . tuberculosis was correlated with inhibition of intracellular or extracellular glutamine synthetase or both forms of the enzyme. To determine the site of the inhibitor's effect, we incubated M . tuberculosis Erdman cultures with l -methionine- S -sulfoximine at 20 μM, a concentration that completely inhibits M . tuberculosis growth over 4 wk, and assayed the levels of intra- and extracellular glutamine synthetase activity by transfer assay at weekly intervals . In the absence of inhibitor, the total detectable glutamine synthetase activity was 58 mU of cell-associated vs. 23 mU of extracellular enzyme activity per 10 8 cells, a ratio of ∼2.5:1, nearly identical to our previously reported results (64 mU of cell-associated vs. 29 mU of extracellular enzyme activity per 10 8 cells, a ratio of 2.2:1 ). In the presence of inhibitor, the level of cell-associated glutamine synthetase activity was minimally affected; activity plateaued at ∼50 mU, a 14% decrease from the uninhibited level . In contrast, the level of extracellular glutamine synthetase activity was reduced from the uninhibited level by 80%, from ∼23 mU to ∼4.5 mU after 4 wk of growth . l -methionine- S -sulfoximine reduced the activity of extracellular glutamine synthetase but had no detectable effect on the quantity of glutamine synthetase protein exported by M . tuberculosis . Coomassie blue–stained polyacrylamide gels showed no reduction in the amount of extracellular or cell-associated glutamine synthetase in the presence of inhibitor (data not shown). To confirm that l -methionine- S -sulfoximine did not have a general effect on protein export, we analyzed its effect on the export and activity of another large, multimeric, leaderless protein, M . tuberculosis superoxide dismutase ( 18 ). Mycobacterial cultures treated with 20 μM l -methionine- S -sulfoximine exhibited no reduction in activity or quantity of extracellular superoxide dismutase compared with untreated cultures, as calculated on a per-cell basis. Moreover, on Coomassie blue–stained polyacrylamide gels, none of the 12 major M . tuberculosis extracellular proteins were reduced in the culture supernate in the presence of inhibitor (data not shown). l -methionine- S -sulfoximine also selectively inhibited extracellular glutamine synthetase activity of a recombinant strain of M . smegmatis harboring a plasmid that allowed the expression and export of recombinant M . tuberculosis glutamine synthetase. In the absence of inhibitor, export of endogenous glutamine synthetase by the parent and recombinant M . smegmatis strain was near the level of detection (<1 mU), whereas export of M . tuberculosis glutamine synthetase by the recombinant strain was 69 mU/10 8 cells; the recombinant and endogenous glutamine synthetases were readily differentiated by NH 2 -terminal amino acid analysis. In the presence of 200 μM l -methionine- S -sulfoximine, the level of extracellular glutamine synthetase activity of the recombinant strain decreased 89%, whereas the level of cell-associated glutamine synthetase activity (sum of endogenous and recombinant enzymes) decreased only 17%; the growth rate of the parent and recombinant M . smegmatis strain was unaffected by the inhibitor. Before studying the effect of the glutamine synthetase inhibitor on M . tuberculosis growing intracellularly in human monocytes, we first evaluated its effect on uninfected host cells. These studies showed that l -methionine- S -sulfoximine at concentrations of 20 and 200 μM had no effect on the morphology, doubling time, or viability of differentiated THP-1 human monocytes. This result was consistent with the relative insensitivity of eukaryotic glutamine synthetases to inhibitors, as demonstrated for sheep brain glutamine synthetase (Table I ). We next analyzed the effect of l -methionine- S -sulfoximine on two different mycobacterial species, M . tuberculosis Erdman and M . avium , growing intracellularly in THP-1 cells . The enumeration of viable intracellular mycobacteria during 5 d of culture showed that, at 200 μM l -methionine- S -sulfoximine, growth of the bacteria was strongly inhibited. However, little if any killing of bacteria occurred. At 2 μM of inhibitor, growth of M . tuberculosis was slowed, but growth of M . avium was uninhibited. The greater sensitivity to l -methionine- S -sulfoximine in human monocytes of M . tuberculosis compared with M . avium parallels the greater sensitivity to this inhibitor under cell-free growth conditions of M . tuberculosis compared with M . avium . This pattern of sensitivity in turn correlates with the finding that M . tuberculosis releases ∼3–4-fold more glutamine synthetase extracellularly than M . avium . Pathogenic, but not nonpathogenic, mycobacteria release abundant amounts of glutamine synthetase and contain a large amount of poly- l -glutamate/glutamine in the bacterial cell wall. The high correlation between glutamine synthetase export and the presence of poly- l -glutamate/glutamine in the bacterial cell wall suggests the possibility that extracellular glutamine synthetase is involved in construction of the poly- l -glutamate/glutamine cell wall structure. If this is true, then inhibition of extracellular glutamine synthetase might reduce the amount of this structure. We investigated this hypothesis by assaying the amount of this cell wall structure in M . tuberculosis organisms grown in the presence of various amounts of the glutamine synthetase inhibitor l -methionine- S -sulfoximine. The mycobacteria were grown for 2 wk without inhibitor (to insure a sufficient number of cells for analysis) and then for 4 wk in the presence of 0, 0.2, 2, 20, and 200 μM inhibitor, after which the amount of the poly- l -glutamate/glutamine structure in the cell walls was analyzed ( 6 ). The purity of the isolated polymer ranged from 90 to 95%. The inhibitor caused a marked reduction in the amount of poly- l -glutamate/glutamine, which decreased in a dose-dependent fashion from 46 μg/10 10 cells in the absence of inhibitor to 3.2 μg/10 10 cells in the presence of 200 μM inhibitor (Table II ). Because the isolation procedure results in the hydrolysis of glutamine, we could not determine the exact ratio of glutamate/glutamine residues. For comparison, we studied the effect of the inhibitor on the amount of alanine in the peptidoglycan fraction, which contains one d - and one l -alanine per peptidoglycan monomer. The purified (>90%) peptidoglycan fraction contained from 21 μg d , l -alanine/10 10 cells at 0 μM inhibitor to 16.6 μg at 200 μM of inhibitor. Thus, in contrast to the pronounced effect of the inhibitor on the amount of poly- l -glutamate/glutamine in the cell wall, the inhibitor had only a minimal effect on the amount of alanine in the cell wall. To confirm that the effect of l -methionine- S -sulfoximine on the poly- l -glutamate/glutamine content of the mycobacterial cell wall was not a nonspecific consequence of inhibition of bacterial growth, we studied the effect of two bactericidal antibiotics, amikacin and rifampin, on the mycobacterial cell wall. As shown in Table II , these antibiotics had little influence on the content of either poly- l -glutamate/ glutamine or d , l -alanine, confirming that their mode of action is quite different from that of l -methionine- S -sulfoximine and that they do not target, directly or indirectly, these two structures in the mycobacterial cell wall. The finding that l -methionine- S -sulfoximine markedly reduces the amount of a cell wall structure that comprises 8–10% of the M . tuberculosis cell wall suggested the possibility that the glutamine synthetase inhibitor affects the integrity of the cell wall. To investigate this idea, we studied the influence of the inhibitor on the sensitivity of M . tuberculosis to conventional antibiotics. We cultured M . tuberculosis in the presence of subinhibitory concentrations of l -methionine- S -sulfoximine (0.02, 0.2, or 2 μM) and subinhibitory concentrations of each of four antibiotics: isoniazid, rifampin, pyrazinamide, or ethambutol. In all cases, bacterial growth in the presence of the combination of l -methionine- S -sulfoximine and antibiotic was significantly less than in the presence of either inhibitor alone. The most pronounced effect on bacterial growth was observed for isoniazid and rifampin at one-tenth their minimal inhibitory concentrations in combination with 0.2 μM l -methionine- S -sulfoximine. The antibiotics had a negligible effect on intracellular or extracellular glutamine synthetase activity. This result is consistent with the hypothesis that the inhibitory effect of l -methionine- S -sulfoximine on the extracellular glutamine synthetase affects the integrity of the M . tuberculosis cell wall so as to allow antibiotics greater access to the bacterial cytoplasm. Our study demonstrates that inhibitors of extracellular glutamine synthetase block the growth of M. tuberculosis and other pathogenic mycobacteria. Remarkably, the inhibitors are selective for pathogenic mycobacteria, which export glutamine synthetase and contain the poly- l -glutamate/glutamine cell wall structure. The correlation between export of glutamine synthetase and the presence of this cell wall structure revealed in our previous study led us to hypothesize that exported glutamine synthetase is involved in the synthesis of this major cell wall component, the function of which remains elusive. The study here, demonstrating that inhibition of extracellular glutamine synthetase is associated with a marked diminution in the cell wall structure, lends further support to this hypothesis. That this in turn is correlated with bacteriostasis suggests that the poly- l -glutamate/glutamine structure plays an important role in the homeostasis of pathogenic mycobacteria. Extracellular glutamine synthetase is clearly a prime target of the irreversible glutamine synthetase inhibitor l -methionine- S -sulfoximine. The inhibitor reduced glutamine synthetase activity in the extracellular milieu of M. tuberculosis cultures by 80% but had little effect on cell-associated glutamine synthetase. Indeed, given the formidable barrier presented by the lipid-rich cell wall of M. tuberculosis , the small amount of cell-associated enzyme that is affected by l -methionine- S -sulfoximine may be an enzyme that has in fact cleared the bacterial cell membrane but not yet been released by the bacterium. That the glutamine synthetase inhibitor l -methionine- S -sulfoximine selectively blocks the growth of mycobacteria that export a large proportion of their glutamine synthetase suggests that inhibition of glutamine synthetase may be directly responsible for the bacteriostatic effect of the inhibitor. However, inactivation of additional extracellular targets by l -methionine- S -sulfoximine, such as enzymes involved in the synthesis and cell wall anchoring of the heteropolymer poly- l -glutamate/glutamine, may contribute to the observed bacteriostatic effect of this inhibitor. In addition to inhibiting glutamine synthetase, l -methionine- S -sulfoximine inhibits γ-glutamylcysteine synthetase, which catalyzes a chemical reaction very similar to the one catalyzed by glutamine synthetase. However, the inhibitor's effect on this enzyme is reversible, in contrast to the case with glutamine synthetase ( 19 ). Whether this typically intracellular enzyme is exported by M. tuberculosis is not known. However, if it is, the amount exported must be very small compared with glutamine synthetase as, unlike glutamine synthetase, γ-glutamylcysteine synthetase is not one of the 12 most abundant proteins exported by M. tuberculosis . New antibiotics are desperately needed to combat the rapidly emerging strains of M. tuberculosis that are resistant to conventional antibiotics. However, M. tuberculosis presents a formidable challenge to the design of antibiotics. First, the antibiotic must penetrate the host cell and reach the organism within its unique intracellular compartment, a specialized, membrane-bound phagosome ( 3 ). Second, the antibiotic must reach its molecular target, generally within the organism, in which case the thick waxy coat of the mycobacterium presents an additional major obstacle. Our study provides an approach to circumventing the second major obstacle, targeting an extracellular enzyme crucial to bacterial cell growth. Specifically, our study demonstrates that treatment of M . tuberculosis with a drug that inactivates extracellular glutamine synthetase inhibits mycobacterial growth. Hence, drugs functionally analogous to l -methionine- S -sulfoximine, but perhaps with even greater specificity for the M . tuberculosis enzyme relative to the mammalian enzyme, have great potential as antibiotics against this pathogen. Our demonstration that l -methionine- S -sulfoximine inhibits M . tuberculosis growing within its host cell, the human mononuclear phagocyte, underscores the feasibility of this approach. More generally, our study suggests that other extracellular enzymes of M . tuberculosis and other pathogenic mycobacteria, and even nonmycobacterial pathogens, may serve as readily accessible targets of antibiotics. In this regard, an obvious mycobacterial target is the 30/32-kD protein complex, the most abundant proteins released by M . tuberculosis ( 2 , 20 ). These unique extracellular enzymes of pathogenic mycobacteria, which have been reported to have mycolyl transferase activity ( 21 ), presumably serve an essential role in microbial physiology and pathogenicity and represent highly specific targets for antibiotics. | Study | biomedical | en | 0.999996 |
10224283 | Female BALB/cJ mice (6–8 wk of age) bought from The Jackson Laboratory were maintained at the Fordham University (Bronx, NY) vivarium. Meth A cells, grown in ascites, and livers from naive BALB/cJ mice served as the source of gp96. Purification of gp96 was performed as described earlier ( 2 ). Two doses of gp96 were administered 1 wk apart and mice were challenged with tumor cells 7 d after the last immunization. Injections were performed using a 1-cm 3 insulin syringe ( Becton Dickinson ) in a volume of 200 μl. Subcutaneous injections were administered under the loose skin fold in the cervical region, dorsally, and intradermal injections were given in the skin on the ventral aspect of the trunk. During intradermal injection, care was taken to ensure that the inoculum was in the intradermal compartment without extravasation into the subcutaneous area. This estimation was made by the presence of a raised bleb confirming intradermal inoculation. Intravenous immunization was given retro-orbitally into the venous plexus. Oral immunization was administered by feeding the mice 200 μl of gp96 solution using the nozzle of a syringe. Intramuscular vaccination was performed in the muscle of the thigh. gp96 was injected intraperitoneally by inserting the needle subcutaneously for a length of 2 mm and then turning at right angles to the long axis of the body to penetrate the muscle and peritoneum. After injecting the requisite amount of inoculum, the needle was withdrawn in the same order. This method ensured that there was no extravasation of the inoculum from the peritoneal cavity, as the entry points in the skin and the peritoneum did not line up. Meth A and CMS5 lines, derived from antigenically distinct, chemically induced murine sarcomas, were used. Tumor challenges comprised 100,000 live cells (Meth A or CMS5), administered intradermally on the shaved dorsal aspect of the mouse. Tumor growth was recorded twice per week using vernier calipers measuring both the longitudinal and the transverse diameter. Average diameters of the two axes were plotted. Spleens were harvested from donor mice and RBCs were removed by incubation of the total cells with a filtered solution containing ammonium chloride and Tris base, pH 7.2. The residual cells were labeled with MACS ® antibodies for CD4 + or CD8 + cells (Miltenyi Biotec) and loaded onto MACS ® VS + separation columns (Miltenyi Biotec). After repeated washings, the cells were eluted off the columns and counted. FACS ® analysis confirmed >90% purity of the cells. As control donors, cells from mice immunized with buffer and the same age as the recipients were used. Cells were tested for >95% viability, suspended in 200 μl plain RPMI, and injected intravenously via the retro-orbital venous plexus of recipient mice. The data in Fig. 1 show the dose-restricted nature of immunogenicity of gp96. BALB/c mice were immunized subcutaneously with Meth A–derived gp96 with 1, 5, 10, or 50 μg per injection (twice, 1 wk apart) and were challenged with 100,000 Meth A cells 1 wk after the last challenge. In accord with a previous report, immunization with 10 μg gp96 was effective at eliciting tumor rejection, whereas lower (1- and 5-μg) and higher (50-μg) doses were ineffective . Doses lower than 1 μg Meth A–derived gp96 were also tested and found to be ineffective (data not shown). Doses between 10 and 50 μg were also tested in independent experiments, and a gradual diminution of activity was observed at higher doses in this range: immunization with 20, 30, or 50 μg of gp96 led to tumor take in 1/5, 3/5, and 4/5 mice, respectively. Thus, the dose restriction was consistent, reproducible, and titratable. In a demonstration of specificity of gp96-elicited immunity, immunization with gp96 derived from normal liver was found ineffective at eliciting immunity to Meth A, and mice immunized with Meth A–derived gp96 remained sensitive to challenges with a syngeneic and antigenically distinct fibrosarcoma CMS5 . When mice were immunized with Meth A–derived gp96 by various routes at various doses (1, 5, 10, and 50 μg per dose, two doses given 1 wk apart), several novel aspects emerged . As little as 1 μg of gp96 administered intradermally imparted tumor protection, whereas a minimum of 10 μg was needed subcutaneously and 50 μg intraperitoneally to elicit corresponding levels of protection. Thus, intradermal immunization was more efficient than subcutaneous, and subcutaneous more efficient than the intraperitoneal route, on a per-microgram basis. Immunization with any dose of gp96 by intramuscular, oral, or intravenous routes showed no protection from tumor challenge (data not shown). Furthermore, although the subcutaneous and intradermal routes were observed to vary quite significantly, dose restriction of activity was observed in both routes . In the case of intradermal immunization, <1 μg gp96 was ineffective, 1 μg was the optimal dose, and 10 μg did not elicit protective immunity; in the case of subcutaneous immunization, <10 μg gp96 was ineffective, 10 μg was the optimal dose, and 50 μg did not elicit protective immunity. Other parameters of immunity elicited by immunization with gp96 by various routes also showed common patterns. Immunity elicited by all routes was exquisitely tumor specific, and mice immunized by all routes developed a memory response. Mice immunized intradermally or subcutaneously with Meth A–derived or liver-derived gp96 were parked for 3 mo following the last immunization and were challenged at the end of that period with live Meth A cells. Meth A–derived gp96, given intradermally, intraperitoneally, or subcutaneously, elicited specific protection from subsequent challenge with Meth A and failed to protect from CMS5 challenge . Liver-derived gp96, delivered by any route, failed to protect from tumor challenge . As higher than optimal doses of gp96 did not immunize, it was difficult to assess the antigen specificity of this nonresponse. However, a method of testing this question was developed based on the phenomenon of concomitant immunity ( 18 ). It has been observed previously that if mice are challenged with a given tumor at one site in the body, and if this tumor is allowed to grow, the mice show resistance to challenge with the same tumor at another anatomical site. Thus, although the animal succumbs to a tumor at one location, it resists the same tumor at another location. This phenomenon is only observed during a narrow time window of 6–9 d after primary tumor transplantation. North and Bursuker ( 18 ) have elegantly demonstrated that concomitant immunity results from the fact that progressively growing tumors elicit an antitumor immune response, which gets rapidly downregulated after a certain period. In the period before downregulation has occurred, mice show systemic protective immunity to challenges with the same tumor as used in the primary challenge but not to other, antigenically distinct tumors ( 18 ). In the present study, we sought to test whether prior immunization of mice with higher than optimal doses of Meth A–derived or unrelated gp96 would or would not abrogate concomitant immunity. The design of this experiment is shown in Fig. 3 A. Mice were preimmunized with gp96 in either tumor-protective doses (1 μg intradermal [i.d.] or 10 μg s.c.) or higher doses (10 μg i.d. or 100 μg s.c.) and subsequently challenged with live tumor cells. 8 d after tumor challenge, the growing tumors were excised. (As shown earlier, all mice develop tumors that grow equally and at a consistent rate for the first 5–10 d, after which they either regress or continue to grow depending upon the immunizing dose.) Mice were allowed to recover for 4–7 d after surgery and were then rechallenged with Meth A tumor cells. The kinetics of tumor growth in each group were monitored. It was observed that mice that had been preimmunized with the larger doses of Meth A gp96 showed a loss of concomitant immunity to the second tumor challenge. Previous immunization with buffer alone or with optimal or larger doses of liver-derived gp96 did not show abrogation of immunity. These observations were noted in mice immunized by the subcutaneous or intradermal routes. The results show that the larger than optimal doses of cognate gp96 elicit an antigen-specific downregulatory influence on immune response. As the loss of tumor immunity elicited by high doses of gp96 would appear similar to immunization with buffer, an indirect assay had to be devised to measure the activity of high doses of gp96. Mice were first immunized with doses of gp96 that elicit tumor immunity. These mice then received sera or T cell subsets from other mice that had been previously immunized with high doses of gp96. Preliminary analysis suggested that a cellular and not a humoral component could transfer downregulation (data not shown). Furthermore, splenic T lymphocytes from mice immunized with high doses of Meth A–derived gp96 were fractionated into CD4 + and CD8 + populations as described in Materials and Methods. The lymphocytes were adoptively transferred to mice that had been previously immunized with the effective dose of gp96. All mice were challenged with Meth A cells, and the kinetics of tumor growth were monitored. It was observed that tumor immunity was abrogated in mice that received CD4 + T lymphocytes from high-dose gp96–immunized mice; in contrast, mice that received CD8 + T lymphocytes from the high-dose gp96–immunized group remained protected, as did the mice that received CD4 + or CD8 + T lymphocytes from buffer-immunized mice. In other control experiments, mice that received buffer or CD4 + or CD8 + T lymphocytes from low-dose gp96–immunized mice remained protected from tumor challenge (data not shown). Intradermal immunization with 1 μg Meth A gp96 given in divided doses at different sites (0.25 μg/site in four sites, targeting different regional lymph nodes) was found to elicit protective tumor immunity. The 0.25-μg dose administered at one site only did not elicit immunity . The degree of protection conferred by immunization with 0.25 μg/site at four sites was comparable to that elicited by 1 μg given at a single site or as a single dose. The higher dose of gp96 (5 μg i.d. at a single site), which mediated active downregulation of immune response when administered at one site in one large dose, also mediated downregulation when given in four divided doses of 1.25 μg each, intradermally, at multiple sites . These observations indicate that gp96-induced immunity shows spatial summation and suggest the existence of a substantial degree of cross-talk between lymph nodes. Studies reported here indicate that immunization with gp96 preparations elicits a complex and highly regulated immune response, which, depending upon the route and the quantity used for the immunization, results in antitumor immune response or its downregulation. The lack of tumor-protective responses by high doses of gp96 is not a null event but an active process that can downregulate tumor immunity elicited by immunization with a growing tumor or by the optimal dose of the cognate gp96. This active process includes generation of a downregulatory CD4 + T lymphocyte population. Although there is precedent for CD4 + T cell populations that can downregulate immune response to cancers and infectious agents ( 19 , 20 ), the mechanisms through which they implement their action have not been elucidated in the present or previous systems. This must await cloning and structural characterization of the downregulatory cells and the cytokines elaborated by and responded to by them. As APCs play the central role in immunogenicity of gp96 preparations ( 5 ), the log scale differences among the efficiencies of the various routes suggest that the differences may reflect the nature and density of the relevant APC populations at various sites. Thus, Langerhans cells, assorted subcutaneous macrophages, and peritoneal macrophages might be involved in processing of gp96–peptide complexes in the intradermal, subcutaneous, and intraperitoneal sites, respectively. Differences in efficiencies of such APCs have indeed been recorded ( 21 ). The primary events of elicitation or downregulation of immune response presumably occur at the level of gp96–APC interaction. Two possibilities may be envisaged. First, activation of an APC by different quantities of gp96 may lead to qualitatively different types of signals, one leading to stimulation of CD8 + T cells and the other to generation of a downregulatory CD4 + T cell population. This possibility, although attractive, is weakened by the observation that stimulation of macrophage in vitro with gp96–peptide complexes over a wide range of quantities leads to uniform stimulation of CD8 + T cells ( 14 ). The second possibility is that a larger quantity of gp96 molecules might interact with a larger number of APCs, leading to generation of an amplified signal, such as a particular cytokine or combination of cytokines, that stimulates CD8 + T cells at lower levels and an inhibitory CD4 + T cell population at higher levels. Presently, we favor the second possibility. It has been postulated that gp96 molecules interact with APCs through a receptor ( 22 ). Inherent in the second possibility is the prediction that the expression level of the putative gp96 receptor on APCs is quite low and limiting, leading to the rather narrow window of the effective immunizing dose of gp96. Interestingly, when mice are immunized with increasing doses of irradiated, intact Meth A tumor cells ranging from 10 million cells to 200 million cells, no restriction of immunizing activity is observed at the higher doses (data not shown). This result provides further support to our previous studies, in which it was shown that the mechanisms through which intact cells elicit immunity are distinct from the mechanism through which gp96 isolated from the same cells becomes immunogenic ( 5 ). The observation that immunogenicity of gp96 is exquisitely sensitive to the abrogation of the function of APCs, whereas immunogenicity of whole cells is not ( 5 ), further points to the APC as the site most likely to be responsible for the dual nature of the specific immunological activity of gp96. Our results with gp96 are also reminiscent of earlier studies describing high-zone tolerance induced by immunization with large quantities of soluble proteins ( 23 ). As gp96 molecules chaperone antigens instead of being antigenic themselves ( 17 ), the differences between the mechanisms of tolerance induction in the two instances would be interesting and instructive. The observations that administration of several fractions of doses is as effective in eliciting or downregulating immune response as administration of a single dose appear to suggest that both events, the immune response and its downregulation elicited by gp96, require a threshold that can be met by events at one microenvironment or collectively at several. The observation that immunization with several suboptimal doses can help meet the threshold to an active immune response is understandable with relative ease. In contrast, the observation that several immunizations, each with an optimal immune-stimulating dose, can lead to a systemic downregulated immune response is surprising. It suggests that even though an immune response at a given site may have a given consequence, it may be overridden by independent events occurring at other sites. Collectively, these results argue for a level of cross-talk among different immunological microenvironments that appears surprising in light of other studies that show a high degree of autonomy for individual lymph nodes ( 24 ). Our observations have a significant implication for therapy. Autologous cancer–derived gp96 preparations have been used for immunizing cancer patients in a pilot clinical trial, and other such trials are underway. The quantity of gp96 to be used in the trials requires careful calibration such that it does not become immune inhibitory. Similar but converse concerns must be kept in mind in considering possible applications of the use of gp96 or other HSPs toward therapy of autoimmune conditions. | Other | biomedical | en | 0.999997 |
10224284 | The pSPIg8 plasmid, which has a 12.7-kb BamHI genomic insert containing the New Zealand black mouse J κ cluster, was provided by B. Van Ness (University of Minnesota, Minneapolis, MN). The following modifications were introduced in defined subclones, and used thereafter to reconstitute the complete BamHI fragment: the lox P-flanked neo R gene, obtained from the pLZ- neo R plasmid (provided by H. Gu, NIAID, National Institutes of Health, Bethesda, MD), was blunt-end inserted in the most 5′ HindIII site. A XhoI site was introduced 127-bp upstream of J κ1 by site-directed mutagenesis (In Vitro Mutagenesis kit; Bio-Rad) and was used thereafter to insert a XhoI-SalI lox P fragment from pLZ- neo R in constructs containing or not containing the KI-KII mutation ( 10 ). The HSV- tk gene obtained from the pIC19R plasmid ( 14 ) was blunt-end ligated in the SalI site of the pSPIg8 plasmid. The final constructs thus contained three lox P sequence elements in the same orientation (verified by sequencing) allowing for the Cre-mediated deletion of either the neo R gene alone (control mice or clones) or of both the neo R gene and the 4-kb fragment . For constructions used in the Abelson virus–transformed 103/4 cell line ( 13 ), a diagnostic EcoRI site was created instead of the natural StyI site between J κ1 and J κ2 by site-directed mutagenesis on both the KI-KII–mutated and the wild-type constructs. Since the 103/4-bcl2 cell line was derived by transfection of a bcl2 construct carrying the neo R gene that is present in our gene targeting constructs, we established a new 103/bcl2-hygro R derivative by transfection of the original 103/4 cell line with the pSFFV bcl2 expression vector (provided by S.J. Korsmeyer, The Rockefeller University Press, New York) in which the neo R gene was replaced by a hygro R gene ( 15 ) driven by the CMV promoter. E14.1 enbryonic stem (ES) cells were cultured, transfected, and selected as previously described ( 10 ). The analysis of recombinant clones before and after Cre-mediated deletion of the neo R gene and/or of the 4-kb DNA fragment was performed by Southern blot analysis similarly for ES and 103-derived cells. The transfection protocol of the 103/4 cell line was as follows: 10 7 cells were electroporated with 30 μg linearized DNA in 800 μl RPMI, 10 mM Hepes, pH 7.4 (300 V, 900 μF). The cells were immediately resuspended in 50 ml RPMI, 10% FCS, 0.05 mM β-ME, and subcloned in 96-well plates at 20,000 cells/well. Selection with 1.5 mg/ml hygromycin B ( Boehringer Mannheim ) or G418 ( GIBCO BRL ) was applied 48 h later. The resistant clones were then subcloned at 0.5 cells/well, grown in culture for 3 wk, and analyzed by Southern blot after expansion. Approximately 10% of each of the transfectants were targeted to the κ locus, but only in a fraction of them did the recombination extend over the 10 kb containing the three lox P sites and the EcoRI restriction site. In a typical experiment, 66 clones were obtained from 10 7 cells, of which 6 were targeted to the κ locus and 1 contained the three lox P modifications. Chimeric mice were obtained after aggregation of gene-targeted ES cells (see below) with CD-1 morulas. Analysis of ES-derived lymphoid cells took advantage of a polymorphism that frequently occurs between the CD-1 and 129 cells at the Ly5 locus. The anti–mouse Ly5.2-PE and Ly5.1-PE antibodies were provided by B. Rocha (INSERM, Faculté de Médicine Necker, Paris, France). Anti–mouse CD19-FITC was purchased from PharMingen . Spleen cells from chimeric mice were prepared and labeled as previously described ( 10 ), and Ly5.2 + , CD19 + , and CD19 − cells (∼50% chimerism in the spleen) were purified using a FACSVantage ® cell sorter ( Becton Dickinson ). Genomic DNA preparation was performed as described ( 10 ). Alkaline Southern blots after digestion of genomic DNA with BamHI and hybridization with a 1.8-kb SphI-PstI random-labeled DNA fragment (probe C) encompassing the mouse J κ cluster were performed as previously described ( 10 ). Germline retention in control mice was carried out by PCR with the primers used in the reverse transcription (RT)-PCR detection of the long germline transcript from the wild-type allele (see below). V κ to J κ1 rearrangement products were amplified with the primers described in references 16 and 17 (our PCR parameters: 1 min at 94°C; 1.5 min at 66°C; 1.5 min at 72°C; 24 cycles). StyI and EcoRI digestions were carried out for 4 h in separate reactions to assess the percentage of rearrangement corresponding to the wild-type and mutated alleles, respectively. Total RNA was extracted with Trizol ( GIBCO BRL ) from 10 6 cells cultured at the permissive (34°C) and the nonpermissive (40°C) temperatures for 6 and 12 h. After reverse transcription with an equimolar mixture of oligo-dT and random primers (Stratagene), cDNA was resuspended in 50 μl of water. For each sample, four different amounts of cDNA (2, 1, 0.5, and 0.25 μl) were amplified in separate reactions for each primer set described below to avoid plateau effects. For the long germline transcript, the 3′ primer was AGCAATTCCCTTCACTCAAACCCCCATAC for both alleles; the 5′ primers were GTCTGAAAGAGGAGTTTACGTCCAGC (hereafter referred to as lox P-5′-primer) for the mutated allele containing a lox P site (1 min 94°C; 1 min 66°C; 1.5 min 72°C; 30 cycles) and CCTATGGAAGAGCAGCGAGTGCC (hereafter referred to as wt-5′-primer) for the wild-type allele (same PCR parameters, but 27 cycles). Normalization was performed with respect to β-actin (mouse β-actin amplimer set; Clontech ). The PCR products were blotted onto nylon membranes and hybridized with the oligonucleotide DAR25 ( 17 ) for the long transcript, and with AACATGGCATTGTTAC for β-actin. Southern blots and PCR hybridizations were exposed to phosphor screens, scanned with a Storm 480 machine (Molecular Dynamics), and analyzed with the public domain NIH Image program (developed at NIH and available at http://rsb.info.nih.gov/nih-image ). Gene-targeted Ly5.2 + B and non-B cells were sorted from spleens of chimeric mice obtained by aggregation of mutated ES cells with Ly5.1 + morulas. In a first series of mice (D5, D6, D7) the sorted cells carried the 4-kb deletion on one κ allele . Analysis by Southern blot of the percentage of wild-type over mutated allele germline retention in B cells of three different mice (14/86, 15/85, and 23/77) versus non-B cells (47/53, 44/56, and 49/51) showed a strong decrease in rearrangement levels for the mutated allele, indicating that a positive regulatory element of rearrangement had been inactivated upon deletion. Germline retention analysis of B cells from a chimera bearing only the two lox P sites (control mice) indicated that these two elements had no effect on rearrangement . A similar analysis was carried out in a second series of mice (DM1 and DM4), which had the KI-KII mutation on the same allele as the 4-kb deletion . We previously showed that the KI-KII mutation results in a very strong reduction of κ rearrangement in cis ( 10 ). The analysis showed that rearrangement of the mutated allele was further reduced when compared with the wild-type allele when both the deletion and the KI-KII mutation were present in cis (7/93 and 3/97 percentage wild-type over mutated allele germline retention in B cells versus 47/53 in non-B cells) . This stronger reduction in rearrangement in the presence of both mutations was confirmed by PCR analysis (data not shown), indicating that deletion of the 4-kb DNA fragment and mutation of the KI-KII motif have cumulative effects. The 103/bcl2-hygro R cell line, transformed with a temperature-sensitive mutant Abelson virus, rearranges the light chain locus when shifted to the nonpermissive temperature (34°C→ 39°C). It was shown that this rearrangement process follows a kinetics where 20–30% of κ loci are rearranged in 48 h; to a lesser degree, the λ loci of these cells also rearrange during this time course. After 4 d most of the cells have rearranged both κ alleles ( 13 ). Upon subcloning after 24 h of induction, we have observed that among 17 clones, 6 had rearranged one κ allele, 2 had rearranged both alleles, and 9 had their κ alleles in germline configuration (data not shown). Rearrangement of the wild-type over mutated alleles was assessed in the 103/bcl2-hygro R –derived clones carrying the following mutations, introduced by homologous recombination on one κ allele: the 4-kb deletion alone, the KI-KII mutation alone with the two lox P sites, the two mutations together, or the control lox P sites only . We have chosen clones where the rearrangement status of the κ loci was close to germline at the permissive temperature, since it has been reported previously that a small percentage of these cells can rearrange spontaneously ( 13 ). Rearrangement levels were quantified by PCR at several time points after induction (Table II ). In the cell line carrying the 4-kb deletion on one κ allele (clone 7.1.18.3), there was a strong decrease of the rearrangement level of the mutated allele when compared to the wild-type allele (24:76 at 48 h). This indicates that the deletion gives similar results in the cell line and in mice. The control lox P clone (7.1.18.9) showed no difference between the two alleles, confirming the neutrality of the two lox P inserted in the κ locus on the induction of rearrangement. The KI-KII mutation alone in clone 19.3.9.10 showed no significant effect on rearrangement of the modified allele 48–96 h after induction. A small difference favoring the KI-KII mutation–bearing allele was seen in this clone 12– 24 h after induction (see Table II ), but these differences did not stay consistent at later points of the assay. Accordingly, in the cell line carrying both the deletion and the KI-KII mutation, the reduction in rearrangement was identical to the one obtained with the sole 4-kb deletion (clone 19.3.9.14, 24:76 at 48 h). Altogether, these results suggest that in this particular in vitro model the 4-kb deletion mimics the effect obtained with the corresponding mutated mice. On the other hand, the regulatory mechanism involving the KI-KII motif could be nonoperative in these conditions. Overall expression levels of long germline transcripts increased upon induction at the nonpermissive temperature at 6 h in the 103/bcl2-hygro R pre-B cell line and started to decrease at 12 h when rearrangement levels start to increase . The 4-kb deleted fragment contains the promoter of the long germline transcript. Long germline transcripts were expressed at similar levels from the wild-type and lox P-bearing alleles in the control clone 19.3.9.10 . Analysis of transcription initiating upstream from the lox P site on the allele carrying the 4-kb deletion in the 19.3.9.14 deletion clone showed negligible amounts of RT-PCR product, indicating that the deletion did not force an ectopical initiation of transcription upstream of the 5′ boundary of the deleted fragment. Moreover, the presence of the lox P site near KI-KII had no effect of transcription of the long germline transcript. Here we show that a deletion of 4 kb immediately upstream of the KI-KII sites in the κ V-J intervening sequence results in a dramatic reduction of rearrangement in cis in mouse spleen B lymphocytes heterozygous for the mutation. When added to the 4-kb deletion, mutations of the KI-KII sites, which had been previously characterized as enhancers of rearrangement, further decreased rearrangement, leading to the almost exclusive use of the unmutated allele. These results clearly demonstrate that a second positive regulatory element of rearrangement is located in the 4-kb fragment adjacent to KI-KII and that both elements must act in concert to enhance rearrangement of the locus. The same mutations were introduced by homologous recombination of one κ allele in the rearrangement-inducible 103/bcl2-hygro R cell line. This pre-B cell line, which has been transformed with a temperature-sensitive Abelson virus mutant, has both heavy chain alleles rearranged nonproductively and initiates rearrangement of the light chain loci when put at a nonpermissive temperature. As in normal pre-B cells, rearrangement initiates in the 103 cell line on one κ allele, but in the absence of a feedback inhibition of the recombinase it then proceeds to the other allele and to the λ locus if the cell is maintained at the nonpermissive temperature for 48 h ( 13 ). A reduction of rearrangement was obtained for the allele carrying the 4-kb deletion in the 103/bcl2-hygro R cell line, quantitatively similar to the reduction observed in mutant mice. Several results have been obtained that imply germline transcription as a prerequisite event to rearrangement. When tested in the 103/bcl2-hygro R cell line, transcription of the long germline transcript could only be induced from the unmutated allele upon induction of rearrangement. Therefore, it is highly probable that the effect we have observed with the 4-kb mutation is due to the deletion of the promoter of the long germline transcript, but one cannot exclude that this segment may contain additional regulatory elements. On the other hand, the KI-KII mutation did not alter the efficiency of rearrangement in the 103 cell line, contrary to the result obtained in vivo. It has been shown that the KI-KII motifs can bind the transcription factor Pax-5 ( 18 ). This factor also binds a locus control region located at the 3′ end of the IgH locus ( 19 , 20 ), and seems to be involved in V H gene accessibility before the V H -DJ H rearrangement step. We have found Pax-5 expression in the 103 cell line (data not shown), but specific cofactors ( 21 ) necessary for the KI-KII enhancement effect may nevertheless be absent from this cell line. In the context of the regulation of allelic exclusion that starts by initiating rearrangement on one chromosome, one can envision that a limited balanced set of positive and negative factors present during a short window of development may control accessibility to one allele only. These factors will bind at the promoter and the enhancer regions but also at some specific sites in the V-(D)-J intervening segments ( 4 , 22 – 24 ) or upstream of the V regions ( 25 , 26 ). Alternatively, only one allele per progenitor could be accessible to the recombinase if it is marked by a modification enzyme as recently proposed ( 27 ) or by a specific anatomical location in the nucleus. It has been shown recently that in double positive thymic cells the excluded β allele was mostly hypomethylated and transcriptionally active ( 28 ). This result would fit better with a repression of the silent allele in the second phase of the allelic exclusion process than with a regulation operating via methylation. The 103/bcl2-hygro R cell line may be a valuable tool to address such issues. | Study | biomedical | en | 0.999995 |
10224285 | A human T lymphoma cell line Jurkat was obtained from Japan Cancer Research Bank and cultured in RPMI 1640 medium containing 10% FCS, 100 μg/ml streptomycin and penicillin, and 2 mM glutamine (culture medium). A mouse B lymphoma cell line 2PK-3 was obtained from American Type Culture Collection and maintained in culture medium. 2PK-3–derived transfectants, hTRAIL/2PK-3 and hFasL/2PK-3, which stably express human TRAIL and human FasL, respectively, were prepared as described previously ( 20 ) and maintained in culture medium. Human RCC-derived cell lines 769P, A498, ACHN, Caki 1, and Caki 2 were obtained from American Type Culture Collection, and KO-RCC-1 was established as described previously ( 25 ). RCC-derived cell lines KN-39, KN-41, and OWR-10 were provided by Dr. Y. Kinoshita (Kobe University, Kobe, Japan). All of these RCC-derived cell lines were maintained in culture medium. Human IFN-γ, TNF-α, IL-4, IL-6, IL-15, and IL-16 were purchased from PharMingen . Human IL-1α, IL-1β, and IL-7 were purchased from Genzyme . Human IL-12 was purchased from R&D Systems. Human IL-2, IFN-α, and IFN-β were provided by Shionogi (Osaka, Japan), Sumitomo Pharma (Osaka, Japan), and Toray (Kamakura, Japan), respectively. Human IL-18 was provided by Drs. H. Tsutsui, H. Okamura, and K. Nakanishi (Hyogo College of Medicine, Nishinomiya, Japan). A synthetic double-stranded RNA, polyinosinic acid:polycytidylic acid (poly IC), and PHA were purchased from Sigma . Concanamycin A (CMA), which inhibits perforin-mediated cytotoxicity ( 26 ), was purchased from Wako Pure Chemicals. An anti-CD28 mAb (CD28.2) was purchased from PharMingen . An anti-CD3 mAb, OKT-3, was prepared from the hybridoma obtained from American Type Culture Collection. A neutralizing anti–human FasL mAb (NOK-2, mouse IgG2a/κ) and a neutralizing anti– human TRAIL mAb (RIK-2, mouse IgG1/κ) were prepared as described previously ( 20 , 27 ). PBMCs were prepared from healthy volunteers by Ficoll-Hypaque ( Sigma ) centrifugation. PBMCs (3 × 10 6 cells/ml) were cultured at 37°C for 24 or 48 h on 24-well plates precoated with 10 μg/ml of anti-CD3 mAb, or in the presence of IL-2 (500 U/ml), PHA (20 μg/ml), or poly IC (20 μg/ml). For preparation of PBT cells, PBMCs were first cultured on plastic dishes for 1 h at 37°C to deplete adherent monocytes. After passage of nonadherent cells through a nylon wool column, PBT cells were isolated by E-rosetting with sheep red blood cells followed by depletion of residual NK and B cells by using anti-CD16 mAb (3G8; PharMingen ), anti-CD20 mAb (2H7; PharMingen ), anti-CD56 mAb (B159; PharMingen ), and anti–mouse Ig immunomagnetic beads ( Dynal ). Purity of PBT cells was >95% CD3 + as determined by flow cytometry. In some experiments, CD4 + and CD8 + T cells were isolated from PBT cells by using anti-CD4 or anti-CD8 immunomagnetic beads ( Dynal ) and DetachaBead ® ( Dynal ). The purity of each population was >98% CD3 + CD4 + or >98% CD3 + CD8 + as estimated by flow cytometry. Purified PBT cells (3 × 10 6 cells/ml) were cultured on plates uncoated or precoated with anti-CD3 mAb (10 μg/ml) in the presence or absence of IFN-α (200 U/ml), IFN-β (200 U/ml), IFN-γ (500 U/ml), TNF-α (100 ng/ml), IL-1α (50 ng/ml), IL-1β (50 ng/ml), IL-2 (500 U/ml), IL-4 (50 ng/ml), IL-6 (200 U/ml), IL-7 (20 ng/ml), IL-12 (20 ng/ml), IL-15 (150 ng/ml), IL-16 (100 ng/ml), or IL-18 (500 ng/ml) at 37°C for 2 or 48 h. In some experiments, soluble anti-CD28 mAb (10 μg/ml) was added to the culture. Cells (10 6 ) were incubated with 1 μg of biotinylated RIK-2 or control mouse IgG1 ( PharMingen ) for 1 h at 4°C followed by PE-labeled avidin ( PharMingen ). After washing with PBS, the cells were analyzed on a FACScan™ ( Becton Dickinson ), and data were processed by using Cell Quest™ software ( Becton Dickinson ). In some experiments, FITC-labeled anti–human CD3 mAb ( PharMingen ) was included to identify T cells in PBMCs. PBT cells (3 × 10 6 cells/ml) were cultured on plates uncoated or precoated with anti-CD3 mAb (10 μg/ml) in the presence or absence of IFN-α (200 U/ml) for 12 h. Total RNA was then extracted from the cells by using RNA STAT-60™ (Tel-test, Inc.) according to the manufacturer's instructions. 10 μg each of denatured RNAs was electrophoresed in a 1.5% agarose gel containing 6.6% formaldehyde, and then transferred to a nylon membrane (Pall). The membrane was hybridized for 4 h with an [α- 32 P]dCTP-labeled 900-bp XhoI-NotI fragment containing human TRAIL cDNA ( 20 ) at 65°C in ExpressHyb™ hybridization solution ( Clontech ), and then washed twice in 2× SSC/0.1% SDS at 65°C for 15 min and twice in 0.5× SSC/0.1% SDS at 65°C for 15 min. The membrane was analyzed by autoradiography . In a similar way, the same membrane was rehybridized with a 1-kb EcoRI-BamHI fragment containing β-actin cDNA ( 28 ). PBT cells (3 × 10 6 cells/ml) were prestimulated for 12 h on plates uncoated or precoated with anti-CD3 mAb (10 μg/ml) in the presence or absence of IFN-α (200 U/ml). After washing twice with culture medium, the cells were used as effector cells. In some experiments, human TRAIL or FasL transfectants were used as effector cells. A 51 Cr-release assay was performed as described previously ( 29 ). In brief, 51 Cr-labeled target cells (10 4 ) and effector cells were mixed in U-bottomed wells of a 96-well microtiter plate at the indicated E/T ratios. After an 8-h incubation, cell-free supernatants were collected and radioactivity was measured in a γ-counter. Percent specific 51 Cr release was calculated as described ( 29 ). In some experiments, the effector cells were pretreated with 20 nM CMA for 2 h to inactivate perforin ( 26 ). Anti-FasL mAb (NOK-2) and/or anti-TRAIL mAb (RIK-2) were added to a final concentration of 10 μg/ml each at the start of the cytotoxic assay. We recently demonstrated that human CD4 + T cell clones constitutively expressed TRAIL on their surface as estimated by staining with neutralizing anti-TRAIL mAbs, RIK-1 and RIK-2 ( 20 ). In contrast, as represented in Fig. 1 , we could not find a detectable level of surface TRAIL on freshly isolated PBT cells. Because the expression of TNF family proteins such as TNF-α and FasL can be induced on activated T cells, we examined whether surface TRAIL expression can be also upregulated on PBT cells by various stimuli. PBMCs were stimulated with immobilized anti-CD3 mAb, PHA, IL-2, or poly IC for 24 or 48 h, and TRAIL expression on CD3 + PBT cells was then examined by flow cytometry with RIK-2 mAb. As represented in Fig. 1 , no detectable level of TRAIL was found on PBT cells when stimulated with PHA or IL-2. In contrast, marginal but significant levels of TRAIL expression were consistently observed upon stimulation with anti-CD3 mAb or poly IC. Since poly IC has been known to be a potent inducer of type I IFNs ( 21 , 22 ), we next examined whether IFN-α can directly affect TRAIL expression on PBT cells. As represented in Fig. 2 A, marginal but significant levels of TRAIL expression were observed on both CD4 + and CD8 + PBT cells at 12 h after stimulation with either anti-CD3 mAb or IFN-α alone. Interestingly, the combination of anti-CD3 mAb and IFN-α greatly upregulated TRAIL expression on both CD4 + and CD8 + PBT cells. Similar results were obtained at 24 or 48 h after each stimulation (data not shown). These results indicated that stimulation with anti-CD3 mAb alone is not sufficient for maximal induction of surface TRAIL expression on PBT cells, and that IFN-α provides a costimulatory signal that synergistically upregulates TRAIL expression. It has been shown that expression of some TNF family proteins such as CD40L on activated T cells was upregulated by a CD28-mediated costimulatory signal ( 30 ). Therefore, we tested whether the CD28-mediated costimulation might affect expression of TRAIL on anti-CD3–stimulated PBT cells. As represented in Fig. 2 A, the addition of an agonistic anti-CD28 mAb barely affected the expression levels of surface TRAIL on anti-CD3– or anti-CD3 plus IFN-α–stimulated PBT cells, indicating that the CD28- mediated costimulatory signal does not contribute to TRAIL induction in PBT cells. We also examined the kinetics of TRAIL expression after stimulation with anti-CD3 plus IFN-α. As represented in Fig. 2 B, TRAIL expression appeared on both CD4 + and CD8 + PBT cells at 2 h after stimulation, and reached a peak at 6 h. Similar levels of surface TRAIL expression were retained on CD4 + and CD8 + PBT cells at 48 h (not shown). We next examined various cytokines for their ability to regulate TRAIL expression on unstimulated or anti-CD3–stimulated PBT cells. IFN-β, another type I IFN that shares the same receptor (IFNAR) with IFN-α ( 21 , 22 ), exhibited a similar effect as IFN-α, which alone induced a marginal TRAIL expression on PBT cells and greatly upregulated TRAIL expression on anti-CD3–stimulated PBT cells . Similar levels of TRAIL expression on anti-CD3–stimulated PBT cells were observed upon costimulation with a lower dose of IFN-α and IFN-β (50 U/ml; not shown). In contrast, the type II IFN (IFN-γ), which stimulates a distinct receptor (IFNGR), exhibited no effect on TRAIL expression on unstimulated or anti-CD3– stimulated PBT cells at 12 h or 24 or 48 h (not shown) even at a high dose of 500 U/ml. We also examined IL-2, IL-15, IL-12, and IL-18, which have been reported to upregulate FasL expression on T and NK cells ( 4 , 31 – 33 ), but observed no effect on TRAIL expression . Similarly, no effect was observed with the other cytokines tested, including TNF-α, IL-1α, IL-1β, IL-4, IL-6, IL-7, and IL-16 (data not shown). These results indicated that the ability to induce TRAIL expression on PBT cells is a unique feature of type I IFNs. It has been reported that TRAIL mRNA was detectable in a variety of tissues and cells ( 1 ). Furthermore, a recent study demonstrated TRAIL mRNA expression in human PBT cells after activation with anti-CD3 mAb or PMA plus ionomycin ( 34 ). Therefore, we examined whether stimulation with anti-CD3 mAb and/or IFN-α upregulates TRAIL expression in PBT cells at the transcriptional level. As represented in Fig. 4 , PBT cells cultured with medium alone expressed only a marginal level of the 1.6-kb TRAIL transcript. Although stimulation with either IFN-α or anti-CD3 mAb alone significantly increased TRAIL mRNA expression, a remarkable increase was observed after stimulation with the combination of anti-CD3 mAb and IFN-α. It was also noted that IFN-α was more effective than anti-CD3 mAb in inducing TRAIL mRNA expression when used alone. Similar results were obtained when PBT cells were stimulated with IFN-β (not shown). These results indicated that type I IFNs regulate TRAIL expression in unstimulated or anti-CD3–stimulated PBT cells at the transcriptional level. Type I IFNs have been used for clinical treatment of various tumors, including melanomas, gliomas, CMLs, and RCCs ( 23 , 24 ). The unique feature of type I IFNs to upregulate TRAIL expression on PBT cells prompted us to examine whether IFN-induced TRAIL on PBT cells might be directly involved in enhanced cytotoxicity against tumor cells. In this respect, we first examined the susceptibility of human RCC cell lines to cytotoxicity by human TRAIL transfectants. We also examined their susceptibility to human FasL, which constitutes another pathway of T cell cytotoxicity. As represented in Fig. 5 , eight out of nine RCC cell lines tested were sensitive to TRAIL-induced cytotoxicity. Some RCC cell lines (A498 and KN-41) exhibited as high sensitivity as Jurkat, which has been commonly used as a highly sensitive target for TRAIL ( 1 – 3 ). On the other hand, the FasL transfectants lysed five out of nine RCC cell lines, all of which were also lysed by the TRAIL transfectants. Some RCC cell lines (Caki 1, KN-39, and KN-41) were sensitive to TRAIL but not to FasL. We then examined the cytotoxic activity of anti-CD3– and/or IFN-α–stimulated PBT cells against RCC cell lines that were sensitive to both TRAIL and FasL (ACHN and A498) or sensitive to TRAIL but resistant to FasL (KN-39 and KN-41). A neutralizing anti-TRAIL mAb (RIK-2 ) and a neutralizing anti-FasL mAb (NOK-2 ) were used to assess the contribution of TRAIL and FasL to the cytotoxicity. CMA pretreatment, which inactivates perforin ( 26 ), was used to assess the contribution of perforin. As shown in Fig. 6 , anti-CD3–stimulated PBT cells exhibited substantial cytotoxic activities against both types of RCC target cells, which were partially inhibited by RIK-2 but not by NOK-2; the residual cytotoxicity was mostly abrogated by CMA treatment, indicating that these cytotoxic activities were mediated by TRAIL and perforin. Although IFN-α stimulation alone did not significantly induce cytotoxicity, costimulation with IFN-α greatly enhanced the cytotoxic activities of anti-CD3–stimulated PBT cells; this enhancement was abrogated by RIK-2 but not by NOK-2, indicating that the IFN-α–enhanced cytotoxicity was predominantly mediated by TRAIL but not by FasL. It was also noted that the TRAIL-independent residual cytotoxic activities, which were abrogated by CMA treatment, were almost comparable between the anti-CD3–stimulated T cells and the anti-CD3 plus IFN-α–stimulated T cells, indicating that IFN-α did not enhance perforin-mediated cytotoxicity. These results indicate that IFN-α can augment the cytotoxic activity of TCR/CD3-stimulated PBT cells against RCC cells by specifically enhancing TRAIL-mediated cytotoxicity. We recently demonstrated that human CD4 + T cell clones constitutively expressed TRAIL, which was fully responsible for spontaneous cytotoxicity against certain tumor cells, such as Jurkat cells ( 20 ). Others have also recently shown that the cytotoxic activity of human CD4 + T cell clones against melanoma cells was at least partly mediated by TRAIL ( 3 ). In this study, we characterized the expression and function of TRAIL on human PBT cells and found that both CD4 + and CD8 + PBT cells express TRAIL upon TCR/CD3 stimulation, especially in the presence of type I IFNs. This indicated that TRAIL-mediated cytotoxicity is not confined to particular T cell clones but can be generally involved in TCR/CD3-mediated, antigen-specific cytotoxicity exerted by human T cells, especially when they are exposed to exogenously administrated or endogenously produced type I IFNs. A previous study by others demonstrated the upregulation of TRAIL mRNA in human PBT cells after activation with anti-CD3 mAb or PMA plus ionomycin ( 34 ). In the present study, we demonstrated that anti-CD3 stimulation induced surface TRAIL expression on human PBT cells but only marginally. We found that IFN-α greatly enhanced TRAIL expression on anti-CD3–stimulated PBT cells, whereas IFN-α solely induced TRAIL expression on PBT cells and only marginally. This enhancement of TRAIL expression was a unique feature of type I IFNs (IFN-α and IFN-β) among the various cytokines so far tested. Other cytokines that have been shown to upregulate FasL expression in T and NK cells (IL-2, IL-12, IL-15, and IL-18 [4, 31–33]) or those enhancing T cell cytotoxicity (IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, and IL-18) did not affect TRAIL expression on unstimulated or anti-CD3–stimulated PBT cells. Northern blot analysis showed that the synergistic effect of anti-CD3 and IFN-α was exerted at the transcriptional level. A higher transcriptional induction by IFN-α alone than that by anti-CD3 alone suggests the possibility that TRAIL gene expression is primarily regulated by IFN-α and that anti-CD3 stimulation may act synergistically by upregulating type I IFN receptors (IFNARs) on PBT cells. Binding of type I IFN to IFNAR leads to activation of the receptor-associated tyrosine kinases, Tyk-2 and Jak-1, which phosphorylate signal transducer and activator of transcription (STAT)-1 and STAT-2. Phosphorylated STAT-1 and STAT-2 form a transcriptionally active heterotrimer complex (ISGF3) together with ISGF3γ/p48, which binds to a cis -acting enhancer sequence termed IFN-stimulated response element (ISRE ). Over 30 proteins have been known to be the products of type I IFN–inducible genes whose transcription can be upregulated by ISRE in the flanking region ( 21 , 22 ). Further studies are needed to characterize the regulatory region of the TRAIL gene and to elucidate the regulatory mechanisms for TRAIL expression at the transcriptional level. Type I IFNs have been commonly used for clinical treatments of certain tumors, including melanomas, gliomas, CMLs, and RCCs ( 23 , 24 ). However, the majority of these tumors are resistant to type I IFN-induced growth arrest or apoptosis in vitro. Previous studies in the mouse system have shown that the antitumor effect of type I IFNs in vivo was mediated by host T cells ( 36 , 37 ). Furthermore, the combination of type I IFNs and adoptive transfer of tumor-specific T cells was effective for rejection of certain tumors ( 38 , 39 ). Although it is well known that type I IFNs exert pleiotropic immunomodulatory effects including upregulation of MHC class I expression and enhancement of CTL cytotoxicity ( 21 , 22 ), the effector molecules responsible for mediating the antitumor effects of type I IFNs have not been well characterized. In this study, we revealed that type I IFNs upregulate TRAIL expression on TCR/CD3-stimulated T cells and augment their cytotoxicity against TRAIL-sensitive tumor cells. We found that almost all of the RCC cell lines tested were TRAIL sensitive, and that IFN-α substantially enhanced the cytotoxic activity of TCR/CD3-stimulated PBT cells against these target cells in a TRAIL-dependent manner. Moreover, a recent study by Thomas and Hersey demonstrated that most of the melanoma cell lines they tested were also sensitive to TRAIL-induced apoptosis ( 3 ). Therefore, it is possible that the enhancement by IFN-α of TRAIL-mediated cytotoxicity by TCR/ CD3-stimulated T cells at least partly accounts for the antitumor effects of type I IFNs against RCCs and melanomas. It is known that melanomas and RCCs are highly antigenic, and tumor-infiltrating T lymphocytes (TILs) are frequently observed in patients with RCCs or melanomas ( 40 , 41 ). In such a situation, the administration of type I IFNs would upregulate TRAIL expression on TCR/CD3-stimulated TILs that are reactive to some tumor antigen and thus augment their cytotoxicity against TRAIL-sensitive tumor cells. Type I IFNs would also upregulate MHC class I expression on tumor cells and thus enhance TCR/CD3- mediated upregulation of TRAIL expression on tumor-reactive T cells. In addition, it has been shown that the combination of type I IFNs and certain chemotherapeutic drugs exerts synergistic antitumor effects ( 23 , 24 ). In this respect, it is noteworthy that the expression of a proapoptotic TRAIL receptor, TRAIL-R2/DR5/killer, can be upregulated in some tumor cells by chemotherapeutic drugs in a p53-dependent manner ( 7 ). Therefore, it is possible that the synergistic antitumor effects of type I IFNs and some chemotherapeutic drugs may be at least partly mediated by TRAIL induction by the former and TRAIL-R2 induction by the latter. It is known that T cell cytotoxicity is mediated by perforin and FasL ( 11 – 13 ). Our present study demonstrated that TRAIL constitutes an additional predominant pathway of T cell cytotoxicity potentially against various tumor cells. However, the expression of these effector molecules in PBT cells is differently regulated. We showed previously that the expression of perforin in PBT cells is confined to a minor population of CD8 + T cells that express CD11b and represent memory effector CTLs ( 29 ). We also demonstrated previously that FasL is not expressed on PBT cells but can be transiently expressed on CD45RO + memory CD4 + and CD8 + T cells upon stimulation with PMA plus ionomycin or anti-CD3 mAb ( 42 ). However, the cell surface expression of FasL on activated T cells is rapidly downregulated by shedding mediated by some proteases ( 27 ). Also in the present study, we examined FasL expression in anti-CD3– and/or IFN-α–stimulated PBT cells (data not shown). Rehybridization of the Northern blot shown in Fig. 4 with a human FasL cDNA probe showed that FasL mRNA expression was induced by anti-CD3 but not by IFN-α, and no enhancement of anti-CD3–induced FasL mRNA expression was observed upon costimulation with IFN-α. However, we could not find a detectable level of cell surface FasL expression on the anti-CD3–stimulated PBT cells, possibly due to the rapid shedding. Consequently, we could not find a substantial contribution of FasL to the cytotoxic activities of anti-CD3– and/or IFN-α–stimulated PBT cells even against the FasL-sensitive target cells . In contrast to this transient nature of FasL expression, TRAIL can be stably expressed on most CD4 + and CD8 + T cells, especially when costimulated with anti-CD3 and IFN-α . This suggests that TRAIL can mediate more long-lasting T cell cytotoxicity than FasL, which would be beneficial for exerting an antitumor effect. In the case of FasL, the rapid downregulation by shedding has been suggested to be beneficial for avoiding normal tissue damage, such as in hepatitis ( 43 , 44 ). In contrast, it has been shown that TRAIL exerts no apparent toxicity in vivo ( 44 ). Therefore, the specific upregulation of TRAIL by type I IFNs can be expected to augment T cell cytotoxicity against tumor cells without harmful effects on normal tissues. Although this study demonstrated a potential role of TRAIL in mediating T cell cytotoxicity against tumor cells, physiological and pathological roles of TRAIL-mediated T cell cytotoxicity remain largely unknown. It is well known that type I IFNs can be inducible by infection of various viruses, bacteria, and mycoplasmas ( 21 , 22 ). In this respect, TRAIL-mediated T cell cytotoxicity may play an important role in clearance of virus- or intracellular organism–infected cells, which has been shown to be dependent on type I IFNs but not on perforin or FasL ( 14 , 45 ). It is also interesting to note that TRAIL might be involved in the pathogenesis of AIDS. Recently, Jeremias et al. reported that PBT cells from HIV-infected patients but not those from healthy individuals were susceptible to TRAIL-induced apoptosis ( 34 ). Since elevated serum levels of IFN-α were found during HIV infection ( 46 ), it is possible that type I IFN-induced, TRAIL-dependent T cell cytotoxicity might be involved in clearance of HIV-infected cells or in the pathogenesis of AIDS and HIV-associated diseases. This notion is further supported by a recent report showing a contribution of TRAIL to anti-CD3–induced apoptosis of PBT cells from HIV-infected patients ( 47 ). Further studies are now underway to address these issues. | Study | biomedical | en | 0.999997 |
10224286 | Human SDF-1α, 12G5 anti-CXCR4, and anti– SDF-1α antibodies were from R & D Systems, Inc.; human serum albumin (HSA) and BSA, FITC-conjugated F(ab′) 2 fragment of goat anti–mouse IgG, and PMA were from Sigma Chemical Co. The M4 mAb anti–chicken IgM has previously been described ( 17 ). Wild type (wt) and Btk- ( 18 ), Syk- ( 19 ), phospholipase C (Plc)γ2- ( 20 ), BLNK- ( 21 ), or IP3R ( 22 )-deficient chicken DT40 cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% chicken serum, 50 mM 2-ME, 2 mM l -glutamine, and antibiotics. The constructions containing wt and SSLKIL→ AALKAA (4A) mutants of human CXCR4 have been described previously ( 23 ). Cells were transfected by electroporation at 250 V and 960 μF in PBS (10 7 cells/0.5 ml). 20 μg expression constructs were cotransfected with 2 μg pBabe-puro r vector ( 24 ). Transfectants were selected in 0.5 μg/ml puromycin 24 h after electroporation. The presence of CXCR4 surface expression was determined by FACS ® analysis with 12G5 mAb and FITC-conjugated secondary antibody. In each condition: DT40-wt + CXCR4 (wt or 4A), Plcg2 −\\− + CXCR4 (wt or 4A). Two clones were analyzed for the experiments; they had comparable and homogenous levels of expression ranging from 120 to 200 arbitrary units (data not shown). DT40 cells (10 6 cells per condition) were washed and resuspended in 100 ml RPMI 1640 and 0.25% HSA and incubated for 1 h at 39°C in the presence of different concentrations of anti-BCR antibodies. Cells were then added to the top chamber of a 6.5-mm diameter, 5-μm pore polycarbonate transwell culture insert (Costar Corp.); the lower chamber contained RPMI 0.25% HSA alone or supplemented with 100 nM SDF-1α. Migration proceeded for 3 h at 39°C. Transmigrated cells were then vigorously suspended and counted with a FACScan™ ( Becton Dickinson ) for 20 s at 60 μl/min, with gating on forward and side scatter to exclude debris. 100% migration was obtained by counting cells added directly to the lower chamber. Cells expressing wt or the 4A mutant of human CXCR4 were resuspended in RPMI 1640 and 0.25% HSA at 10 7 cells/ml. They were then diluted twice with the same buffer or with medium supplemented with 200 nM SDF-1α, 200 nM PMA, or 20 μg/ml anti-BCR mAb and kept either at 4°C (for T = 0) or incubated at 39°C for 1 or 2 h. All subsequent steps were carried out at 4°C. Cells were washed once in staining buffer (PBS, 0.5% BSA, 0.05% azide, and 5% normal goat serum) and incubated in the presence of 12G5 anti-CXCR4 antibodies for 1 h. After two washes, primary antibodies were detected using a FITC-conjugated F(ab′) 2 fragment of goat anti–mouse IgG. Signals were acquired on a FACScan™. Results are given as percentage of controls, 100% corresponding to cells incubated in medium alone. No inhibition of 12G5 binding was found when cells were preincubated with SDF-1α, PMA, or anti-BCR at 4°C, showing that modulation of 12G5 binding was the consequence of an active process. Further controls included absence of staining of nontransfected cells by 12G5 mAb (data not shown) or of CXCR4-transfected cells by an isotype control primary antibody . To determine if the chicken B cell line DT40 was an accurate model for SDF-1α–dependent migration and BCR-induced arrest, we characterized the ability of these cells to migrate in response to this chemokine. DT40 cells were placed on the upper side of a transwell apparatus, and human SDF-1α was placed on the opposite side. DT40 cells migrated efficiently to this chemokine. The migration was specifically inhibited by anti– SDF-1α antibody but not by an irrelevant antibody (not shown), demonstrating the specificity of this migration effect. Cross-linking of the BCR on DT40 cells with the murine mAb M4 for 1 h resulted in a dose-dependent inhibition of SDF-1α–mediated migration, resulting in full inhibition at 5 μg/ml. The ability of this chicken cell line to respond to the human chemokine further confirms that SDF-1α and its receptor are highly conserved among diverse species. Although the chicken version of SDF-1α and its receptor have not been characterized, it is likely that a high degree of sequence conservation will be found for this species as well. A series of homozygous cell lines deficient in specific BCR signaling components has been generated in DT40 cells ( 18 – 22 ). These mutant lines were tested for their ability to migrate in response to SDF-1α and to arrest upon BCR cross-linking. As seen in Fig. 2 , all of the mutants tested, whether deficient in either early (syk) or late (Btk, BLNK, Plcγ2, IP 3 R) components of BCR-induced signaling, migrated in response to SDF-1α. In contrast, the BCR-induced arrest of SDF-1α–directed migration was observed in some, but not all, of the mutants tested. Although cells deficient for molecules involved in Plcγ2 activation such as syk, BLNK, Btk, and Plcγ2 were unable to mediate BCR-induced arrest of SDF-1 migration, IP 3 R-deficient cells (generated by deletion of the three IP 3 receptor genes) retained their ability to migrate in response to SDF-1α and arrest upon BCR cross-linking. DT40 cells have three IP 3 receptors that mediate the efflux of calcium from intracellular stores in response to IP 3 ( 22 ). A triple knockout of these receptors is unable to trigger intracellular calcium release in response to BCR-induced IP 3 stimulation yet maintains its ability to arrest SDF-1α–mediated migration. IP 3 and diacylglycerol are both produced in response to Plcγ2 activation. As BCR-induced arrest is Plcγ2 dependent but IP 3 R independent, it implies that the pathway triggered by Plcγ2, which is affected in BCR- induced arrest of SDF-1α migration, is diacylglycerol dependent, which, in turn, activates PKC. Thus, the dependence on Plcγ2 in the absence of IP 3 -stimulated release of calcium implies that the mechanism by which BCR cross-linking results in migration arrest to SDF-1α may be dependent upon PKC activation through Plcγ2. One mechanism by which BCR activation could lead to arrest of SDF-1α– mediated migration might result from BCR-induced downregulation of the SDF-1α receptor from the cell surface by a PKC-dependent internalization of the SDF-1α receptor. Previous studies have demonstrated that the SDF-1 receptor, CXCR4, is rapidly internalized upon activation of PKC by phorbol esters that, in turn, can be blocked by inhibitors of PKC ( 25 , 26 ). To determine if a BCR-induced, PKC- dependent internalization of the CXCR4 pathway is present in DT40 cells, the human SDF-1α receptor, CXCR4, was stably transfected into wt and Plcγ2-mutant DT40 cells. Cell surface expression of human CXCR4 on DT40 cells is downregulated in response to BCR cross-linking, phorbol ester treatment, or SDF-1α exposure . However, BCR-induced downregulation of CXCR4 is blocked in the Plcγ2-deficient DT40 background, which correlates with the inability of this mutant to display BCR-mediated arrest of SDF-1α migration. To determine if Plcγ2 is upstream, downstream, or pleiotropic in relation to CXCR4, this mutant was tested for its ability to respond to phorbol esters or SDF-1α. CXCR4 surface expression is downregulated normally in Plcγ2-deficient cells in response to phorbol esters or SDF-1α , indicating that Plcγ2 lies upstream of CXCR4 in the BCR-induced internalization pathway and that SDF-1α–induced internalization is independent of Plcγ2 activation. These results thus suggest that the BCR-induced arrest of SDF-1α–directed migration may be due in part to CXCR4 internalization triggered by BCR-mediated stimulation of Plcγ2 and PKC activation. In addition, they show that SDF-1α and BCR activation lead to CXCR4 surface downregulation through different pathways in DT40 B cells. Signoret et al. ( 23 ) have demonstrated that a SSXXIL motif, similar to that required for endocytosis of CD4 and the TCR complex, is required for phorbol ester–induced, but not ligand-induced, internalization of CXCR4. To determine the contribution of this motif to the BCR-induced internalization of CXCR4 in DT40 cells, we generated stable transfectants of DT40 wt or Plcγ2-mutant cells expressing a CXCR4 mutant in which the SSLKIL sequence was replaced by AALKAA. As seen in Figs. 3 and 4 , wt DT40 cells expressing the wt human CXCR4 receptor internalize this receptor in response to BCR cross-linking, SDF-1α treatment, and PMA stimulation. In contrast, the SSLKIL→ AALKAA mutant CXCR4 receptor , whether expressed in wt or Plcγ2-mutant DT40 cells, was incapable of BCR- or PMA-induced internalization but retained significant receptor downmodulation in response to SDF-1α. The 4A mutant CXCR4 was unable to migrate in response to SDF-1α, either in the presence or absence of BCR cross-linking (data not shown). The basis for this migration defect has not been determined. BCR- and Plcγ2-dependent internalization of CXCR4 thus appears to utilize the same pathway as PMA, a PKC-dependent downmodulation of this receptor. AgR signaling determines B cell maturation, selection, and orientation within lymphoid organs. Progression from newly generated B cells into MZ and follicular B cells is driven by BCR signaling and is associated with specific anatomic localization inside the spleen. Supra-threshold AgR engagement redirects B cells from follicles, MZ, or blood circulation toward the periarteriolar lymphoid sheath. Depending on their ability to direct cognate interaction with T cells, a humoral response will emerge or B cells will die in a few days ( 2 , 5 , 6 , 27 ). It is now clearly established that chemokines play an important role in these relocalization processes. Thus, the expression of BLR1 is associated with follicular B cell maturation and is required for their tropism in the spleen and Peyer's patch, whereas SDF-1α and SLE responses are rapidly regulated upon BCR engagement ( 12 , 14 , 16 ). The BCR-induced downregulation of CXCR4 demonstrated here offers a first example in which differential AgR engagement might promote differential responsiveness to a chemokine and allow repertoire-based interclonal competition for migration toward a restricted, chemokine-secreting environment ( 28 ). However, as seen in Fig. 3 , BCR cross-linking results in a twofold reduction in CXCR4 expression. Although this change in expression may account for some of the migration inhibition seen, it suggests that other pathways may be involved as well. The generation of CXCR4 mutants that are deficient in BCR-induced downregulation yet retain chemotactic response to SDF-1α will allow further dissection of the contribution of this pathway to the antigen-driven compartmentalization of lymphocytes. In addition, the definition of SDF-1α secretion sites will provide important clues toward the understanding of B cell migration and selection. In vitro studies have shown that pro-B cells are dependent on contact with stromal cells and cytokines for survival, whereas cells expressing the pre-B cell receptor are only dependent on soluble factors ( 29 , 30 ). Bone marrow stromal cells produce SDF-1α, and pro-B cells respond to this chemokine ( 13 , 31 ). It is tempting to transpose our data from the early steps of pro-B to pre-B cell transition. Thus, like BCR, pre-BCR signaling might induce the downregulation of CXCR4 and block SDF-1α–dependent migration of pre-B cells toward stromal cells. Therefore, CXCR4 downregulation might allow B cells to lose stromal cell tropism upon successful rearrangement of their IgH gene and signaling through the pre-B cell receptor. This mechanism could guarantee the restriction of rare niches to pro-B cells. In agreement with the importance of SDF-1 during early B cell differentiation, SDF-1 and CXCR4 knockout mice show a profound defect in pro-B cell production ( 32 – 34 ). The present definition of a pathway from BCR to the CXCR4 receptor and of a motif responsible for this coupling may allow the construction of mutants to directly test the role of this pathway in vivo. Such analysis might provide insights that will define how Ag-dependent competitive migration participates in B cell maturation and response to Ag. | Study | biomedical | en | 0.999995 |
10224287 | A 7-kb PCR product amplified from murine 129/Sv genomic DNA and containing exon 2 of the Lfa-1 gene was subcloned into pSP72. A selection cassette containing stop codons in all three frames, an independent ribosomal entry sequence (IRES) followed by the LacZ gene with an SV40 polyadenylation signal, and a neomycin phosphotransferase gene was introduced into exon 2. Culture and transfection of GK129 embryonic stem (ES) cells was done as described previously ( 27 ). EcoRI-digested ES cell DNA was analyzed by Southern blot analysis using probe A, and homologous recombination was confirmed using probe B . Blastocyst injection and breeding have been described previously ( 27 ). Mice were genotyped from EcoRI-digested tail DNA by Southern blot (data not shown) or PCR . A multiplex PCR using primers on either side of the selection cassette and one within the neomycin resistance gene yielded fragments of 90 bp (wild-type) and 660 bp (LFA-1–deficient), respectively. All mice were kept under specific pathogen–free conditions and in accordance with United Kingdom Home Office regulations. The mice were monitored on a six-monthly basis for a wide selection of parasites, bacteria, and fungi and were regularly found to be pathogen free. LN sections from sentinel mice belonging to litters used in the reported experiments were inspected for the presence of germinal centers by hematoxylin and eosin staining and for positive MAdCAM-1 staining in pLNs ( 28 ). These latter indicators of an inflammatory response were routinely negative for mice housed in London and Hamburg. The following purified rat mAbs were used in this study: LFA-1 α subunit mAbs H35.89.9 (IgG2b) and H68 (IgG2a), both obtained from Dr. Michel Pierres (Centre d'Immunologie, INSERM-CNRS de Marseille-Luminy, Marseille, France); α4 subunit mAb PS/2 (IgG2b); anti–VCAM-1 mAb MK2.7 (IgG1); anti–MAdCAM-1 mAb MECA-367 (IgG2a), obtained from American Type Culture Collection; anti-PNAd mAb MECA-79 (IgM; obtained from Drs. Mark Singer and Steve Rosen, University of California, San Francisco, CA); MECA-325 (IgG1), obtained from Dr. A. Duijvestijn (University of Limburg, Maastricht, Netherlands); CD54 mAb YN1/1.7 (IgG2a; obtained from Dr. Fumio Takei, Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada); CD4, CD8, and B220 mAbs ( PharMingen ); and control rat IgG1 mAb PyLT-1 (IgG1; Imperial Cancer Research Fund) and control null rat IgG1 and IgG2a mAbs ( PharMingen ). Secondary detection antibodies were FITC-conjugated goat anti–rat Ig (Jackson ImmunoResearch Laboratories) and biotinylated rabbit anti–rat Ig (Vector Laboratories). Fab fragments were prepared by papain digestion using either the ImmunoPure ® Fab kit ( Pierce Chemical Co. ) or HPLC to remove any residual intact IgG ( 7 ). Single cell suspensions from 8–12-wk-old mice were prepared by mincing or rubbing between glass slides with frequent rinsing with 5% FCS/RPMI or 0.2% BSA/PBS. Bone marrow cells were harvested by flushing with 5% FCS/RPMI via cut ends of tibias and femurs, followed by disaggregation and filtering through nylon gauze. Cells were washed and incubated at 5 × 10 6 cells/ml with specific primary mAbs, then FITC-conjugated goat anti–rat Ig (1:200; Jackson ImmunoResearch Laboratories) at 4°C for 30 min. Cells were fixed in 1% HCHO/PBS before analysis on a FACScan™ instrument ( Becton Dickinson ). Lymphocytes from pLNs and mLNs of LFA-1 +/+ and LFA-1 −/− mice were prepared and adjusted to 5 × 10 6 cells/ml in 5% FCS/RPMI for labeling as described ( 29 ). LFA-1 +/+ lymphocytes were incubated with 0.2 mM CellTracker™ (CT) Green 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes) and LFA-1 −/− lymphocytes with 5 mM CT Orange 5-chloromethyl tetramethylrhodamine (CMTMR) at 37°C for 45 min. The two cell preparations were washed and counted, then mixed together to achieve a 1:1 ratio of differentially labeled LFA-1 +/+ and LFA-1 −/− lymphocytes for injection into recipient 8–12-wk-old C57BL/6 mice for a period of 1 h. A correction factor was applied to the subsequent data if the lymphocyte ratio was not precisely 1:1. Reversing the CT dyes had no effect on results. The lymphocytes were frequently tested for activation status before and after the labeling procedure by measuring the level of L-selectin that can be enzymatically cleaved after an activating stimulus ( 30 ). In general, a total of 2 × 10 7 cells in a volume of 200 μl was injected per tail vein. In experiments involving antibody blocking, the cells and mAbs were coinjected. Fab fragments were used in amounts of 250–400 μg per mouse. Irrelevant control Fab fragments were without effect. Lymphocytes obtained as above were labeled with 20 μCi 51 Cr/ml as described previously ( 29 ), and dead cells removed by centrifugation on a 17% Nycodenz cushion (Nycomed). 5 × 10 6 lymphocytes in 200 μl were injected into the tail vein of recipient 8–12-wk-old C57BL/6 mice. mAbs were coinjected as Fab fragments and at 300 μg/200 μl. Sample groups of four mice were used in each condition. The mice were killed after 1 h, and the distribution of radioactivity was determined for the different organs and for the residual body mass. PBLs were calculated for a blood volume of 1.5 ml. LN cells were labeled by dissolving 1 mg digoxigenin in 0.5 ml DMSO and incubating the lymphocytes for 15 min at a final concentration of 5 × 10 7 cells/ml with 60 μg digoxigenin. After washing, 0.8 × 10 7 cells were injected intravenously, and pLNs were removed 30 min later and frozen in liquid nitrogen. To localize the donor cells in the HEVs, two antigens were revealed simultaneously as described ( 31 ). In brief, cryostat sections were incubated with the anti-HEV mAb MECA-325, which was revealed by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique (blue ). The donor cells were identified by a peroxidase-conjugated antidigoxigenin antibody (brown) as described previously ( 31 ). The area of the HEVs (excluding the lumen) together with the area within 4-cell diameters surrounding the vessel was then determined ( 31 ), and the number of injected cells adhering to and within the endothelium, and within the tissue was analyzed. Sections of 6 μm were cut from snap-frozen tissues of pLN, mLN, and PP and placed on silane-coated slides which were fixed in acetone for 10 min and allowed to air dry. Slides were washed in standard Tris-buffered saline (TBS), pH 7.6, and primary mAb was added for 1 h at room temperature. mAbs were used at optimal dilution: 10 μg/ml for anti– VCAM-1 mAb MK2.7 (IgG1) and IgG1 isotype control mAb PyLT-1, MECA-79 at 4 μg/ml, and MECA-367 as tissue culture supernatant. Sections were also tested with null IgG1 and IgG2a mAbs ( PharMingen ; data not shown). Biotinylated rabbit anti–rat Ig (1:100; Vector Laboratories) was added for 35 min. A tertiary stage was carried out using the StreptABComplex/HRP kit following the manufacturer's instructions (K377; Dako), and the slides were incubated again for 35 min before exposure to 1 mg/ml 3,3′diaminobenzidine. Lfa-1 , the gene for the LFA-1 α subunit, was mutated in ES cells using a replacement-type targeting vector and the strategy shown in Fig. 1 a. G418-resistant colonies were identified by Southern blot analysis of EcoR1-digested ES cell genomic DNA using the probe shown in Fig. 1 a. Of 400 G418-resistant ES cell colonies, 10 correctly targeted colonies were identified. Three different homologously targeted ES cell clones were injected into C57BL/6 blastocysts, and all of them were transmitted through the germline. Southern blot analysis of mouse tail DNA enabled identification of LFA-1 wild-type (+/+), heterozygous (+/−), and gene-deleted status (−/−) (data not shown). This was confirmed by PCR analysis . To establish that the capacity for LFA-1 synthesis had been completely ablated, leukocyte populations were examined for LFA-1 expression and function. Thymocytes from LFA-1 −/− mice were compared with LFA-1 +/+ littermates and shown to be devoid of LFA-1 expression . The lack of LFA-1 surface expression was also demonstrated for leukocytes from other lymphoid tissue, including pLN, mLN, PP, spleen, bone marrow, and blood (data not shown). To test for absence of LFA-1 function, control phorbol ester–treated LFA-1 +/+ thymocytes were adhered to murine ICAM-1–transfected COS-7 cells, and this adhesion could be blocked with anti–LFA-1 mAb H68 (data not shown). In contrast, no adhesion was evident with LFA-1 −/− thymocytes. Therefore, testing for both expression and function of the LFA-1 receptor provided further proof that the LFA-1 −/− mice were totally deficient in this β2 integrin. Analysis of LFA-1 −/− compared with LFA-1 +/+ mice revealed an increase in spleen size and a decrease in size of the pLNs, as observed previously ( 25 ). For 30-g male mice ( n = 29), the LFA-1 −/− spleens were 1.7 times larger than those of LFA-1 +/+ mice (182.0 ± 56.4 compared with 107.8 ± 30.0 mg). For 25-g female mice ( n = 37), the same comparison yielded a 1.2-fold increase in weight (137.6 ± 33.0 compared with 118.5 ± 42.3 mg). Second, the pLNs from LFA-1 −/− mice were smaller than those from LFA-1 +/+ mice, with an average decreased lymphocyte number for LFA-1 −/− mice of ∼30% that of the LFA-1 +/+ littermates (3.54 ± 0.74 × 10 6 compared with 12.15 ± 2.90 × 10 6 ; n = 9). To discover whether alteration had occurred in lymphocyte numbers in general or in a particular subtype, we next analyzed CD4 and CD8 T cell subsets and used mAb B220 to detect B cells. In the pLNs, substantial loss in numbers of CD4 and CD8 T cells as well as a deficiency in B cells was observed . Furthermore, there was no difference in naive versus memory phenotype in LFA-1 −/− and LFA-1 +/+ mice as indicated by expression levels of L-selectin, CD44, and CD45RB antigens (data not shown). In the other LNs, wild-type and LFA-1–deficient mice were similar in terms of lymphocyte numbers and subsets, as was expected from the fact that these LNs were comparable in size between the two types of mice. In spleen, there was a significant increase in CD4 T cells and CD8 cells . To gain further information about the diminished cellularity of the pLNs and to investigate trafficking capabilities of LFA-1–deficient lymphocytes, short-term migration studies were performed. The approach taken was to label LFA-1 −/− and LFA-1 +/+ lymphocytes with two distinguishable CT orange and green fluorescent dyes and inject equivalent numbers into the tail vein of C57BL/6 recipients. This allowed direct comparison of the homing activity of the lymphocytes within each LN setting ( 29 ). An example of the methodology is illustrated in Fig. 3 a. When the pLN lymphocytes were examined for proportions of CT orange to green after 1 h of homing, it was observed that ∼13% of LFA-1 −/− cells compared with LFA-1 +/+ cells had migrated into the pLNs . We then examined the relative ability of LFA-1 −/− cells to gain entry into other secondary lymphoid tissues . When compared with LFA-1 +/+ , the LFA-1 −/− cells were most poorly represented in pLNs (0.21 ± 0.01), followed by mLNs (0.51 ± 0.02) and PPs (0.68 ± 0.03). In contrast, there was a marked increase in the LFA-1 −/− cells in the spleen (1.29 ± 0.04) which is likely due to redistribution of lymphocytes from LN to spleen. These findings on the effect of LFA-1 absence on the trafficking of lymphocytes were confirmed using the technique of tracking 51 Cr-labeled lymphocytes to compare homing in LFA-1 −/− and LFA-1 +/+ mice (data not shown). When CD4, CD8, and B220 + lymphocyte subsets were analyzed separately, the ratio of migrated to injected lymphocytes was identical among the subsets whether derived from wild-type or LFA-1 −/− mice, indicating that the lack of LFA-1 caused equal difficulty for all types of lymphocytes to gain entry into the LNs (data not shown). The above findings showed that LFA-1 −/− cells migrated less effectively, particularly to the pLNs, within the 1-h period but did not reveal the stage at which the lack of LFA-1 had its effect. To discover where LFA-1 deficiency caused difficulty, we turned to histochemistry and examined pLN tissue sections from mice injected with LFA-1 −/− and LFA-1 +/+ lymphocytes for 30 min. As shown in Fig. 4 , the LFA-1–deficient lymphocytes bound less well to HEVs than the LFA-1-expressing lymphocytes. The total numbers of lymphocytes at the HEV level and surrounding 4-cell diameters were 285 ± 70 cells/mm 2 for LFA-1 +/+ and 85 ± 10 cells/mm 2 for LFA-1 −/− lymphocytes ( n = 3). These data suggest that loss of LFA-1 expression reduces lymphocyte adherence to and transmigration across HEVs by 70%, thereby causing substantially diminished recruitment into pLNs. When the cells were divided between those adhering to and within the HEVs and those found within 4-cell diameters beyond HEVs, the following proportions were observed: for LFA-1 +/+ lymphocytes, 73 ± 7 and 27± 2%; for LFA-1 −/− lymphocytes, 78 ± 1 and 22 ± 1%, respectively. These data suggest that the major block is at the level of the HEVs. If the deficiency had been acting selectively at the level of the transmigration step, a larger accumulation of lymphocytes at the HEV level would have been expected. Although LFA-1 was obviously playing a critical role at the HEV level, a proportion of lymphocytes remained capable of migrating into lymphoid tissue . Of other adhesion receptors that might substitute for LFA-1, the α4 integrins were attractive candidates, as they were active in migration in other situations. In the next series of experiments, the migratory behavior of CT-labeled LFA-1 −/− and LFA-1 +/+ cells was compared in host animals simultaneously injected with Fab fragments of α4 mAb or α4β7-specific mAb. The α4 mAb completely prevented the migration of the residual numbers of LFA-1 −/− lymphocytes homing to pLNs, mLNs, and PPs . In addition, the α4β7-specific mAb blocked lymphocyte entry into pLNs to ∼22% and completely prevented migration into mLNs and PPs. There was no significant effect of α4 or α4β7 mAbs on migration into spleen. These results were duplicated in a 51 Cr lymphocyte labeling experiment in which α4 mAb abolished all entry into the pLNs, mLNs, and PPs of LFA-1 −/− lymphocytes (data not shown). The α4β7 mAb blocked entry into mLNs and PPs and inhibited entry into pLNs to ∼35% (data not shown). It was possible that the α4-dependent LFA-1 −/− cells migrating into pLNs were a specific subset of lymphocytes expanded in the LFA-1–deficient environment. However, phenotyping of these cells using a third fluorescent tag of Tricolor-conjugated anti–rat Ig showed the migrated LFA-1 −/− and LFA-1 +/+ lymphocytes to have identical phenotypic profiles with regard to their levels of α4 and α4β7 integrins and L-selectin (data not shown). The suggestion that α4 integrins, α4β7 acting together with α4β1, have a role in migration to the pLNs has not previously been recognized. To further confirm the findings and to gain information about tissues other than secondary LNs, 51 Cr-labeled lymphocytes from normal BALB/c mice were coinjected with 300 μg of Fab fragments from either α4 mAb, LFA-1 mAb, or both into host BALB/c mice. The findings with the single mAbs were as reported previously , but the combination of α4 and LFA-1 mAbs totally prevented migration into pLNs as well as mLNs and PPs . There was also an appreciable decrease in migration into intestinal tissue and into the “body” (see below). This mAb blockade provoked an increase in circulating blood cells as well as an increase in migration into the spleen. Put together, these observations provide evidence that the α4 integrins operating together with LFA-1 have an essential role in migration to pLNs, as found previously for other secondary LNs. The identification of the ligand(s) recognized by the α4 integrins, particularly in pLNs, was next explored. Using a MAdCAM-1–specific mAb, homing to both PPs and mLNs was substantially prevented, as described previously . However, the mAb had no significant effect on migration of LFA-1 −/− lymphocytes into pLNs and spleen. VCAM-1 is another ligand recognized by both α4β1 and α4β7 integrins ( 32 , 33 ), and in the present experiments, the anti–VCAM-1 mAb MK2.7 completely eliminated homing of LFA-1 −/− lymphocytes to pLNs and also substantially blocked entry into both mLNs and PPs although not into spleen . The foregoing experiments showed that VCAM-1 has a role in the trafficking into LNs, suggesting that this ligand is indeed expressed on the HEVs. To further address this question, immunohistochemical staining was performed using fresh frozen tissue sections of pLNs, mLNs, and PPs from LFA-1 −/− and LFA-1 +/+ mice and normal C57BL/6 mice. In pLNs, anti–VCAM-1 mAb MK2.7 (IgG1) was identified to label the same HEVs as PNAd-specific mAb MECA-79 . Of interest was the observation that the MAdCAM-1 mAb MECA-367 was negative, as was an IgG1 isotype control mAb, PyLT-1 . In mLNs where both MAdCAM-1 and PNAd are chiefly coexpressed on HEVs ( 34 ), VCAM-1 is also present, whereas the isotype control mAb was negative . In a preliminary quantitative study of mLNs, complete overlap was observed in HEV staining of VCAM-1 with one other HEV ligand, MAdCAM-1 (data not shown). The level of VCAM-1 expression was comparable on HEVs from all LNs and from LFA-1 +/+ , LFA-1 −/− , and C57BL/6 mice (data not shown). Therefore the absence of LFA-1 did not induce a compensatory increase in expression of VCAM-1. To confirm that VCAM-1 is expressed on the luminal surface of the endothelium and therefore accessible to recirculating lymphocytes, C57BL/6 mice were intravenously injected for 10 min with rat anti–VCAM-1 mAb MK2.7 or anti-HEV mAb MECA-325. On tissue sections stained with anti–rat Ig, both the pLN and mLN HEVs scored positive to a similar degree for both the anti– VCAM-1 and the anti-HEV mAbs (data not shown). This experiment provided a second type of histochemical proof that VCAM-1 is luminally expressed on the HEVs of the LNs. In the migration experiments with 51 Cr-labeled lymphocytes, significantly fewer lymphocytes distributed into the mouse carcass after treatment with a combination of LFA-1 and α4 mAbs . To discover the identity of the relevant tissue compartment, individual body parts of muscle, heart, kidney, thymus, and bone were isolated and counted separately. The target tissue with diminished lymphocyte migration was determined to be the limb bones, indicating that lymphocyte recirculation was occurring in the bone marrow (data not shown). Within 1 h, a single femur recruited ∼1% of 51 Cr-labeled lymphocytes, suggesting that the total bone marrow compartment attracted lymphocytes at a rate comparable to the 5–10% lymphocyte entry into the combined LNs. Which lymphocytes were migrating into bone marrow was tested by using CT-labeled lymphocytes and subsequent staining with subset-specific mAbs detected with Tricolor-conjugated anti–rat Ig. The ratio of migrated to injected CD4/CD8/B220 subsets was ∼0.5/ 1.0/2.0. This advantage for B cell and disadvantage for CD4 T cells were independent of LFA-1 expression status. LFA-1 +/+ and LFA-1 −/− cells did not significantly differ in their trafficking to bone marrow (ratio of LFA-1 −/− to LFA-1 +/+ was 1.1; data not shown), suggesting that the migration was not solely reliant on LFA-1. However, when LFA-1 −/− lymphocytes were coinjected with α4 mAb, the trafficking to bone marrow was completely abolished . These results suggest that migration into bone marrow can be accomplished either by LFA-1 or in its absence by an α4 integrin. As the α4β7 mAb had a partial effect on migration, whereas α4 mAb blocked migration completely, we conclude that in bone marrow, in contrast to the migration into LNs, α4β1 dominates α4β7. Finally, the VCAM-1 mAb, but not MAdCAM-1 mAb, had an inhibitory effect. Considered together, these results demonstrate that migration of recirculating lymphocytes into bone marrow can be mediated by either LFA-1 or the α4 integrins, and, as in pLNs, that VCAM-1 serves as the chief ligand for the latter receptors. This study shows that LFA-1 has a key role in migration to the pLNs, other LNs, and bone marrow but not into the spleen. Also revealed is a hitherto unrecognized ability of the α4 integrins, α4β7 and α4β1, to compensate for the lack of LFA-1 in lymphocyte trafficking to pLNs and other lymphoid tissues, including bone marrow. In general, these findings highlight common features between lymphocyte homing and the response to inflammatory stimuli and extend the validity of the multistep model of adhesion and transmigration. The LFA-1 −/− mouse described in this report has, as its key phenotypic characteristic, pLNs that contain ∼30% normal lymphocyte numbers, as also noted previously ( 25 ). The decreased trafficking of LFA-1 −/− lymphocytes to the pLNs to ∼20–30% of wild-type lymphocytes suggested that LFA-1 −/− lymphocytes, irrespective of which subset, had difficulty gaining entry to the LNs. This was confirmed by microscopic studies showing that circulating LFA-1 −/− lymphocytes bound poorly to HEVs. Migration into mLNs and PPs was also depressed, but LN cell counts were normal, suggesting compensatory measures were in operation in these tissues but not in pLNs. The decrease in migration of lymphocytes to pLNs is consistent with previous work using function-blocking LFA-1 mAbs ( 10 ) and has recently been reported in another study using LFA-1–deficient mice ( 35 ). Thus, the general importance of LFA-1 in mature lymphocyte trafficking to secondary lymphoid tissue is confirmed. In our studies, LFA-1 −/− lymphocytes were able to gain entry into the pLNs, albeit at a lower level, suggesting involvement of further receptors. We here demonstrate these additional receptors to be the α4 integrins. Skewed receptor usage towards increased expression of α4 integrins in LFA-1 −/− mice as a possible cause of experimental bias was excluded (data not shown). The fact that the presence of mAbs to both LFA-1 and α4 integrins caused complete blockade of lymphocyte entry into normal pLNs and other LNs strongly implies that α4 has a critical role in migration of normal lymphocytes into pLNs. That this role for α4 integrin has not previously been observed might be because LFA-1 has a larger role than α4 integrin in adherence to pLN HEVs than HEVs of other organs. This is in good agreement with intravital microscopy studies which show LFA-1 and L-selectin are the essential codependent pLN-specific adhesion pair for lymphocyte adherence to pLN vessels ( 36 ). Put together, the data suggest that α4 integrins have a less prominent role in adherence to pLN HEVs, but potentially a larger role in the transmigration step. Our data also suggest unexpectedly that α4β7 acts as the major α4 receptor in conjunction with α4β1 in lymphocyte recirculation into pLNs. There has previously been no evidence to link α4β7 with pLN migration, although its dominant role in trafficking into mucosal tissue of the mLNs and PPs is well documented ( 3 , 17 ) and is confirmed here. The integrin α4β1 has usually been associated with inflammatory responses ( 18 – 20 ) rather than with lymphocyte recirculation. However, as these two α4 integrins might be acting either in synergy or in sequence, the extent of the contribution of α4β1 must await a potent murine CD29 blocking mAb. The results imply that the succession of adhesive events in lymphocyte recirculation is similar to that in inflammatory responses, and that the α4 integrins in cooperation with L-selectin and LFA-1 have a more specific and necessary role than previously perceived. The data presented here identify VCAM-1 and not MAdCAM-1 as ligand on pLN HEVs for α4 integrins in spite of α4β7 involvement. The result was unexpected, as α4β1 has been identified as the major VCAM-1 binding integrin in inflammatory responses ( 37 ) in studies using transfectant ( 38 ) and cell lines ( 17 ). VCAM-1 also made a significant contribution to trafficking into mLNs and, to a lesser extent, PPs. This cooperative activity with MAdCAM-1 was also unexpected, as it has previously been considered that entry into these LNs was regulated only by MAdCAM-1. The findings presented here suggest that potential roles for α4β7 in addition to α4β1 should be sought in other circumstances where VCAM-1 is the major ligand. One explanation for having overlooked the importance of VCAM-1 as an HEV ligand is its reported absence from normal lymphoid tissue ( 22 , 23 , 39 ), with the exception of a report on its expression on rat HEVs ( 40 ). In the present study, expression occurred at comparable levels on HEVs in pLNs, mLNs, and PP LNs and without obvious difference between LFA-1 −/− mice, their wild-type littermates, C57BL/6, or BALB/c mice. Such broad and constant presence of VCAM-1 implies it is constitutively expressed rather than as a consequence of an inflammatory signal or a compensatory mechanism in LFA-1 −/− mice. The involvement of VCAM-1 raises the issue as to whether lymphocytes can use this ligand for migration across HEVs into the LN as well as adhesion to the luminal surface of HEVs. In this study, the lack of LFA-1 decreased migration across HEVs by ∼25% only, suggesting involvement of other receptor/ligand pair(s) (data not shown). For HUVECs, VCAM-1 is reported to be restricted to an apical location and therefore available for adhesion but not for transendothelial migration ( 41 ). However, monocytes can transmigrate HUVECs, making use of α4/VCAM-1 independently of β2 integrins ( 42 ). In mice, two major forms of VCAM-1 have been identified, the common seven-domain form and also a three-domain glycosylphosphatidylinositol (GPI)-linked splice variant which is induced by inflammation ( 43 , 44 ). It is of interest that in polarized epithelial cells, the GPI-linked VCAM-1 was found apically, whereas the seven-domain VCAM-1 was localized to the basolateral surface, where it could theoretically serve as a ligand for transmigration ( 45 ). The issue of where VCAM-1 is expressed, particularly in murine HEVs, warrants further investigation. Migration of recirculating lymphocytes to bone marrow, a primary lymphoid tissue, has been noted in the past ( 46 ). Bone marrow serves as a site of hematopoiesis and for B cell maturation in the mammal. It can also act as a reservoir for a primary immune response in the absence of a spleen and presence of L-selectin mAb ( 47 ). In this study, we find an unexpectedly high degree of mature lymphocyte recirculation in which B cells dominate into bone marrow. We suggest here that bone marrow, but not the thymus (data not shown), should be considered as a major part of the lymphocyte recirculation network. Migration to bone marrow is restricted by adhesion mechanisms. Both human ( 48 ) and murine ( 49 ) hematopoietic progenitors make use of α4 integrin to lodge in the bone marrow. The α4 integrins are also reported to be necessary for correct lymphocyte development ( 50 ). We show that normal lymphocyte recirculation through the bone marrow is regulated by LFA-1 and the α4 integrins and, in contrast to the LNs, α4β1 substantially dominates α4β7. In this context, it is again VCAM-1 and not MAdCAM-1 which acts as ligand for the α4 integrins. VCAM-1 is expressed constitutively by bone marrow sinusoidal vasculature ( 51 , 52 ), and as progenitor cells have been demonstrated to roll on bone marrow endothelial VCAM-1 ( 48 ), it can be speculated that other lymphocytes might behave similarly. However VCAM-1 is also expressed constitutively by stromal reticular cells ( 53 , 54 ), and it is possible that lymphocytes may be retarded not only at the level of the endothelium but also within the bone marrow matrix. Factors produced by the stromal cells might be beneficial for the maintenance of the recirculating lymphocytes. For example, the interaction with VCAM-1 might suppress lymphocyte apoptosis ( 55 ). On the other hand, the incoming cells might contribute to the regulation of hematopoiesis. In summary, the observations presented here extend and validate the concept of a multistage adhesion response requiring selectins and integrins. Rather than exclusive LN-specific use of receptors such as LFA-1 and L-selectin in trafficking to pLNs or α4β7 to mucosal sites, we have presented evidence that LFA-1 and the α4 integrins can operate in migration of unstimulated lymphocytes to the pLNs as well as other secondary LNs and bone marrow. These general features, including the use of VCAM-1 as an α4 integrin ligand, resemble the response to an inflammatory signal. Our findings suggest a unifying hypothesis for migration of lymphocytes across HEVs into secondary lymphoid tissue and also establish further parallels between the activities of adhesion receptors in the “homing” context and the mechanisms used in the more sporadic responses to tissue injury and inflammation. | Study | biomedical | en | 0.999995 |
10224288 | The wild-type S . typhimurium strain SL1344 and its isogenic mutant derivative strain SB161, which carries a nonpolar mutation in the invG gene, have been described previously ( 35 ). Plasmid J3HmPAK-3 encoding HA epitope–tagged mPAK-3 has been described previously ( 27 ). Plasmid pSB961 was constructed by subcloning the 1.7-kb BamHI fragment from J3HmPAK-3KR plasmid into the EcoRI site of pSB936. The resulting plasmid encodes the mPAK-3 kinase– defective mutant at the first cistron and the green fluorescent protein (GFP) at the second cistron. Plasmids pSB969, pSB970, pSB971, and pSB972 were constructed by subcloning the 1.7-kb BamHI fragments from pGEM-P1PAK (encoding PAK A12A14 ), pGEM-P2PAK (encoding PAK A36A39 ), pGEM-P3PAK (encoding PAK A165A168 ), and pGEM-P4PAK (encoding PAK A213A216 ), respectively, into the EcoRV site of the dicistronic expression vector pSB965. The resulting plasmids encode the different PAK mutants at the first cistron and GFP at the second cistron. Plasmid pSB974, which encodes Cdc42Hs C40 , was constructed by introducing a point mutation (codon 40 of Cdc42Hs changed from TAT to TGT) into the coding sequence of wild-type Cdc42Hs encoded by plasmid pSB944. The resulting plasmid encodes Cdc42Hs C40 at the first cistron and GFP at the second cistron. COS-1 cells were grown to subconfluence on glass coverslips placed in 24-well culture dishes and transfected by the calcium phosphate method ( 36 ) using a total of 1 μg of DNA per well. For PAK localization studies, COS cells were infected with wild-type S . typhimurium with a multiplicity of infection (moi) of 20. At different times after infection, cells were fixed in 3.7% formaldehyde in PBS for 1 h, permeabilized in the presence of 0.15% Triton X-100 for 5 min, incubated for 1 h in blocking buffer (PBS, 5% milk), and stained as described above using a rabbit polyclonal antibody that recognizes all isoforms of PAK ( Santa Cruz Biotech, Inc. ). Rhodamine-conjugated phalloidin (1 U/ml in PBS; Molecular Probes) was used to visualize the actin cytoskeleton, and 4′,6′-diamidino-2-phenylindole (DAPI) to stain DNA. Coverslips were mounted onto slides with Vectashield mounting solution (Vector Labs, Inc.) and visualized under a 40× objective in a Nikon Diaphot fluorescence microscope. Images were captured with a Hamamatsu 75i CCD camera and pseudocolored using an Argus 20 image processor. Bacterial internalization was measured as described elsewhere ( 8 ). In brief, COS-1 cells grown on glass coverslips were transfected with a total of 1 μg of DNA of dicistronic vectors expressing different forms of PAK or Cdc42Hs in the first cistron and GFP in the second cistron. 48 h after transfection, the cells were washed and infected at an moi of 40 with wild-type S . typhimurium . After 1 h of infection, cells were washed and internalized bacteria were detected using a staining protocol that allows the distinction between extracellular and intracellular bacteria ( 8 ). Cells expressing the different PAK or Cdc42Hs constructs were identified by the coupled expression of GFP. COS-1 cells were grown in 6-cm tissue culture dishes and transfected by the calcium phosphate method using a total of 10 μg of DNA. When appropriate, 48 h after transfection cells were infected with wild-type S . typhimurium or the isogenic invG mutant strain SB161 with an moi of 20. At different times after infection, cells were lysed in lysis buffer (1% NP-40, 40 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 25 mM NaF, 1 mM sodium vanadate), and the levels of JNK or PAK activity were measured by an immunocomplex kinase assay as described elsewhere ( 37 ). The relative amounts of substrate phosphorylation were quantitated with a PhosphorImager (Storm; Molecular Dynamics). Readings were standardized relative to a given sample that was assigned the value 1. The levels of Flag-JNK-1, HA-PAK, or M45-SopE in cell lysates were determined by immunoblotting with the respective antibodies. To gain insight into the role of downstream effectors of Cdc42 in the S . typhimurium – induced cytoskeletal and nuclear responses, we investigated whether S . typhimurium infection of cultured cells would result in PAK activation. COS-1 cells were transfected with HA epitope–tagged mPAK-3, a ubiquitously distributed isoform of the PAK family of protein kinases. Transfected cells were then infected with either wild-type S . typhimurium or an isogenic derivative strain carrying a null mutation in invG . InvG is an essential component of the type III secretion apparatus, and therefore failure to express this protein results in a strain that is unable to induce cellular responses dependent on this system. At different times after infection, the PAK activity in infected cells was measured in an immunocomplex kinase assay as described in Materials and Methods. As shown in Fig. 1 , wild-type S . typhimurium induced significant activation of PAK. PAK activation was observed as early as 5 min after infection, reaching a maximum at ∼10 min after infection and rapidly decreasing over time. In contrast, the signaling- defective S . typhimurium invG mutant strain failed to induce PAK activation even after 60 min of infection. These results indicate that S . typhimurium interaction with host cells results in the activation of PAK, and such activation is strictly dependent on the function of the signaling-associated type III secretion system. We then tested whether S . typhimurium infection of cultured cells resulted in a redistribution of endogenous PAK. It has been previously shown that recruitment of PAK to the cell membrane results in its activation ( 38 ). COS cells were infected with wild-type S . typhimurium for various periods of time, then fixed and stained with a polyclonal anti-PAK antibody and rhodamine-labeled phalloidin to visualize the actin cytoskeleton. As shown in Fig. 2 , A and B, S . typhimurium infection resulted in the rapid recruitment of PAK to the bacterial-stimulated membrane ruffles. Ruffles stained by the PAK antibodies were seen as early as 5 min after infection. Interestingly, such a recruitment was seen in only a subset of the membrane ruffles stimulated by S . typhimurium . In fact, the recruitment of PAK to the membrane ruffles appears to be transient, as later (30 min) in infection the proportion of ruffles exhibiting PAK staining significantly decreased. The recruitment of PAK to only a subset of agonist-induced membrane ruffles has been previously reported ( 39 ). Infection of COS cells with the invG mutant strain did not result in any detectable change in the localization of endogenous PAK (data not shown). Taken together, these results indicate that S . typhimurium is capable of changing the distribution of PAK in infected cells through the function of its signaling-associated type III protein secretion and translocation system. SopE is an effector protein delivered by S . typhimurium into host cells via its type III secretion system ( 40 ). We have shown previously that transient expression of this protein results in membrane ruffling and JNK activation as a result of the direct stimulation of Cdc42 and Rac-1 by SopE ( 9 ). Therefore, we examined the distribution of PAK in COS cells transiently expressing the bacterial effector SopE. As shown in Fig. 2 , C and D, PAK was also recruited to the SopE-stimulated membrane ruffles. We have previously shown that S . typhimurium stimulates the stress-activated protein kinase JNK in a Cdc42-dependent manner ( 8 ). The PAK family of proteins has been implicated in the Cdc42- and Rac-1–mediated activation of both JNK and p38 protein kinases. The finding that S . typhimurium infection of host cells leads to the activation of PAK prompted us to examine the role of this kinase in S . typhimurium –induced JNK activation. COS-1 cells were cotransfected with a vector encoding Flag epitope–tagged JNK-1 and a vector encoding either wild-type PAK, the kinase-defective PAK R297 mutant, or the empty vector control. Transfected cells were infected with wild-type S . typhimurium , and the activity of JNK was measured in an immunocomplex kinase assay as described in Materials and Methods. As shown in Fig. 3 A, expression of the kinase-defective PAK R297 mutant blocked S . typhimurium –induced JNK activation. Expression of wild-type PAK did not result in significant inhibition of bacteria-induced JNK activation (data not shown). Since expression of PAK R297 did not result in inhibition of other Cdc42-dependent events (see below), the inhibitory effect of this mutant cannot be explained by nonspecific sequestration of Cdc42. Therefore, these results indicate that PAK is required for S . typhimurium –induced nuclear responses. We also tested whether the activation of JNK induced by the bacterial effector SopE ( 40 ) was also dependent on the function of PAK. COS-1 cells were transfected with a vector encoding Flag epitope–tagged JNK-1, the SopE 78–240 effector protein along with wild-type PAK, the kinase- defective mutant PAK R297 , or the empty vector control. Cotransfection of SopE with the kinase-defective PAK R297 mutant effectively blocked JNK activation . In contrast, cotransfection of SopE 78–240 with wild-type PAK did not result in significant inhibition of SopE-mediated JNK activation. Taken together, these results implicate PAK in the nuclear responses stimulated by wild-type S . typhimurium and its effector protein SopE. In addition to the stimulation of the stress-activated kinases JNK and p38, the PAK family of protein kinases has been implicated in the organization of the actin cytoskeleton ( 26 , 28 ). Furthermore, we showed that PAK is transiently recruited to the S . typhimurium – and SopE-induced membrane ruffles . Therefore, we investigated the role of the kinase activity of this effector molecule in the actin cytoskeleton–mediated S . typhimurium internalization into cultured cells. A kinase-defective PAK mutant (PAK R297 ) was expressed in COS-1 cells using a dicistronic expression system in which the cells expressing PAK R297 could be identified by the coupled expression of GFP. Transfected cells were infected with wild-type S . typhimurium , and bacterial internalization was quantified by a staining protocol that distinguishes extracellular and intracellular bacteria, as described in Materials and Methods. As shown in Fig. 4 , expression of a kinase-defective PAK did not inhibit bacterial entry into host cells. As previously shown, expression of dominant-negative Cdc42Hs (Cdc42Hs N17 ) effectively blocked bacterial internalization. These results indicate that the kinase activity of PAK is not required for actin cytoskeleton–mediated S . typhimurium internalization into host cells. Since this activity is required for bacteria-induced JNK activation (see above), these results also show that the S . typhimurium stimulation of actin cytoskeleton reorganization and nuclear responses are mediated by different downstream effector activities of Cdc42 signaling. In addition to the conserved kinase domain, the PAK family of proteins exhibits other highly conserved structural features, such as the presence at its NH 2 terminus of several proline-rich regions resembling SH3-binding domains ( 27 ). At least one of these domains has been implicated in regulating the formation of polarized membrane ruffles and focal complexes and in the binding of PAK to the SH3-containing adapter protein Nck ( 41 ). To examine the potential involvement of the NH 2 -terminal proline-rich regions of PAK in S . typhimurium internalization into host cells, we transiently expressed in COS-1 cells mutants of PAK (PAK A12A14 , PAK A36A39 , PAK A165A168 , and PAK A213A216 ) containing changes in the conserved proline-rich NH 2 -terminal domains. Although not formally investigated, we made the assumption that if any of these domains were required for S . typhimurium –induced cytoskeletal responses, transient expression of these mutants might result in a dominant-negative effect. Western blot analysis showed that all mutant forms of PAK were expressed in the transfected cells (data not shown). Transfected cells were infected with wild-type S . typhimurium , and the number of internalized bacteria in cells expressing the different mutant PAKs was determined as described in Materials and Methods. As shown in Fig. 4 , bacterial internalization was not affected by the expression of any of the PAK mutants tested. These results suggest that the SH3-binding domains of PAK may not be required for actin cytoskeleton–mediated S . typhimurium internalization into host cells. However, the presence of multiple SH3-binding domains may prevent observation of the potential dominant-negative effect resulting from the expression of single SH3-binding domain mutants. In addition to PAK, Cdc42 binds to several putative effector proteins that contain a conserved 16–amino acid domain, termed CRIB or p21-binding domain (PBD) ( 24 ). Effector domain mutation analysis of Cdc42 has identified a critical residue for binding to this domain. Thus, Cdc42 carrying a Y to C mutation at residue 40 was unable to bind to all CRIB domain– containing proteins tested, including PAK, Wiskott-Aldrich syndrome protein (WASP), MSE55, and the Caenorhabditis elegans protein F09F7 ( 32 ). Since this effector loop mutant is unable to bind CRIB-containing effector proteins, we reasoned that if any of these effectors were required for S . typhimurium –induced responses, such a mutant should act as dominant interfering by nonproductively binding the bacterial effector SopE and thereby effectively titrating it out. Therefore, to investigate the potential role of CRIB domain–containing Cdc42 effector proteins in S . typhimurium – stimulated cellular responses, we transiently expressed in COS-1 cells the effector domain binding mutant Cdc42Hs C40 . We first examined the effect of expression of Cdc42Hs C40 in S . typhimurium –induced JNK activation. COS-1 cells were cotransfected with a vector encoding Flag epitope–tagged JNK-1 and a vector encoding Cdc42Hs C40 , Cdc42Hs N17 , or the empty vector control. Transfected cells were infected with wild-type S . typhimurium , and the activity of JNK was measured in an immunocomplex kinase assay. As shown in Fig. 5 A, the expression of Cdc42Hs C40 effectively blocked S . typhimurium –induced JNK activation. The inhibitory effect of Cdc42Hs C40 was comparable to that of Cdc42Hs N17 . In contrast, transfection of wild-type Cdc42Hs or the empty vector control did not result in any measurable inhibition of bacteria-induced JNK activation . These results indicate that a Cdc42 effector protein(s) that binds to the CRIB-binding domain of Cdc42 is required for S . typhimurium –induced JNK activation. Expression of Cdc42Hs C40 also blocked S . typhimurium –induced PAK activation, which is consistent with the involvement of this effector in bacteria-induced nuclear responses . The inhibiting effect of Cdc42Hs C40 was equivalent to that of Cdc42Hs N17 . These results also demonstrate that Cdc42Hs C40 can effectively exert a dominant interfering effect on S . typhimurium –induced signaling. We then tested the effect of the expression of Cdc42Hs C40 on the actin cytoskeleton reorganization and membrane ruffling induced by S . typhimurium or the transient expression of its effector SopE. COS-1 cells were transfected with a double cistronic vector expressing Cdc42Hs C40 and GFP or the empty vector control. Transfected cells were then infected with wild-type S . typhimurium , and the actin cytoskeleton rearrangements resulting from bacterial infection were examined by rhodamine-phalloidin staining. Alternatively, internalized bacteria were enumerated as described in Materials and Methods. Expression of Cdc42Hs C40 effectively prevented both S . typhimurium –induced actin cytoskeleton rearrangements and bacterial internalization . Similarly, expression of Cdc42Hs C40 also blocked the cytoskeletal rearrangements induced by the transient expression of SopE . In contrast, expression of the constitutively active effector loop mutant Cdc42Hs L61C40 did not inhibit bacterial internalization . Cdc42Hs L61 does not efficiently bind the bacterial effector SopE ( 9 ); therefore, introduction of the activating mutation relieves the dominant-negative effect conferred by the C40 effector loop mutation, since this mutant is unable to sequester the bacterial effector. Taken together, these results indicate that a CRIB domain–containing effector protein(s) (such as PAK) or another effector protein(s) that binds to the same effector loop of Cdc42 is required for bacterial internalization as well as S . typhimurium – and SopE-induced actin cytoskeleton reorganization and nuclear responses. S . typhimurium induces nuclear and morphological responses in infected cells in a manner that is absolutely dependent on the function of the small GTP-binding protein Cdc42 ( 8 ). The related GTPase Rac-1 also plays a significant but clearly less important role in these responses. It is now apparent that S . typhimurium triggers these cellular responses by delivering into the host cell cytosol at least one bacterial effector protein that directly stimulates GDP/GTP nucleotide exchange on these Rho GTPases ( 9 ). The delivery of the effector proteins is carried out by a complex specialized protein secretion and translocation apparatus termed type III, encoded at centisome 63 of the S . typhimurium chromosome ( 3 ). Small GTPases of the Rho subfamily have been implicated in a wide variety of cellular functions, including the organization of the actin cytoskeleton, the assembly of focal adhesion complexes, cytokinesis, and cell growth and differentiation ( 42 ). The actual mechanisms by which this family of small G proteins modulates such a large variety of cellular functions are poorly understood, although it is assumed that they exert their various functions by engaging different downstream effectors. Several putative effectors of Cdc42 and Rac have been identified using a variety of biochemical or genetic approaches. In most instances, these putative effectors have been identified by exploiting their ability to bind these Rho GTPases in a GTP-dependent manner. The identified putative effectors are either protein kinases such as PAK, activated Cdc42- associated kinase (ACK), and mixed lineage kinase 3 (MLK3), or, as in the case of IQGAP and WASP, proteins that contain domains suggestive of their involvement in signal transduction by protein–protein interactions ( 16 ). The identification of putative effector proteins has been complemented by the definition of specific domains or effector loops in the GTPases themselves that are thought to specifically mediate their functional linkage to specific downstream signaling pathways or cellular responses. In this report, we have investigated the potential role of PAK, a putative effector of Cdc42, in S . typhimurium –induced cellular responses. Infection of cultured cells with wild-type S . typhimurium resulted in a significant stimulation of PAK activity. The stimulation was rapid and short-lived, with peak kinase activity 10 min after infection and a rapid decline shortly thereafter. PAK activation was strictly dependent on the delivery of effector proteins through the type III protein secretion system, since a S . typhimurium invG mutant, which is deficient for this system, failed to activate PAK activity. Expression of a dominant-negative kinase-deficient PAK mutant blocked JNK activation, indicating that the kinase activity of PAK is required for S . typhimurium –induced nuclear responses. The inhibitory effect of the kinase-defective mutant is unlikely to have been due to a nonspecific sequestration of Cdc42, since the same construct did not block other Cdc42-dependent responses, such as bacterial internalization. The specificity of this effect is further demonstrated by the finding that expression of a kinase-defective mutant of MLK3, another effector target of Cdc42, did not significantly block S . typhimurium – induced JNK activation (Chen, L.-M., and J.E. Galán, unpublished results). The S . typhimurium –induced JNK activation as a consequence of the stimulation of PAK is consistent with previous reports that have shown that expression of constitutively active PAK resulted in JNK activation ( 37 , 43 ). In contrast to the nuclear responses, the actin cytoskeleton rearrangements induced by S . typhimurium were not dependent on the kinase activity of PAK. Expression of a kinase-defective PAK did not result in inhibition of actin cytoskeleton–mediated S . typhimurium internalization into host cells. These results clearly demonstrate that the S . typhimurium –induced cellular responses are dependent on different downstream Cdc42 effector activities. However, previous reports have shown that PAK modulates the organization of the actin cytoskeleton via kinase-independent mechanisms ( 28 ). Those reports have implicated certain proline-rich domains at the NH 2 terminus of PAK that are postulated to be involved in the binding of SH3 domains in downstream effector proteins. In particular, studies have identified a proline-rich motif between amino acids 11 and 16 of PAK that is essential for the modulation of actin cytoskeletal organization. This motif has also been implicated in the binding of the SH3-containing adapter protein Nck ( 41 ). Therefore, we investigated the potential role of these NH 2 -terminal proline-rich domains of PAK in S . typhimurium internalization into host cells. Expression of PAK mutants carrying specific mutations in each of these proline-rich regions did not impair actin cytoskeleton–mediated bacterial internalization. These results suggest that PAK may not be required for actin cytoskeleton responses stimulated by S . typhimurium . However, it is possible that expression of such mutants may not result in an adequate dominant-negative effect, or PAK may contain other domains that may be involved in bacteria-induced cytoskeletal responses. Transient expression of an effector loop mutant of Cdc42 (Cdc42 C40 ) unable to bind CRIB domain–containing proteins resulted in effective inhibition of both S . typhimurium – induced nuclear and actin cytoskeleton responses. These results indicate that both responses required effectors that interact with this domain of Cdc42. PAK contains a CRIB domain and is therefore impaired in binding to this effector loop mutant of Cdc42. Thus, the dominant-negative effect of Cdc42Hs C40 on the nuclear responses induced by S . typhimurium is consistent with our findings that PAK activity is required for bacteria-induced JNK activation. However, it is unclear whether a potential requirement for PAK may explain the effect of Cdc42Hs C40 on S . typhimurium –induced cytoskeletal rearrangements, as we failed to demonstrate the involvement of this kinase on the bacteria-induced morphological response. Further studies will be required to address this question and to identify other effectors of Cdc42 that may be required for nuclear and cytoskeletal responses. Our results showing the requirement of the CRIB-binding domain of Cdc42 for the actin cytoskeleton reorganization induced by S . typhimurium are not in full agreement with previous studies with effector loop mutations of Cdc42 that have argued that CRIB-containing effector proteins do not mediate actin cytoskeleton responses modulated by this small G protein ( 32 ). However, this discrepancy may be due to the different experimental set-ups. In our studies, the activation of Cdc42 to induce cellular responses is mediated by a bacterial effector that directly stimulates this small G protein. In contrast, other studies have made use of a constitutively active Cdc42 mutant carrying the effector loop substitutions (e.g., Cdc42Hs L61C40 ; reference 32 ). Most likely, the constitutive activation of this GTPase is not equivalent to the S . typhimurium –mediated stimulation of Cdc42, which is transient. Thus, activation of this GTPase mediated by the bacterial agonist may lead to interactions with downstream effectors that are different from those resulting from its constitutive, irreversible activation by introduction of an activating mutation. The results described here show that PAK is activated upon S . typhimurium infection of host cells. This activity is required for bacteria-induced nuclear responses, as expression of a kinase-defective PAK R297 mutant blocked both S . typhimurium – and SopE-mediated JNK activation. In contrast, this mutant did not block actin cytoskeleton–mediated S . typhimurium entry into host cells, indicating that the nuclear and morphological responses stimulated by the bacteria are mediated by different Cdc42Hs downstream effector activities. Expression of Cdc42Hs C40 , which is defective for binding to PAK and other effectors containing a CRIB domain, blocked both S . typhimurium nuclear and cytoskeletal responses, implicating this effector loop of Cdc42 in mediating both responses. | Study | biomedical | en | 0.999997 |
10224289 | Generation of Jurkat cell lines constitutively expressing CD8 tag or CD8-Nef chimeras was described recently ( 8 , 26 ). Jurkat, J.CaM.1 (Lck − ), J.45.01 (CD45 − ), and J.RT3-T3.5 (TCR − ) cells were provided by Arthur Weiss (University of California, San Francisco, CA ). J.RT3-T3.5 cells expressing various CD16ζ constructs or coexpressing CD8-Nef were generated by electroporation using puromycin and neomycin for selection. Stable clones were enriched for protein expression by magnetic anti-CD16/anti-CD8 beads. The mAbs against the AU-5/ AU-1 epitope and FasL (NOK-1) were purchased from Hiss Diagnostics and PharMingen . Generation of the CD8-Nef (SF2) and CD16ζ/ε chimeras as well as COOH-terminal–tagged Nef (AU-1) was described previously ( 8 , 26 , 28 ). Fusion proteins between CD16 and individual ζ ITAMs (ITAM 1, amino acids [aa] 1–70; ITAM 2, aa 70–110; ITAM 3, aa 110–141) were generated as described previously ( 28 ). The mutations in CD16ζ as well as in Nef/CN.94 were generated by a two-step PCR procedure and cloned into the pRcCMV expression vector (Invitrogen). In CD16ζmu, the tyrosine residues in three ITAM motifs (two tyrosine residues in each ITAM) were mutated to alanines. In CN.94PXmu/Nef.PXmu, the FPVR motif of Nef (aa 72–75) was mutated to VRIT. Construction of the proviral clone NL4-3, containing the SF2 nef gene (NL4-3.SF2Nef), as well as the Nef-negative construct (NL4-3ΔNef), was described previously ( 26 ). For the generation of recombinant baculoviruses, the “bac to bac” system was used (Bio-Rad Laboratories). Transfections into 293T cells, metabolic labeling with 35 S-Translabel, immunoprecipitation, Western blot, and in vitro kinase assays were performed as described previously ( 8 , 26 ). The immunoprecipitates were washed three times (wash buffer: 1% NP-40, 450 mM NaCl, 50 mM Tris-HCl [pH 8], 1 mM EDTA). To show an interaction between Nef and ζ, extraction and washing buffers contained 1% Brij instead of 1% NP-40. FasL promoter activity was tested as described previously ( 29 ) by cotransfection of pFasL-Luc (provided by Xiangdong Liu, Department of Virus and Cancer, Aarhus, Denmark) with CD8-Nef constructs or pCTax as positive control (provided by Ralph Grassman, Institute of Virology, Erlangen, Germany). All transfections were performed in duplicate by mixing 6 μl of liposome reagent (DMRIE-C; GIBCO BRL ) and 2 μg of each plasmid for 2 × 10 6 Jurkat or Jurkat mutant cells. Cells (5 × 10 6 ) were superinfected with 1 ml of HIV IIIB (1.6 × 10 4 cpm/ml, reverse transcriptase [RT] activity), NL4-3.SF2.Nef (2.4 × 10 4 cpm/ml, RT activity), or NL4-3ΔNef (2.5 × 10 4 cpm/ml, RT activity) for 2 h. After infection, cells were washed and adjusted to a concentration of 10 6 /ml and incubated for an additional 48 h. Cell culture supernatants were collected on day 5 for analysis of p24 by ELISA or RT activity by Quan-T-RT kit ( Amersham Pharmacia Biotech ). To assess cell surface FasL expression on HIV-infected cells or transiently transfected Jurkat TAg cells, the metalloprotease inhibitor BB2116 (British Biotech ) was added to the medium 4–6 h before the assay to enhance cell surface FasL expression. In brief, cells were stained with 20 μl of biotin-conjugated anti–human FasL mAb (NOK-1; PharMingen ) followed by 5 μl of PE-conjugated streptavidin ( Sigma ). Labeled cells were analyzed on a FACScan™ ( Becton Dickinson ). Isotype-specific mAbs of irrelevant specificity were used as negative controls (Dako Diagnostics). To assess expression of whole FasL protein, 35 S-labeled cells (5 × 10 6 ) were immunopreciptatied for FasL using anti-FasL–specific mAb (NOK-1) as described previously ( 30 ). As shown previously, Nef associates with a serine kinase, termed p62 or Nef-associated kinase (NAK ). The Nef–NAK interaction is complex: Nef stimulates the phosphorylation/activation of NAK, and it is only in this activated form that NAK can bind Nef ( 32 ). This suggests that Nef must act upstream of NAK to promote NAK activation. Our previous results showing that Nef interfered with early signals emanating from the TCR suggested it may interact with a component of the TCR signaling complex. This prompted us to study Nef-mediated NAK/p62 activation in cell lines with TCR signaling defects. CD8-Nef chimeras (CD8-Nef), containing the extracellular domain of CD8α fused to Nef, were stably transfected into wild-type Jurkat and a variety of Jurkat mutant cell lines lacking either Lck (J.CaM.1), CD45 (CD45 − ), or the entire TCR signaling complex (RT3.T3.5). Expression of CD8-Nef in these cell lines was verified by metabolic labeling and immunoprecipitation . The Nef chimeras from these transfectants were immunoprecipitated and subjected to an in vitro kinase assay. NAK/p62 association was observed in all cell lines except the TCR − cells . The latter result was confirmed in a second, independently transfected cell clone (data not shown). Next we asked whether NAK binding could be restored in cells lacking the TCR complex by stable transfection with TCR-ζ or CD3ε fused to the extracellular domain of CD16 (CD16ζ and CD16ε). These TCR subcomponents contain signaling motifs (immunoreceptor tyrosine-based T cell activation motifs [ITAMs]), which are required and sufficient for T cell activation ( 28 , 33 ). After obtaining single cell clones, expression of the chimeras was verified by metabolic protein labeling and FACS ® analysis (data not shown). In several attempts, we were unable to coexpress CD8-Nef (CN) with CD16ζ in TCR − cells. Cell clones that were obtained either showed no detectable CN or CD16 expression or died rapidly. The effect resembled activation-induced cell death (AICD) by Nef as reported previously ( 8 ). Coexpression of CN and CD16ε was achieved; however, the obtained cell clones had a low CN as well as CD16 surface expression . Therefore, we constructed ζ chimeras containing the three individual ζ ITAMs in isolation (CD16ζ1, 2, or 3; see Materials and Methods for details). In a seperate construct, the tyrosine residues in all three YXXL motifs of CD16ζ were mutated to alanines (CD16ζmu). We failed to coexpress the first ζ ITAM with Nef. However, NAK/p62 binding to Nef was reestablished in the TCR − cells by coexpression with the second or third ITAM of ζ . In these latter cell lines, expression of CD16ζ1 and 2 as well as CD8-Nef decreased significantly over time (data not shown), indicating that coexpression of both proteins was not favorable. The difficulties regarding the coexpression of the individual ζ ITAMs with CD8-Nef may be explained by studies published by Combadiere et al. ( 34 ) showing that in particular the first ζ ITAM but much less the second and third are capable of inducing apoptosis when activated. The signaling-defective ζ chain (CD16ζmu) expressed well, but NAK binding to Nef was greatly reduced (lane 6). No NAK/p62 binding was observed by coexpression of CD3ε (lane 3). Since NAK binding to Nef was not completely negative with CD16ζmu, the Nef–ζ complex may recruit additional signaling molecules to the plasma membrane which are important for NAK activation. Assuming that the effects of the first ζ ITAM would be similar to ITAM 2 and 3, it appeared that at least one functional ITAM of the CD3 ζ chain was required for binding of p62/NAK to Nef. The functional link between Nef, ζ, and NAK was confirmed by transient transfection assays in a heterologous system. As shown in Fig. 1 E, cotransfection of CD16ζ (lanes 4 and 5) but not CD16ε (lanes 2 and 3) significantly increased binding of p62/NAK to Nef. A minimal increase was seen after cotransfection of CD16ζmu (lanes 6 and 7), which paralleled the small effect seen in Fig. 1 C, lane 6. Thus, no other T cell–specific components except the functional ITAM(s) from the TCR ζ chain, were required for NAK activation and NAK/Nef association in 293T cells. Full-length CD8-Nef when expressed at the cell membrane promotes AICD. Upon stable transfection, cell clones are preferentially selected in which Nef is predominantly expressed in the cytoplasm, where it does not exert such a detrimental effect on cell survival. In contrast, NH 2 -terminal fragments of Nef are expressed at high levels at the plasma membrane where TCR-ζ is located ( 8 ). These NH 2 -terminal fragments can recruit a complex of proteins to form the NH 2 -terminal kinase complex, which binds between amino acids 20 and 35, and may contribute to T cell activation ( 26 ). The NH 2 terminus also has a conserved domain containing a proline-rich motif (PxxP, aa 73–82) known to associate with SH3 domains of tyrosine kinases ( 35 ). We reasoned that these domains/motifs could interact with and bind TCR-ζ. To prove a direct interaction, coimmunoprecipitation experiments were performed using stable cell lines with different CD8-Nef chimeras . The only interaction was seen with a construct expressing the NH 2 -terminal 94 amino acids of Nef containing the PxxP motif (lane 4). Underscoring the importance of the PxxP motif for ζ binding, we found that point mutations in the PxxP motif of the 94–amino acid Nef construct (CN.94.PXmu) almost completely abolished ζ binding (lane 5). Further evidence for an interaction between Nef and ζ was obtained by coimmunoprecipitation of Nef and ζ from Hi5 insect cells coinfected with recombinant baculoviruses . CD16ζ was found to coprecipitate with Nef , but not with Nef.PXmu (lane 6). To confirm the specificity of the interaction, aliquots of the anti-ζ immunoprecipitates were incubated with Jurkat (ζ-containing) or the TCR − (ζ-lacking) cytoplasmic lysates. Wild-type Jurkat competed for Nef binding (lane 4), whereas the TCR-ζ–negative cytoplasmic lysates did not (lane 5). The reduced Nef signal in lane 5 may be explained by the reduced amount of immunoprecipitated ζ . Additional evidence for the interaction of both proteins was obtained by coimmunoprecipitation after transient transfection into COS cells and subsequent in vitro kinase assay (data not presented). We have previously shown that the upregulation of FasL in SIV infection requires an intact nef gene ( 22 ). In general, the level of cell surface FasL expression is quite low when analyzed by FACS ® even when metalloproteinase inhibitors are used which block cleavage of FasL from the cell surface. In view of this difficulty, we used additional experimental approaches to analyze Nef-mediated FasL expression (see below). Since stimulation of TCR-ζ effectively upregulates FasL expression ( 34 , 36 ), we speculated that the interaction of Nef with TCR-ζ would lead to a similar effect. First, to show that HIV-Nef is required for FasL upregulation, we infected Jurkat with wild-type HIV (NL4-3.SF2Nef) or a mutant lacking the nef gene (NL4-3ΔNef) . Little if any FasL is seen on cells infected with Nef-deleted HIV, thus confirming our previous results with SIV. The level of viral replication in Jurkat cells was comparable, as determined by RT activity (NL4-3.SF2Nef, 4.6 × 10 3 cpm; NL4-3ΔNef, 5.8 × 10 3 cpm). Upregulation of FasL by HIV is also lost in mutant Jurkat cells lacking the TCR complex, whereas cells reconstituted with ζ but not with the ζ mutant restored the FasL expression upon HIV infection as determined by both immunoprecipitation and FACS ® analysis . Viral replication assessed by p24 assay indicated that these cell lines were comparably infected (wild-type, 3.5 ± 0.5; TCR − , 3.8 ± 1.0; CD16ζ, 3.8 ± 0.5; CD16ζmu, 3.1 ± 0.25 ng/ml). To investigate a direct upregulation of FasL by Nef, a CD8-Nef construct not capable of binding ζ and CD8-Nef were transiently expressed in Jurkat cells and analyzed for FasL upregulation. CD8-Nef but not the Nef mutant led to a significant cell surface expression of FasL . Next, FasL upregulation was studied in Jurkat and TCR mutant cell lines using a FasL promotor/luciferase reporter construct. The latter has been shown to be stimulated in transient assays by the HTLV I Tax protein ( 29 ). Nef stimulated the FasL promotor in Jurkat and TCR − mutant cells reconstituted with the TCR ζ chain. No effect was seen using the Nef.PXmu construct or the TCR − Jurkat cell line . These assays confirmed that a functional Nef protein and the TCR ζ chain were both required and sufficient to upregulate FasL in T cells. In general, the interaction between Fas and FasL plays a important role in the homeostatic regulation of normal immune responses ( 37 ). Stimulation of the TCR–CD3 complex in T cells causes upregulation of FasL and eventually leads to AICD or apoptosis ( 23 – 25 ). A key molecule in this process is the TCR ζ chain and the three ITAMs contained therein. Cross-linking of the ζ chain or constructs containing individual ζ ITAMs alone were found to be sufficient to induce T cell activation and Fas-mediated apoptosis ( 28 , 35 , 36 ). In agreement with these findings, we have shown here that TCR-ζ as well as the functional integrity of the ITAM signaling motifs of ζ were required for HIV-mediated upregulation of FasL. However, these findings further implied that HIV targets the TCR ζ chain directly through a viral protein. To date, several lines of evidence indicated that the Nef protein exerted such a role. First, Nef-mediated activation of T cells has been demonstrated in a number of reports (8– 10). Second, expression of Nef in the cytoplasm of T cells interferes with early T cell signaling events emanating from the TCR–CD3 complex, including hypophosphorylation of TCR-ζ, whereas expression of a plasma membrane– associated form of Nef causes AICD in Jurkat cells ( 8 ). Third, a very aggressive form of Nef from SIV, SIV-YE-Nef, basically functions like an ITAM domain of TCR-ζ ( 38 ). Finally, SIV-induced upregulation of FasL in T cells depends on the expression of an intact Nef protein ( 22 ), and Nef from a lethal SIV strain (smmPBj14) alone can directly cause FasL upregulation ( 39 ). Thus, it appeared very likely that Nef acted at the level of the TCR. Indeed, our study confirms this assumption by showing that Nef can directly interact with the TCR ζ chain. Strong evidence for the interaction of Nef with ζ came from a second, surprising finding. In Jurkat cells lacking the TCR, binding of the Nef-associated serine kinase p62/NAK was abolished. Conversely, reconstitution of these cells with the ζ ITAM 2 and 3 restored binding of p62/NAK with Nef. Furthermore, the integrity of the ITAM motif appeared to be important, since mutation of the ζ ITAMs greatly reduced the effect. As shown previously, the p62/NAK kinase has to be activated in order to bind to Nef ( 32 ). These results suggest a dynamic interaction of Nef with the ζ ITAMs, ultimately resulting in the activation of p62/NAK, which in turn binds to Nef. In view of our and other studies, it is likely that activation of p62/NAK is part of Nef-mediated stimulation of T cell signaling pathways; however, at this point it is not clear whether p62/NAK has a role in the Nef-mediated upregulation of FasL. Notably, Nef binds to p62/NAK in cells lacking a TCR (31; e.g., COS cells). In these cells, the TCR ζ chain may be functionally replaced by other receptors, possibly containing ITAMs. This would explain why Nef has effects in cells usually not infected by HIV (40; e.g., NIH 3T3 cells). More recently, Howe et al. showed that Nef from SIV or HIV-2 associated with the TCR ζ chain but failed to show an interaction with HIV-1 Nef ( 41 ). Our study differs from that of Howe et al. in at least two respects. First we made constructs to target Nef to the plasma membrane where the TCR is located. Second, we have established functional consequence of the Nef–ζ interation which may have relevence to the pathogenesis of HIV interaction. Induction of cell death by HIV could be mediated by different viral proteins. Cross-linking of CD4 by HIVgp120 in the presence of Tat protein can induce FasL expression and apoptosis of uninfected T cells ( 42 ). Additionally, interaction of HIVgp120 with chemokine receptor CXCR4 on macrophages leads to death of CD8 + T cells mediated by TNF–TNFRII interaction ( 15 ). In this study, we report an additional important mechanism of HIV-mediated apoptosis by demonstrating that Nef directly interacts with TCR-ζ and that both molecules are required for HIV-mediated upregulation of FasL. The interaction between Nef and TCR-ζ forms a signaling complex, bypassing the requirement for TCR ligation by antigen, and allowing HIV/SIV to activate T cells and upregulate FasL expression on the infected cells. Thus, upregulation of FasL by Nef on HIV- or SIV- infected cells may, like FasL expression at sites of immune privilege and on some tumors, allow infected cells to evade the immune response. In addition, the effect of immune evasion is enhanced by Nef-mediated downregulation of surface MHC class I and CD4 expression . Taken together, our results provide additional insights into the molecular mechanism whereby the HIV accessory protein Nef regulates T cell activity and contributes to the pathogenesis of HIV. | Study | biomedical | en | 0.999995 |
10224290 | TiO 2 and Fe 2 O 3 were provided by Dr. J. Brain (Harvard School of Public Health, Boston, MA). These particles have been shown to be heterogeneous in size, with a median diameter of 1.3 μm ( 23 ). Latex beads (1.0 μm in diameter; sulfated polystyrene) that show green fluorescence after excitation at 488 nm were obtained from Interfacial Dynamics Co. All particles were suspended in balanced salt solution (BSS − [124 mM NaCl, 5.8 mM KCl, 10 mM dextrose, and 20 mM Hepes]) as stock solutions and sonicated ∼30 s before use. Anti-TNP ( PharMingen ), anti-heparan sulfate mAbs (Seikagaku Corp.), anti– human β2 microglobulin mAb ( PharMingen ), and a nonspecific rat IgG1 ( PharMingen ) were used as isotype-matched controls. The anti–human MARCO antibody was raised in rabbits as described ( 18 ). The anti–mouse MARCO mAb, ED31 (IgG1), was provided by Dr. G. Kraal (Vrije Universiteit, Amsterdam, The Netherlands) ( 21 ). All reagents not otherwise specified were obtained from Sigma Chemical Co. SR-A–deficient mice were prepared as reported ( 24 ). Mice or hamsters were killed by intraperitoneal pentobarbital injection. AMs obtained by repeated lung lavage with BSS − were centrifuged at 150 g and resuspended in BSS + . AMs (2 × 10 5 in 100 μl BSS + ) were preincubated with mAbs (100 μl hybridoma supernatant or 10 μg/ml mAb) or inhibitors (10 μg/ml) and 2.5 μg/ml cytochalasin D for 5 min on ice in a 1-ml microfuge tube. After the addition of probe sonicated particles or beads, the tubes were rotated at 37°C for 30 min, placed on ice, and analyzed by flow cytometry. Flow cytometry was performed using an Ortho 2150 cytofluorograph as previously described ( 25 ). AM uptake of particles was measured using the increase in the mean right angle scatter (RAS) caused by these granular materials ( 25 ). Latex bead binding is expressed as relative fluorescence. Fluorescent-labeled, heat-killed bacteria ( Escherichia coli and Staphylococcus aureus ) and yeast (Zymosan) were purchased from PharMingen . The bacteria binding assay was performed exactly as described above, except that AMs were incubated with either bacteria (5 × 10 7 ) or yeast (2 × 10 5 ) instead of particles. Binding was measured by detecting AM-associated fluorescence by flow cytometry. Mice were anaesthetized with halothane (4%), and 125 μl TiO 2 (50 μg/ml) in BSS + was instilled into the lungs using a 22-gauge canula. Bronchoalveolar lavage (BAL) was performed 30 min after instillation, and aliquots of BAL were immediately analyzed by flow cytometry to quantitate cell-associated particles. BALB/c mice were immunized by intraperitoneal injection of 2 × 10 7 hamster AMs. After 3 wk, mice received another injection of 2 × 10 7 AMs i.p., and 3 d later, spleens of the mice were removed. The splenocytes were fused with a nonsecreting mouse myeloma, P3U1, using polyethylene glycol 4000 and cultured in DMEM containing hypoxanthine, aminopterin, and thymidine. After 2 wk, supernatants from hybridoma cultures were screened for their ability to inhibit the adhesion of TiO 2 to AMs. Hamster AM cell surface proteins were labeled with sulfo-NHS-LC-biotin ( Pierce Chemical Co. ) as per the manufacturer's suggested protocol and resuspended at a concentration of 4 × 10 7 cells/ml in a 1% extraction buffer (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM iodoacetamide supplemented with 40 μg/ml PMSF, 2 μg/ml aprotinin, and 10 μg/ml phenanthroline as protease inhibitors). The lysates were precleared with rat anti–mouse IgM magnetic beads (Dynabeads ® ; Dynal Inc. ). Aliquots of lysate were incubated with mAbs PAL-1 or RP-3 bound to rat anti–mouse IgM magnetic beads overnight at 4°C. The immunoprecipitates were washed in cold lysis buffer (without protease inhibitors), subjected to SDS-PAGE, electroblotted to membrane filters, probed with avidin– HRP conjugate ( Pierce Chemical Co. ), and developed using chemiluminence reagent (Supersignal ® ; Pierce Chemical Co. ). Cryostat sections of hamster tissues were fixed in buffered 2% paraformaldehyde for 10 min, followed by 10 min in 100% cold (−20°C) methanol. After rinsing, immunostaining was performed by sequential application of primary antibody (mAb PAL-1 [5 μg/ml] or anti-heparan sulfate mAb [5 μg/ml], goat anti–mouse IgG [1:50; Sternberger Monoclonals, Inc.], and mouse peroxidase–antiperoxidase complex [1:100; Sternberger Monoclonals, Inc.]), followed by labeling with chromogen diaminobenzidine and H 2 O 2 . To increase sensitivity, an additional cycle of secondary and peroxidase–antiperoxidase complex was applied to sections, followed by a second chromogen reaction. Paraffin sections of human lung tissue were immunostained using an avidin–biotin complex protocol as previously described ( 26 ). The slides were washed with water, counterstained with hematoxylin, dehydrated, and mounted for light microscopy. A hamster AM cDNA library prepared in the pcDM8 vector (provided by Dr. B. Seed, Harvard Medical School, Boston, MA; Paulauskis, J., unpublished data) was divided into small pools and transfected into COS cells. The transiently transfected COS cells were isolated by “panning” on mAb PAL-1–coated plates, and plasmid DNA was reisolated and amplified as described ( 27 ). A receptor-positive pool was identified by PAL-1 immunostaining of transfected COS cells after six rounds of screening. This pool was subdivided repeatedly until we obtained a single functional plasmid that upon transfection conferred mAb PAL-1 reactivity to COS cells. Both strands of the cDNA insert were sequenced at the Harvard University Biopolymers Laboratory. Human AMs were collected by BAL from healthy adults under an institutionally reviewed and approved protocol. AMs were washed and lysates prepared as described above (see Immunoprecipitation ). The lysates were mixed with 6× reducing or nonreducing SDS–solubilization buffer, subjected to SDS-PAGE, electroblotted to membrane filters, probed with either control rabbit IgG or purified rabbit anti–human MARCO antibody followed by avidin–HRP conjugate ( Pierce Chemical Co. ), and developed using chemiluminence reagent (Supersignal ® ; Pierce Chemical Co). Data were analyzed using ANOVA and paired t test components of a statistical software package (Statview; Abacus Concepts). Significance was accepted when P < 0.05. To determine whether SR-A (I/II) receptors mediate AM binding of unopsonized particles, the binding of TiO 2 by SR-A (I/II)–deficient AMs (SR-A −/− ) was tested and compared with the binding of TiO 2 by AMs from wild-type mice (SR-A +/+ ). Microscopic evaluation of treated AMs showed similar robust binding of TiO 2 by both SR-A −/− and SR-A +/+ AMs . Quantitation by flow cytometric analysis of RAS increases showed that SR-A −/− and SR-A +/+ AMs demonstrated essentially identical particle binding . SR-A −/− AMs also bound unopsonized ferric oxide and fluorescent latex beads with comparable avidity (data not shown). The SR ligand PI inhibited the adhesion of TiO 2 to both SR-A −/− and SR +/+ AMs by 59 ± 1% and 58 ± 4%, respectively. The control polyanion, chondroitin sulfate (CS), had no effect on particle adhesion. To determine if the in vitro particle binding reflected in vivo events, we measured particle binding to AMs after intratracheal instillation of TiO 2 . SR-A–deficient or wild-type mice were instilled with buffer alone or buffer containing TiO 2 . After 30 min, mice were killed, BAL performed, and AM uptake of TiO 2 quantified by flow cytometry. As shown in Fig. 1 C, both SR-A–deficient AMs and wild-type AMs bound TiO 2 in vivo to a comparable degree. Thus, SR-A deficiency does not alter unopsonized particle binding by AMs. These results suggested that SRs other than SR-A are involved in unopsonized particle binding to AMs. To develop an mAb to the receptor that mediates particle binding, mice were immunized with hamster AMs, and hybridomas were prepared and screened for mAbs that block AM binding of TiO 2 . As shown in Fig. 2 and reported previously ( 10 ), the scavenger receptor ligand, PI, blocked AM binding of TiO 2 and served as a positive control for these assays. A new mAb, PAL-1, inhibited AM binding of TiO 2 by 67 ± 5% ( n = 10). An isotype-matched control mAb (anti-TNP) had no effect on AM binding of TiO 2 . We next examined the effect of mAb PAL-1 on AM binding of other environmental particles such as Fe 2 O 3 or quartz (SiO 2 ) and the surrogate particle, latex beads. As shown in Table I , PAL-1 inhibited AM binding of Fe 2 O 3 , SiO 2, and latex beads by 78 ± 2, 52 ± 24, and 85 ± 4%, respectively. Thus, mAb PAL-1 substantially inhibits AM binding of a broad range of particles. To identify the protein(s) recognized by mAb PAL-1, immunoprecipitation experiments were carried out. AMs were surface-labeled by sulfo-NHS-biotin ( Pierce Chemical Co. ), and the cell surface molecules bound by PAL-1 were analyzed by SDS-PAGE . In both reducing and nonreducing conditions, a single band with an apparent molecular mass of 70 kD was detected in lysates of normal AMs . This band was absent from cells precipitated with an isotype-matched (IgM) negative control antibody. Immunostaining of frozen sections from a range of normal hamster tissues with PAL-1 showed that it reacted strongly with most or all AMs, macrophages of lymph node sinuses, and von Kupffer cells of the liver . Macrophages in other sites were also positive (e.g., splenic red pulp, intestinal Peyer's patch, thymus). Cross-reactions with other tissue structures such as endothelial cells were not observed; numerous other nonlymphoid tissues were negative (skin, brain, heart, stomach, prostate, and kidney; data not shown). Flow cytometric analysis showed that PAL-1 staining is detected on AMs. Lymphocytes isolated from different lymph nodes and thymocytes were negative . Peritoneal macrophages, neutrophils, and monocytes were predominantly negative (data not shown). An expression cloning protocol was used to identify the receptor mediating AM binding of particles. When transfected into COS cells, a cDNA clone conferred mAb PAL-1 reactivity . Both strands of the 1.6-kb insert were sequenced. The sequence, including part of the 5′- and 3′-untranslated regions, is shown in Fig. 6 A. From the first ATG, we predict an open reading frame of 1452 bp, which yields a protein of 483 amino acid residues with a predicted molecular mass of 49.5 kD. GenBank searches of the nucleotide and protein sequences revealed significant homology to murine and human MARCO (84 and 77% nucleotide identity with mouse and human MARCO cDNA, respectively; 18, 19). We refer to this clone as hamster MARCO. Amino acid identity between hamster, murine, and human MARCO was 77 and 65%, respectively . As shown in Table II , amino acid identity between hamster, mouse, and human MARCO was highest in the collagenous domain. The intracellular domain had 80% identity between hamster and mouse MARCO, as compared with 50% identity between hamster and human MARCO. Hamster MARCO, like human MARCO, does not have a cysteine residue in the spacer domain, but like mouse MARCO, it retains two cysteines in the cytoplasmic domain. The two potential carbohydrate attachment sites in the spacer domain, the interruption of Gly-Xaa-Yaa repeats in the collagenous domain, and all six cysteine residues in the scavenger receptor cysteine-rich domain (SRCR) are conserved between all three species . Hamster MARCO differs from both murine and human MARCO in containing a shorter collagenous domain (237 amino acids). To confirm the role of MARCO in particle binding, we transfected COS cells with hamster MARCO cDNA. As shown in Fig. 5 C, COS cells transfected with MARCO cDNA bound TiO 2 (4.6-fold increase in RAS). TiO 2 binding by MARCO-transfected COS cells but not untransfected COS cells was significantly inhibited by the anti-MARCO mAb PAL-1 . Controls, including untransfected COS cells and COS cells transfected with a plasmid encoding the cDNA for hamster CD44, exhibited binding of TiO 2 that was inhibited by both PI and heparin (an agent that does not inhibit AM binding of TiO 2 ) but not mAb PAL-1 (data not shown). The constitutive heparin-sensitive particle-binding receptor on COS cells is different from MARCO and remains to be identified (see Discussion). To further investigate the range of ligands for hamster MARCO, we tested the effect of mAb PAL-1 on AM binding of unopsonized microorganisms. As shown in Fig. 7 , mAb PAL-1 inhibited AM binding of E. coli and S. aureus by 67 ± 5% and 47 ± 4%, respectively. PAL-1 had no effect on AM binding of Zymosan. An isotype-matched control antibody did not inhibit bacteria and yeast binding. Thus hamster MARCO, like human and mouse MARCO ( 18 , 19 ), mediates AM binding of bacteria but not yeast. The original report of human MARCO did not detect MARCO expression in the lungs of a limited number of perinatal autopsy specimens ( 18 ). To evaluate whether adult human AMs express MARCO, we performed immunologic analysis using a polyclonal antibody to a peptide from domain V of human MARCO ( 18 ). Histochemical analysis of human BAL cells (>90% AMs) and lung tissue showed that the anti-MARCO antibody detects antigen on AMs but not on other cells or surrounding tissue structures . We prepared lysates from human AMs ( n = 5) and performed SDS/Western blot analysis. The anti–human MARCO antibody reacted specifically with a discrete band in all AM lysates, which ran at a relative molecular mass of ∼70 kD . No reactivity was seen with an irrelevant control antibody. The same pattern was repeated when the analysis was performed in nonreducing conditions (data not shown). Thus, adult human AMs express MARCO. An mAb to mouse MARCO has been shown to block bacteria binding by mouse macrophages ( 19 ). Although expression of MARCO was not detected in mouse lungs in the original report, we found significant immunostaining (albeit weaker than that seen in human and hamster AMs) of normal mouse AMs by the anti-MARCO mAb ED31 (data not shown; see Discussion) ( 19 ). We therefore examined whether ED31 would block TiO 2 binding by mouse AMs. In these experiments, the SR inhibitor, PI, reduced TiO 2 binding by 44 ± 12 and 52 ± 11% in SR −/− and SR +/+ mice, respectively; the control polyanion CS had no significant effect ( n ≥ 3). Treatment of SR −/− AMs with ED31 blocked binding of TiO 2 by 40 ± 11% ( n = 4). TiO 2 binding by SR +/+ was also inhibited by anti-MARCO mAb treatment by 25 ± 4% ( n = 3). Control IgG1 had no effect on binding. Similar inhibition of mouse AM binding of unopsonized fluorescent latex beads and bacteria was seen (data not shown). Thus, MARCO also functions to bind environmental particles and bacteria in mouse AMs. In this study, we have identified MARCO as a major receptor on AMs for binding of unopsonized environmental particles and certain microorganisms. The lung is constantly exposed to environmental substances such as microbes, pollutant particles, and allergens. The levels of ambient air particles in the environment have been correlated with increased morbidity and mortality ( 28 – 31 ). The clearance of inhaled particulate matter is primarily mediated by AMs through the process of phagocytosis ( 1 , 2 ). AMs have been previously shown to avidly phagocytose unopsonized particles ( 5 , 6 ). However, the receptors on the AMs that recognize and bind particles are not known. Here we have generated an mAb, PAL-1, that blocked particle and bacteria binding by AMs. Using mAb PAL-1 as a probe, we have cloned the cDNA encoding for the receptor from an AM cDNA library. Transfection of COS cells conferred mAb PAL-1 reactivity and mAb PAL-1–inhibitable binding of TiO 2 . By sequence homology and functional similarity, we conclude that the receptor is the hamster homologue of MARCO. The recognition of phagocytic targets is mediated by specific receptors on the phagocytes that either recognize serum components (opsonins) bound to the particle or directly recognize molecular determinants on the target. Thus, based on the mechanism of particle recognition, phagocytosis is either opsonin-dependent or opsonin-independent. The best characterized opsonin-dependent phagocytosis receptors are the Fcγ receptor and the complement receptor CR3 ( 3 , 32 ). Recent advances have highlighted the significance of other receptors, such as the collectin receptor, C1q, in opsonin-dependent phagocytosis ( 33 , 34 ). The opsonin-independent recognition of microorganisms and apoptotic cells is mediated by receptors such as the scavenger receptor, mannose receptor, vitronectin receptor, asialoglycoprotein receptor, and the β2 integrins (35– 37). Based on the high expression of SRs on macrophages and their broad specificity, we considered this class of receptors as likely suspects for interaction with inhaled particles. Indeed, we found that AM phagocytosis of unopsonized environmental dusts (TiO 2 , Fe 2 O 3 , SiO 2 ) or fluorescent latex beads is strongly inhibited by the SR blocker, PI ( 10 ). To more precisely determine the role of SR-A in this interaction, we examined the ability of AMs from SR-A (I/II)–deficient mice ( 24 ) to bind particles. We showed that both wild-type and SR-A–deficient macrophages bound unopsonized TiO 2 , Fe 2 O 3 , and latex beads essentially identically (data not shown). The scavenger receptor ligand PI inhibited the binding of particles to SR-A–deficient macrophages, suggesting that AMs express additional SR-like molecules that mediate opsonin-independent phagocytosis of particulate matter. To identify this receptor, we generated a monoclonal antibody, PAL-1, which blocked AM binding of particles . Using this antibody, we have cloned the cDNA encoding this receptor. Transfection of COS cells with this cDNA clone confers mAb PAL-1 reactivity . Importantly, transfection confers PAL-1–inhibitable binding of TiO 2 by COS cells . Interpretation of the COS cell transfection experiments was complicated by the observation that COS cells constitutively bind particles to a substantial degree. This indicated the presence of endogenous particle adhesion receptor(s). However, transfection of COS cells with MARCO but not control plasmid or cDNA resulted in mAb PAL-1–inhibitable particle binding . Importantly, the constitutive COS cell receptor(s) for particles is sensitive to heparin inhibition, whereas AM receptor–mediated particle binding is not heparin sensitive ( 10 ). Interestingly, transfection with MARCO, in addition to conferring mAb PAL-1–inhibitable particle binding, substantially diminished the component that was heparin inhibitable (data not shown). We speculate that the transfected cDNA competes with the endogenous COS cell particle receptor(s) for either surface expression or binding function. Existence of a particle-binding receptor on nonphagocytes such as COS cells is not unprecedented. The lung epithelial cell line A549 binds and ingests unopsonized TiO 2 , and this binding can also be inhibited by PI and heparin ( 38 ). The particle-binding receptor on hamster AMs is similar to murine and human MARCO . Murine MARCO was originally identified by screening a murine macrophage cDNA library with a human type XIII collagen probe. Murine MARCO is a 210-kD trimer made up of three disulfide-linked, 52-kD monomers and is expressed only on macrophages in spleen and lymph nodes ( 19 ). Human MARCO was recently cloned by screening human liver and spleen cDNA libraries with a murine MARCO probe ( 18 ). Human MARCO shares 68% sequence identity with mouse MARCO. Hamster MARCO had higher sequence identity to mouse than to human MARCO across all domains (Table II ). Interestingly, hamster MARCO has a shorter collagenous domain (less 34 amino acids) than both human and mouse MARCO. Also, like human MARCO, hamster MARCO does not have a cysteine residue in the spacer domain. The six cysteines in the cysteine-rich domain are conserved in all three species . The cysteine-rich domain defines a recently identified family of proteins ( 39 ). The true function of the SRCR is not clear. Hamster MARCO, like mouse and human MARCO, binds bacteria but not yeast . The expression of MARCO on normal AMs merits discussion. Initially, both human and mouse MARCO were not detected in normal lung ( 18 , 19 ). Using Western blot and immunolabeling techniques, we detected MARCO expression in normal lavaged AMs and AMs presented in diseased lungs (surgical specimens) . The use of adult AMs may explain the difference between this finding and the absence of mRNA in neonatal lungs ( 18 ). In mouse AMs, we have detected faint but reproducible immunoperoxidase labeling, using mAb ED31, of normal BAL AMs. This antibody also partially blocks mouse AM binding of particles, indicating that low levels of ED31 (MARCO) are present and functional on these cells. In these studies, we used normal, healthy looking mice housed in conventional facilities, not a “clean room” barrier facility. Expression of MARCO is induced on lung and liver macrophages in mice infected with Klebsiella pneumoniae ( 21 ). It is possible that our mice expressed MARCO because of low-level activation from their less clean environmental surroundings, a possibility yet to be formally tested. It is also noteworthy that both PI and anti-MARCO (ED31) cause lower levels of inhibition of unopsonized particle binding by mouse AMs compared with that seen with hamster AMs. Whether this reflects a significant difference (e.g., the presence of other receptor[s]), minor species, or technical variables remains to be determined. Our finding that MARCO mediates AM binding of particles defines a novel and immunologically important function for MARCO. As MARCO mediates binding of both inert particles and potentially pathogenic microorganisms, it will be interesting to determine how binding by MARCO modulates AM bactericidal functions. Specifically, the laudable absence of AM activation that accompanies binding (and ingestion) of inert environmental dusts may prove harmful for encounters with viable pathogens. Additional studies of the function of MARCO may provide more insight into the role of AMs in the initial, innate response of the lung to inhaled particles and pathogens. | Study | biomedical | en | 0.999998 |
10224291 | Phage displaying IgE-binding proteins were enriched from an A . fumigatus cDNA library constructed in phagemid pJuFo ( 19 ) and displayed on the surface of filamentous phage M13 as described ( 5 , 6 ). Serum IgE from A . fumigatus –sensitized individuals was captured in microtiter plates coated with anti– human mAb TN 142 and used as ligand for selective enrichment of allergen-displaying phage ( 6 ). Sera used for screening were selected according to case history, skin test reactivity to commercial A . fumigatus extracts, and specific IgE to A . fumigatus determined by radioallergosorbent test (RAST) ( 8 ). The screening procedure yielded a wide variety of phage able to bind specifically to human serum IgE and thus displaying allergenic molecules ( 5 , 6 ). Inserts carried by phage displaying IgE-binding proteins differing in length were sequenced as described ( 23 ) on an ABI prism 373A sequencer using the d -rhodamine terminator cycle sequencing kit ( Perkin-Elmer ) according to the manufacturer's instructions. Both DNA strands were sequenced using vector-derived primers. Homology searches and sequence comparisons were performed with BLAST and the Genetics Computer Group program FASTA ( 24 ). One clone revealed strong homology with nucleotide sequences encoding acidic ribosomal phosphoprotein type 2 . The cDNA encoding human P 2 protein was amplified by PCR from a commercial human lung cDNA library (Stratagene) using the following primers: 5′-primer, 5′-GCGGATCCATGCGCTACGTCGCCTCCTACC-3′; 3′-primer, 5′-GCTCTAGATTAATCAAAAAGGCCAAATCCC-3′. The complete cDNA coding for the putative A . fumigatus P 2 protein was amplified from the original clone by PCR using the following primers: 5′-primer, 5′-GCGGATCCATGAAGTACCTCGCAGCTTTCC-3′; 3′-primer, 5′-CCCGGACTTTAAGTCGAAGAGACCGAAGCCC-3′. PCR cycling conditions were 30 cycles of 95°C for 60 s, 57°C for 60 s, and 72°C for 60 s, followed by a terminal extension cycle at 72°C for 10 min. The amplification products were purified using a commercial kit (QIAquick; QIAGEN, Inc.), digested with BamHI and XbaI or BamHI and HindIII, respectively, and ligated to appropriately restricted pHis 6 –DHFR (dihydrofolate reductase) vector ( 4 ). Ligation mixtures were transformed into Escherichia coli strain M 15; transformants were grown in liquid to verify the nucleotide sequence ( 23 ) and used to produce hexahistidine-tagged recombinant proteins ( 4 , 6 ). After a single-step purification over Ni 2+ –chelate affinity columns ( 6 ), molecular size and purity of the recombinant proteins were analyzed by polyacrylamide gradient gels (4.5–20%) and 1-mg samples lyophilized for long term storage ( 14 ). The specific binding properties of serum IgE from A . fumigatus –sensitized individuals to recombinant fungal and human P 2 protein were analyzed by an allergen-specific ELISA ( 8 ). Absorbency was measured at 405 nm with a Molecular Devices reader and optical densities converted into arbitrary ELISA units (EU/ml) calibrated against an in-house serum pool arbitrarily defined as 100 EU/ml ( 8 , 11 , 12 ). Values below 1 EU/ml were set as 1 EU/ml for graphic display and nonparametric statistical analysis (Mann–Whitney U test). For IgE immunoblots, proteins were separated on SDS–polyacrylamide gradient gels (4–20%), transferred to nitrocellulose, incubated with patient sera diluted 1:10, and processed as described ( 16 , 25 ). PBMCs were isolated from heparinized peripheral venous blood by Ficoll density gradient centrifugation, washed three times, and resuspended in RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM l -glutamine, 50 μM 2-ME, 1% MEM nonessential amino acids and vitamins, 100 μg/ml streptomycin, 100 U/ml penicillin (all from Life Technologies), and 10% heat-inactivated FCS (Sera-Lab). Samples of 5 × 10 5 cells/100 μl were stimulated with different concentrations of recombinant A . fumigatus or human P 2 protein or with A . fumigatus extract in triplicate for 7 d. Proliferation was measured as incorporation of tritiated thymidine ( DuPont-NEN ) during the final 16 h of culture. A stimulation index >3 was considered positive. Recombinant proteins were dissolved in 0.9% saline at concentrations ranging from 10 −5 to 1 μg/ml. Intradermal skin tests were performed on the patient's back by injecting 100 μl test solution containing 1 ng recombinant protein. If no positive reaction could be observed after 15 min, testing was continued by injecting a 10-fold higher amount of protein. The test was stopped and considered positive when the wheal surface reached at least half of the histamine wheal size ( 8 , 16 ) or after injection of 1 μg protein. 0.01% histamine dihydrochloride and 0.9% saline were used as positive and negative controls, respectively ( 8 ). An ethical approval for skin testing human subjects with recombinant proteins was obtained from the local ethics committee (Davos, Switzerland). A full explanation of the procedure was given to all participants, and their written consent was obtained before testing. The cloning technology based on phage surface display of expression products from cDNA libraries ( 5 , 6 , 19 ) is particularly suitable for selective isolation of cDNAs encoding IgE-binding proteins from complex allergenic systems ( 26 ). Starting from a phage surface–displayed cDNA library generated from mRNA of A . fumigatus ( 6 ), we selectively enriched phage able to bind human serum IgE from individuals sensitized to the fungus. cDNAs isolated from single phagemids after four rounds of affinity selection, carrying inserts of different lengths, were sequenced and shown to code for different allergenic proteins ( 10 ). Among these, a clone containing an open reading frame spanning 333 bp revealed strong homology with sequences encoding eukaryotic type 2 acidic ribosomal phosphoproteins. The deduced amino acid sequence of this cDNA clone was homologous to P 2 proteins, showing a high sequence identity to the human (62%), C. herbarum (71%), and A. alternata (72%) P 2 proteins . Acidic ribosomal phosphorylated (P) proteins have been isolated and characterized from a variety of eukaryotic cells and share significant sequence identity and similarity ( 20 ). Both complete cDNAs encoding the putative A. fumigatus P 2 protein and the human P 2 protein were amplified by PCR, subcloned into the high level expression plasmid pHis 6 –DHFR, verified by sequencing, and used to produce hexahistidine-tagged P 2 proteins (see Materials and Methods). The constructs yielded, after single-step purification by Ni 2+ –chelate affinity chromatography, 33 and 28 mg/liter E. coli culture of virtually pure A. fumigatus and human P 2 protein, respectively. Purity was analyzed by reducing, denaturing SDS-PAGE, and Coomassie blue staining. In each preparation, only one band with estimated molecular mass in agreement with the calculated values of ∼12.6 kD for the hexahistidine-tagged A. fumigatus and human P 2 protein was visible (data not shown). As expected from the selection procedure devoted to isolate allergens from a phage surface display library, the A . fumigatus P 2 protein was able to bind IgE present in the serum used for screening. However, both A . fumigatus and human P 2 proteins were identified as allergens by ELISA with sera from individuals allergic to A . fumigatus and by IgE immunoblots (data not shown). Inhibition experiments showed that increasing amounts of human recombinant P 2 protein added to the fluid phase were able to inhibit the binding of serum IgE from patients sensitized to A . fumigatus P 2 protein , demonstrating that the proteins share common IgE-binding epitopes. 14/92 patients studied that were suffering from ABPA and 6/75 patients that were allergic to A . fumigatus and suffering from severe atopic dermatitis studied showed relevant levels of serum IgE antibodies to the A . fumigatus P 2 protein, resulting in an incidence of sensitization in the range of 15 and 8%, respectively. Sensitization to P 2 protein was not observed in A . fumigatus –sensitized individuals with mild forms of atopic dermatitis or in patients without ABPA. T cell help is required for the production of allergen-specific IgE ( 27 ). Therefore, we measured the proliferative responses of mononuclear cells from six individuals sensitized to the A . fumigatus P 2 protein to fungal extract, recombinant A . fumigatus , and human P 2 protein. The mean proliferative responses to optimal concentrations of extract, fungal, and human P 2 protein were 39,500; 20,092; and 11,790 cpm, respectively (mean SI 11.9, 6.5, and 5.7). The mean proliferative responses of 15,608; 1,601; and 1,273 cpm (mean SI 12.6, 0.8, and 1) to fungal extract, A . fumigatus , and human P 2 protein, respectively, obtained for four individuals sensitized to A . fumigatus lacking IgE antibodies against the fungal P 2 protein indicate that the recombinant antigens did not induce nonspecific effects. Three control individuals did not respond to any of the antigen preparations (mean SI < 1). Comparison of the values by the rank sum test indicates highly significant differences ( P < 0.01). The proliferative responses induced by human P 2 protein in individuals sensitized to the A . fumigatus P 2 protein indicate a pathogenesis related to autoreactive T cells. Intense local inflammatory responses to A . fumigatus occurring in the lungs of patients suffering from ABPA ( 11 , 12 ) and in the skin of patients suffering from severe atopic dermatitis might result in release of autoantigens as a consequence of tissue damage due to the inflammatory process ( 28 ). Exposure to autoantigens containing cross-reactive determinants (molecular mimicry) can recruit the memory T cell repertoire at the site of inflammation where lymphokine expression is upregulated. These lymphokines can induce expression of MHC II on naive T cells and upregulate accessory molecules that function as costimulatory signals for T cell activation, creating a microenvironment in which all requirements for priming a T cell response are present ( 29 ). Molecular mimicry at the T cell level could be a possible pathogenic mechanism to explain autoaggression remaining confined to the local area of inflammation ( 29 ). The ability of a protein to bind IgE in ELISA and Western blots provides strong evidence for the allergenicity of the protein. However, the final demonstration that a protein preparation acts as an allergen and therefore possesses biological activity in vivo is its ability to elicit a type I skin reaction in sensitized individuals. We have investigated whether the IgE-mediated cross-reactivity against A . fumigatus and human P 2 protein shown in vitro is sufficient to provoke allergic reactions in vivo through skin tests ( 8 , 16 ). Four individuals with high IgE levels against A . fumigatus P 2 protein, four individuals allergic to the fungus lacking IgE responses to the P 2 protein, and two nonallergic control individuals were investigated for their ability to respond to intradermal challenge with recombinant A . fumigatus and human P 2 proteins. As expected, none of the individuals without detectable IgE antibodies to A . fumigatus P 2 protein reacted against the recombinant protein preparations. A positive skin reaction to A . fumigatus P 2 protein was detected only in individuals who had IgE levels >10 EU/ml to the fungal protein. The amounts of recombinant protein needed to elicit a classical type I reaction ranged from 1 to 10 ng, depending on the subject. All individuals reacting to A . fumigatus P 2 protein also showed strong skin reaction to challenges with comparable amounts of human P 2 protein . These results show that human P 2 protein can cross-link IgE on mast cells in vivo ( 30 ) and suggest humoral autoimmune response in some patients suffering from mold allergy. IgE reactivity to fungal and human P 2 protein was, however, only detectable in individuals sensitized to A . fumigatus suffering from ABPA or severe atopic dermatitis. This was also the case for the IgE autoreactivity to human MnSOD described earlier ( 16 ). Therefore, these two human proteins may serve as a tool to study the role of IgE autoreactivity in tissue damage and release of autoantigens at the site of inflammation. | Study | biomedical | en | 0.999997 |
10224292 | All reagents were purchased from Sigma unless stated otherwise. C57BL/6, BALB/c, and CD22-deficient mice on a C57BL/6 background were obtained from our breeding facility. For immunocytochemical staining experiments, female BALB/c mice were purchased from Charles River. Most experiments were done with mice at 6–8 wk of age. Fc proteins used in this study were composed of the first three NH 2 -terminal extracellular Ig-like domains of siglecs fused to the Fc portion of human IgG1. The cDNAs encoding CD22-Fc, the mutant CD22(R130E)-Fc, sialoadhesin (Sn)-Fc, and myelin-associated glycoprotein (MAG)-Fc have been described previously ( 8 , 17 ). CD22-Fc, MAG-Fc, and Sn-Fc proteins were produced in CHO cells stably transfected using a glutamine synthetase expression system ( 18 ) and purified as described ( 8 ). In the comparative staining experiment with CD22(R130E)-Fc and CD22-Fc, concentrated tissue culture supernatants were used that had been predetermined by ELISA to contain immunoreactive Fc protein at 0.15 mg/ml ( 17 ). Femoral bone marrow plugs and other tissues were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature, transferred sequentially for 30 min each into 5, 15, and 30% sucrose in PBS, and then frozen in OCT (Miles, Inc.). 7-μm cryostat sections were treated with methanol plus 0.3% H 2 O 2 and then incubated for 1 h with Fc proteins at 10 μg/ml, followed sequentially by biotinylated anti–human Fc, ABC reagent, and diaminobenzidine (Vector Laboratories). Staining with biotin– Sambucus nigra agglutinin (SNA; Vector Laboratories) at 1 μg/ml was performed similarly. Before staining in some experiments, sections were pretreated for 3 h at 37°C with 0.2 U/ml Arthrobacter ureafaciens sialidase (ICN) in 0.1 M sodium acetate buffer, pH 5.0, in the presence or absence of 20 mM 2,3-dehydro-2-deoxy N -acetyl neuraminic acid (2,3-DDN; Boehringer Mannheim ). Bone marrow fragments were fixed for 1 h in 2% paraformaldehyde in 0.1 M phosphate buffer, washed extensively, and incubated for 12 h at 4°C with CD22-Fc or MAG-Fc (20 μg/ml) followed by goat anti–human Ig-peroxidase. Diaminobenzidine was used for visualization of reaction sites. The samples were postfixed in osmium tetroxide in 0.1 M phosphate buffer, dehydrated, and embedded in Spurr's epoxy resin. Thin sections were cut and examined in a Jeol 1200EX electron microscope. (a) BALB/c mice were injected intravenously with 0.15 mg of CD22-Fc or 0.15 mg MAG-Fc as a control, and killed 2 h later. Bone marrow plugs and spleens were processed and stained as described above, except that no primary antibody was used. (b) BALB/c or C57BL/6 mice (four mice per group) were injected once intravenously with 0.5 mg CD22-Fc or 0.5 mg Sn-Fc as a control, killed 24 h later, and cells were analyzed by flow cytometry. Samples of blood were taken after 12, 24, and 72 h, and the concentration of CD22-Fc was determined by an ELISA, as described ( 17 ). (c) Every 3 d over a period of 2 wk, 4-wk-old C57BL/6 mice were injected intraperitoneally with 0.5 mg of affinity-purified polyclonal rabbit anti-CD22 or with control rabbit IgG that did not bind to the CD22-Fc affinity column during purification ( 8 ). The mice were then killed, and the cellular composition in spleens and bone marrow of individual mice was analyzed by flow cytometry. Flow cytometry was performed as described ( 3 ) using three-color staining of lymphocytes. The following antibodies and reagents were used: FITC- and biotin-rat anti-IgD (11-26C; our hybridoma collection), FITC- and PE-rat anti-B220 (RA3-6B2; PharMingen ), PE-goat anti–mouse F(ab′) 2 IgM (Medac), PE-mouse anti-CD22 (Cy34.1; PharMingen ), and streptavidin-Red 670 (Life Technologies). An enzyme-linked immunospot (Elispot) assay was carried out as described ( 19 ). Bone marrow and spleen cells from three CD22-deficient mice and three age-matched control mice were prepared and pooled for each group, and the Elispot was performed in triplicate. Plates were coated with goat anti– mouse IgM or with goat anti–mouse IgG (Southern Biotechnology). After overnight incubation of cells plated at varying concentrations, they were washed off, and the spots were revealed with alkaline phosphatase–conjugated goat anti–mouse IgM or IgG antibodies (Southern Biotechnology). If the lectin activity of CD22 is directly involved in bone marrow homing of recirculating B cells, we reasoned that ligands for CD22 should be expressed within this tissue. This was investigated by staining sections of bone marrow with CD22-Fc, a soluble Fc chimera containing the NH 2 -terminal three Ig-like domains of mouse CD22. In the bone marrow, CD22-Fc stained tubular structures corresponding to the network of branched sinusoids . A few rare scattered cells in the hematopoietic spaces were also stained. In contrast, no vascular structures were stained in other organs examined, including spleen , lymph node, heart, and liver (not shown). However, in spleen and lymph node (not shown), CD22-Fc gave clear staining of B lymphocytes in follicles, together with scattered T cells, as shown previously ( 20 ). No staining of any tissues was observed with the related proteins, MAG-Fc or Sn-Fc (not shown). Specific staining of endothelial cells by CD22-Fc was confirmed by immunoelectron microscopy . The label was predominantly located along the luminal surface of the sinusoidal endothelial cell that is normally exposed to blood elements, with reduced labeling of the basal surface . In contrast, endothelial cells lining arterioles and venules were unstained (not shown), as were the great majority of hematopoietic cells. Two lines of evidence indicated that the binding of CD22-Fc to bone marrow endothelium was Sia dependent. First, no staining was observed with CD22(R130E)-Fc, which carries an inactivating mutation within the Sia binding site . Second, treatment of bone marrow and spleen sections with A . ureafaciens sialidase was found to abolish binding of CD22-Fc, and this could be reversed by addition of the sialidase inhibitor, 2,3-DDN (not shown). We next compared CD22-Fc staining with that of SNA, a plant lectin with a well-defined specificity for oligosaccharides carrying α2,6-linked Sia ( 21 ). Although the spleen staining was comparable with both reagents (not shown), the bone marrow showed striking differences, in particular the apparent lack of staining of sinusoidal endothelium by SNA . However, SNA labeled a major subset of cells in the hematopoietic spaces, most of which were unlabeled by CD22-Fc. These results suggest that bone marrow ligands recognized by CD22-Fc and SNA are distinct. It was important to determine if CD22 is able to interact with the sialylated bone marrow ligands in vivo , since plasma is rich in α2,6-sialoglycoproteins which could compete for binding. As shown in Fig. 1 H, intravenous injection of CD22-Fc resulted in a staining pattern in bone marrow similar to that observed after in vitro staining , whereas injection of MAG-Fc did not result in detectable labeling . The half-life of CD22-Fc in the circulation was found to be ∼56 h (data not shown). To determine if circulating CD22-Fc could interfere with localization of mature B cells to the bone marrow by masking CD22 ligands, mice were given a single injection of CD22-Fc, and B cell numbers were assayed after 24 h. Compared with either Sn-Fc or PBS (not shown) used as negative controls, injection of CD22-Fc led to a 50% reduction in the population of bone marrow IgD + B cells , whereas immature (IgM lo IgD − ) and transitional (IgM hi IgD lo ) B cells were unaffected . There was no effect on B cell numbers in the spleen . In an attempt to block the Sia binding site of CD22 in vivo, 4-wk-old mice were injected with polyclonal rabbit anti-CD22 IgG over a 2-wk period during which long-lived B cells populate the marrow. Antibody treatment resulted in a selective reduction in the numbers of recirculating IgD + cells in the bone marrow, at ∼50% of the cell numbers present in control mice injected with nonspecific rabbit IgG . This reduction was not found in the spleen , and there was no effect on numbers of immature or transitional B cells (not shown). Unexpectedly, however, the treatment with anti-CD22 led to a downmodulation of the molecule from the surface , presumably due to internalization, and this may have contributed to the reduction of mature B cells in the bone marrow. The bone marrow is known to be a major site of Ig secretion by plasma cells ( 22 ). To address the question of whether the lack of recirculating B cells in the bone marrow of CD22-deficient mice would also affect plasma cells, we determined their number in the bone marrow and spleen of CD22-deficient and control mice. In the bone marrow of CD22-deficient mice, there was a significant reduction of IgM-secreting plasma cells, whereas these cells were increased in the spleen compared with wild-type mice . In this experiment, the number of IgG- secreting plasma cells was also reduced in the bone marrow. In two other experiments, the reduction of IgG-secreting plasma cells in the bone marrow of CD22-deficient mice was less pronounced, whereas IgM plasma cells were consistently reduced (not shown). Here we present evidence that CD22 is a specific receptor involved in the homing of long-lived recirculating B cells to the bone marrow. Our demonstration that CD22 ligands are constitutively expressed on endothelial cells in the bone marrow, but not in other organs, raises the attractive possibility that CD22 can function as a classical homing receptor for the bone marrow by targeting cells to the appropriate microenvironment. It has been reported that IgD + recirculating cells are found in the extravascular space, mainly in perisinusoidal locations ( 15 ). The interaction with CD22 ligands on endothelial cells could be an important first step before transmigration of B cells into the bone marrow parenchyma. Another possibility is that CD22 serves as a retention signal, preventing the reexit of B cells from the bone marrow. The reason for the incomplete block in B cell homing after the in vivo treatments could be related to the relatively low affinity of the carbohydrate binding region of CD22 ( 10 ). Alternatively, CD22-independent homing pathways may exist. Although most of the long-lived IgD + cells are thought to result from a maturation process in the peripheral lymphoid tissue ( 15 , 16 ), it is possible that some of these cells are produced locally in the bone marrow. In support of this, CD22-deficient mice show an age-dependent accumulation of IgD + cells in the bone marrow (3; our unpublished observations). Expression of CD22 ligands is regulated by α2,6-sialyltransferases, especially ST6GalI ( 10 ). Recently, a mouse line deficient in ST6GalI was reported which had a normal composition of B cells in the bone marrow, including mature recirculating B cells ( 23 ). If ST6GalI is the enzyme responsible for creation of the CD22 ligands implicated in B cell homing, we would have expected a reduction of mature B cells in the bone marrow. The discrepancy could be due to expression of an alternative α2,6-sialyltransferase in bone marrow endothelial cells or the use of compensatory homing mechanisms in ST6GalI-deficient mice. One obvious question is how CD22 on B cells is able to bind ligands on other cells when its lectin binding site is largely masked ( 13 ). Interestingly, we have recently found that a significant subset of IgD + B cells in murine bone marrow is able to bind CD22 ligands, whereas only a minor subset of these cells binds in the spleen or lymph nodes (Floyd, H., L. Nitschke, and P.R. Crocker, unpublished observations). One interpretation of this observation is that “lectin-competent” IgD + B cells become selectively enriched in the bone marrow due to interactions with CD22 ligands expressed on bone marrow endothelial cells. In both mice and humans, most of the IgD + recirculating B cells carry rearranged Ig genes with only a small number of somatic mutations ( 24 ). However, there is also evidence that the pool of IgD + B cells in the human bone marrow contains memory B cells with a large number of somatic mutations ( 25 ). Isotype-switched plasma cells are thought to be derived from precursors that encounter antigen in germinal centers and then migrate to the bone marrow, which is a major site of Ig production. However, in the case of IgM-secreting plasma cells, the locus of final maturation is not well defined ( 22 ). It is possible that the bone marrow is a site for the final maturation of these cells from the pool of IgD + recirculating B cells. This would be consistent with our finding that CD22-deficient mice have a reduced number of IgM-secreting plasma cells in this tissue. An alternative explanation is that freshly activated, IgM-secreting B cells are dependent on CD22 for homing to the bone marrow. Finally, an important question that remains is how the adhesive activity of CD22 is coupled to its signaling function. Binding of the lectin domain to ligands in the bone marrow microenvironment would be expected to influence the B cell signaling threshold and thereby determine the fate of the B cell. The issue of how the signaling function is coupled to the lectin function of CD22 will be addressed in future experiments. | Other | biomedical | en | 0.999996 |
10225944 | Similar to steroid and thyroid hormones, vitamin D3, and retinoic acid, it appears that GFs may be present and function in cell nuclei. In different target cells, nuclear association was shown for FGF, EGF, NGF, PDGF, insulin, etc. . Although the idea of nuclear GFs is more or less accepted, the functional significance is generally debated based on some reports. For example, activation of the Raf-MAPK pathway was shown to be sufficient and necessary for transduction of the aFGF mitogenic signal in BaF3 hematopoietic cells . Still, several data indicate that nuclear localization of FGFs may be required for the mitogenic effect in certain conditions in different cell types. The presence of radiolabeled, externally added aFGF in the nuclear fraction appeared to correlate with stimulation of DNA synthesis in a concentration-dependent manner in NIH 3T3 cells (with a submaximal [ 3 H]thymidine incorporation value at 10 ng/ml FGF-1). Correlation between nuclear association of aFGF and DNA synthesis was demonstrated also in diphtheria toxin–resistant U2 Os Dr1 cells. Although these cells lack aFGF receptors, they were able to internalize aFGF via their cell surface toxin receptors, if the GF was fused to the diphtheria toxin fragments. After extracellular administration, the aFGF-toxin label was detected in the nuclear fraction. At the same time, DNA synthesis was found to rise about fourfold (at a fairly low, 5 ng/ml aFGF-toxin). However, no significant increase in the number of cells was observed. Therefore, it appears that although nuclear action of aFGF seems to be sufficient for triggering DNA synthesis, FGFR is indispensable for other processes of cell proliferation . Consistent with this idea, DNA synthesis was accompanied by cell proliferation only in cells which possess aFGF receptors or if the toxin-resistant cells were transfected with a FGFR . However, one has to keep in mind that cell lines, transfected cells, and tumor cells most probably do not behave and cannot be considered as normal cells. Nonetheless, cell proliferation rate and nuclear association of bFGF was reported to change in parallel not only in glioma cells for example, which express transfection-derived endogenous FGF-2, but also in primary cultures of human astrocytes stimulated with extracellular bFGF (concentration range: 0.09–2.5 nM) . These observations support the idea that nuclear translocation of GFs could be related to mitogenesis in normal, nontransformed cells as well. Uptake of extracellular bFGF to the nucleus and to the nucleolus was found to occur only in late G 1 phase of the cell cycle in growing aortic endothelial (ABAE) cells, both by immunocytochemistry and by analysis of radioiodinated cell fractions . Nuclear association of FGF-2 was also observed in mid-late G 1 phase in proliferating epiphyseal plate chondrocytes , suggesting a controlled nuclear entry of GFs around the restriction point of the cell cycle. It is important to note that autocrine and intracrine FGF types can have different effects, which are related to their partially different sequence and to their characteristic site of action. From the four different forms of human FGF-2, the low molecular mass form (with 18 kD) is an autocrine/ paracrine one. The three high molecular mass forms (with 21–22, 22.5, and 24 kD, respectively) are the intracrine ones generated by alternative translation initiation at CUG codon, through an internal ribosome entry process regulated by a cis-acting mechanism . These intracrine forms, which have a longer, arginine-rich NH 2 -terminal with at least two possible short nuclear localization sequences (NLSs) (Gly-Arg-Gly-Arg-Gly-Arg), are preferentially targeted to the nucleus . In contrast, the 18-kD form has only a weak or cryptic short NLS, and is found predominantly in the cytoplasm . Only the short bFGF form can be released from the cell, and can, therefore, interact with the plasma membrane FGFR. Surprisingly, when intracrine bFGF types were expressed in NIH 3T3 cells, high proliferation rates and growth in soft agar were observed, even in the presence of mutant cell surface bFGF receptors lacking the Tyr kinase domain . This reflects a plasma membrane receptor–independent pathway, presumably via formation of complexes between intracrine GFs and intracellular receptors (see below). In vascular smooth muscle cell lines expressing different human bFGFs, the intracrine bFGF forms appeared to be significantly more effective in augmenting the rate of DNA synthesis than the autocrine one . Moreover, the continuous proliferation of two glioma cell lines is suggested to be related to the constitutive presence of endogenous FGF-2 in nuclei; these cells were nonresponsive to extracellular GFs . Synthesis of CUG-initiated forms could be induced also in primary human skin fibroblasts, producing normally the short bFGF form almost exclusively, by heat shock (45°C, 15–60 min) and by oxidative stress, which is probably due to translational activation . Developmental studies indicated that FGF-2, known as a maternal signal involved in mesoderm induction in amphibians, brings about mesoderm induction via Src-kinase Laloo and MAP kinase . However, nuclear bFGF may be involved in other specific developmental phenomena, since nuclear association of bFGF becomes restricted to some cell populations during embryogenesis. In the mid-blastula stage, FGF-2 was demonstrated clearly in the nuclei of the animal hemisphere of Xenopus ; in the prelarval embryo, nuclear bFGF was shown in most head regions (including the brain) and particularly in some muscle cells of the trunk region . This is consistent with the well-known stimulatory effects of bFGF on myoblast proliferation and on proliferation plus differentiation of neuroblasts and glial precursor cells . In early chicken embryos, nuclear FGF-2 isoforms were observed in most cells of the prestreak blastodiscs during hypoblast formation and mesoderm induction. Only the hypoblasts and the blastocoelic cells seemed to maintain their nuclear immunostaining during primitive streak formation and with the onset of gastrulation . In later phases, only a small proportion of limb bud cells, most likely migrating myoblasts, and differentiating kidney podocytes were shown to have considerable nuclear FGF-2 . Recently, several data have accumulated which support the idea of GF receptor translocation to the nucleus. For example, three FGFR-1 variants (with 145, 118, and 103 kD, respectively) were detected in the nucleoplasmic and in the nuclear matrix fractions of human astrocytes and bovine adrenal medullary cells. In the majority of cells, the immunofluorescence signals of FGF-2 and FGFR-1 appeared to colocalize in the nuclei . So, how could the GFR gain access into the nucleus? According to the emerging view, NLS-bearing GFs like FGFs presumably facilitate the nuclear import of their receptors. Theoretically, GFs do not need NLS to enter the nucleus, since the molecular “sieves” of nuclear pores demand it only from compounds >40–45 kD. Possession of NLSs by low molecular mass GFs implies that this may be necessary for the nuclear import of their receptors which can be transported “piggyback” to the nucleus in association with NLS-bearing ligands . The concept that plasma membrane GFRs could enter the nucleus upon extracellular GF stimulation is supported by the accurate study of Maher , who demonstrated a dose- and time-dependent increase of nucleus-associated FGFR-1 immunoreactivity in Swiss 3T3 fibroblasts (onset: within 10 min, max: 1 h; concentration: 5–15–50 ng/ml). Moreover, the FGFR-1 in the nuclear fraction was shown to bear the impermeable biotin label of the cell surface proteins and was proven to be of full length, verifying its plasma membrane origin. Even intracrine FGFs may enter the nucleus in complex with intracellular receptors. Consistent with this idea, several truncated forms of FGFR-1 and FGFR-2 have been described, which are devoid of the transmembrane region . Furthermore, a truncated FGFR3 variant missing the transmembrane part and half of the final Ig-like domain was shown to be characteristically associated with cell nuclei in breast epithelial cell lines by immunocytochemistry . Considering the role of high-affinity GF receptors in nuclear targeting, they are probably prerequisite for the intracellular transport of GFs to the perinuclear region during receptor-mediated endocytosis. According to the studies of Prudovsky et al. on transfected L6 myoblasts, the first Ig-like loop in type 1 FGFRs may facilitate the transport of exogenous FGF-1 to the perinuclear area, as mostly the α, 3-loop receptor isoforms possessing this domain (and not the β isoforms lacking it) were demonstrated in the nuclear/perinuclear fraction. N-glycosylation seems to be also important, as tunicamycin treatment significantly reduced the presence of the α receptor forms in the nuclear/perinuclear fraction. This can be interpreted on the basis of NLS-independent, but sugar-dependent nuclear import mechanism described by Duverger et al. , as GFRs are glycoproteins . Regarding FGFs, possible involvement of low-affinity saccharide receptors in nuclear translocation cannot be ruled out. Heparan sulfate proteoglycans (HSPGs) with highly O-sulfated oligosaccharide chains are well known to play a crucial role in the formation and in the maintenance of the active FGFR–a/bFGF complexes at the plasma membrane . Perlecan, a basal lamina proteoglycan , syndicans, and glypican proved to be effective in stimulating FGF–FGFR interaction. It is thought that HSPG and extracellular bFGF bound to FGFR might be cotranslocated to the nucleus; HSPG could stabilize the complex and protect it from degradation in the endocytotic vesicles and in the lysosomes . Since glypican was observed in association with cell nuclei (in rat neurons and in glioma cells) and, moreover, it was shown to have functional NLS , this gives further credence to the mentioned idea. In NIH 3T3 cells, the constitutively activated FGFR-3 mutant kinase domains in linkage with the plasma membrane appeared to be sufficient to trigger cell proliferation and transformation, in contrast to wild-type kinase domains, or to activated kinase domains targeted to the nucleus or to the cytoplasm . However, in astrocyte and glioma cultures, cell proliferation appeared to correlate with the nuclear presence of FGFR-1. Continuously proliferating glioma cells, unresponsive to external FGF, displayed constitutive nuclear association of FGFR-1. In contrast, astrocytes had decreasing nuclear appearance of FGFR-1 in parallel to increasing cell density in cultures approaching confluency. Furthermore, enhanced cell proliferation rate could be achieved in glioma cells lacking FGFR-1 by transfecting them with the full-length receptor cDNA; thereafter, immunoreactivity of FGFR-1 was seen predominantly in association with the nucleus . Both in astrocytes and in bovine adrenal medullary cells, the nuclear FGFR was shown to retain kinase activity. With this observation, we arrived at a basic, but currently unresolved question. How could GFs and GFRs act in the nucleus? Nuclear FGFR kinase activity is thought to have no significant role in the induction of cell proliferation . However, bFGF may be involved in induction of ribosomal gene transcription via stimulation of casein kinase-2, which is known to regulate nucleolin, a major component of the nucleolus implicated in ribosome biogenesis. Using nuclear extracts of FM3A cells and purified proteins, FGF-2 was shown to bind CK-2 and stimulate its activity, resulting in an increased phosphorylation of nucleolin . (Enhancement of CK-2 activity reached its maximum at 10 -7 M FGF-2 concentration, which was calculated to be a possible bFGF concentration in the nucleus.) Supporting these observations, rRNA was found to increase severalfold upon addition of bFGF (0.1–1 nM) to isolated nuclei from quiescent ABAE cells . Furthermore, GF–GFR complexes may cotransport intranuclearly acting molecules to the nucleus, via binding to the receptor, as was suggested for IFN-γ–IFN receptor complex and STAT by Johnson et al. . According to the classic view, extracellular GFs stimulate their receptor-mediated endocytosis, which leads to degradation (or to recycling) of GF receptors. However, it is plausible to suppose that a portion of GFs and GFRs can escape from the endosomes or lysosomes and may reach the nucleus. Growth hormone was demonstrated to undergo a receptor-dependent nuclear translocation via the endosomes in rat hepatocytes ; its nuclear uptake could be significantly increased upon the addition of some lysosome inhibitors, indicating an escape route from the lysosomes. It is noteworthy that FGFR-1 could be detected only in few regions at the nuclear envelope and displayed a patchy distribution within the nucleus of bovine adrenal medullary cells; this may reflect nuclear entry at determined membrane pores and controlled transport to special nuclear sites . It is intriguing to hypothesize that actin is involved not only in endocytosis and in the transport of endosomes to the perinuclear area , but also in the precise nuclear targeting of GF–GFR complexes from the perinuclear cytoplasm, since actin is known to be present in abundance in cell nuclei, both in the chromatin and in the nuclear matrix fraction . Furthermore, there is evidence that a GF receptor, the EGFR, is a (direct) actin-binding protein ; other GFRs may be linked to actin indirectly, via actin-binding proteins, during their nuclear translocation. It should be noted in this context that extracellular matrix-dependent cytoskeletal organization supervises GF action on proliferation of normal cells, reflected in the well-known phenomenon of anchorage-dependent growth. Cell division is generally preceded by extensive cell spreading . Spreading is probably necessary for nuclear translocation of GF–GFR complex in normal cells, since only astrocytes in subconfluent cultures (and not the ones in confluent cultures in short of extracellular surface) were observed to have nuclear-associated bFGF and FGFR-1. On the contrary, in continuously growing glioma cells, nuclear appearance of FGF-2 and FGFR-1 was constitutive and was largely independent of cell density . Finally, bFGF gene is continuously activated in glioma cells irrespective of cell density, whereas in astrocytes bFGF transcription is induced by subconfluency . All in all, it seems that a portion of internalized exogenous FGFs plus their receptors may escape degradation and could be transported to the cell nucleus. Nuclear GF– GFR complexes appear to stimulate cell proliferation in certain conditions in several cell types; in addition, activation of cell line–specific genes may occur in some differentiating cells. Continuous proliferation of transformed cells could be partially due to the continuous nuclear presence of GFs and GFRs. Obviously, much work has to be done to elucidate details of the nuclear targeting of GF–GFR complexes and to be able to understand their nuclear action fully. | Study | biomedical | en | 0.999996 |
10225945 | The clones of two Mad2 ESTs (expressed sequence tags) were gifts from Pioneer Hi-Bred. At the time the EST database was queried, there were 80,000 sequences available. One clone, CGEUZ35 is described here (the other clone, CDPEE81 may identify a second locus but further studies are needed). Complete sequencing revealed CGEUZ35 to be a full-length Mad2 cDNA. To express MAD2 in E . coli , KpnI and XbaI restriction sites were incorporated into the 5′ and 3′ ends of the open reading frame by PCR, and the fragment inserted into the pThioHisC vector (Invitrogen). The pThioHisC vector is designed to fuse thioredoxin to the NH 2 terminus of the expressed protein. Thioredoxin-MAD2 fusion protein was induced using 10 mM IPTG in E . coli TOP10 cultures grown at room temperature. The recombinant protein was partially purified on a nickel column (Invitrogen), and then purified to near-homogeneity by ion-exchange chromatography (Macro-Prep DEAE Support; BioRad). Polyclonal antibodies against the purified thioredoxin-MAD2 protein were produced in rabbits by the UGA polyclonal antibody facility. The resulting antibodies were either blot affinity purified or column affinity purified against the recombinant maize MAD2 using an UltraLink Immobilization Kit (Pierce). Affinity-purified antibodies were extensively dialyzed in PBS at 4°C, and concentrated to ∼1 mg/ml with Centriplus concentrators (Amicon). Different maize tissues from the W23 inbred, including young tassels (∼5 cm), young ears (∼8 cm), young leaves, and root tips (∼1–2 cm) were ground in liquid nitrogen and resuspended in 20 mM Tris-HCl (pH 6.8), 20 mM EDTA, 200 mM NaCl, and 1 mM PMSF. Standard SDS-PAGE was performed and proteins were blotted to nitrocellulose . The protein blot was blocked with 5% Carnation nonfat milk and incubated with affinity-purified anti-MAD2 antibodies (final concentration ∼0.2 μg/ml) for at least 2 h at room temperature. After washing 3× in TBST, the membrane was incubated with peroxidase-conjugated goat anti–rabbit antibodies ( Amersham ) for one hour at room temperature, and chemiluminescent immunodetection carried out using an ECL Western-blotting kit ( Amersham ). When the affinity-purified antiserum was preincubated with the purified thioredoxin-MAD2 before use, no bands were detected on maize protein blots (data not shown). The culture and data collection from live microsporocytes was performed as before . Meiocytes at the appropriate stages were dissected from anthers into a modified rye culture medium, stained with Syto 12 at ∼2.5 μM, and visualized using the DeltaVision system (described below). Four-dimensional data sets (three-dimensions over time) were collected and analyzed. Seeds from the KYS inbred line were germinated in a moist chamber at 26°C. 3-d-old seedlings with root tip lengths of ∼1 cm were treated with 1 μM oryzalin (Chem Service), a concentration that is sufficient to depolymerize all of the mitotic microtubule arrays in oat . Oryzalin-treated root tips were harvested at 0, 4, and 8 h, and processed for immunocytochemistry as described below. Meiocytes from the maize inbred W23 were extruded into PHEMS containing 3% paraformaldehyde and 0.05% Triton X-100. In the experiments involving the 3F3/2 antibody, 100 nM microcystin (a phosphatase inhibitor; Sigma ) was added to the fixation buffer. Cells were transferred to polylysine-coated coverslips for ∼15 min to allow fixation. Fixed cells were washed 3× in PBS before immunocytological experiments. Mitotic cells (untreated or oryzalin-treated) were prepared from the seedlings of W23 and KYS inbreds that had been cultured in a moist chamber at 26°C. Root tips ∼1 cm in length were cut from 3-d-old seedlings and transferred to the same buffer used to fix meiocytes for ∼30 min at room temperature. Fixed root tips were washed 2× in PBS and quickly frozen in PolyFreeze (PolySciences) using liquid nitrogen. Sections ∼10 μm thick were prepared on a cryostat at −20°C. The root tip sections were transferred to polylysine-coated slides for further study. In double-labeling experiments for MAD2 and tubulin, or MAD2 and the 3F3/2 epitope, it was possible to use indirect immunolocalization. The procedure was performed essentially as before except that the rabbit antiserum was detected with rhodamine-conjugated secondary antibodies (111-095-144; Jackson ImmunoResearch Laboratories). The mouse monoclonal antibody to α-tubulin, a generous gift of David Asai , and the monoclonal antibody 3F3/2, a generous gift of Gary Gorbsky (University of Virginia, Charlottesville), were detected by FITC-conjugated secondary antibodies (115-095-146; Jackson ImmunoResearch Laboratories). The 3F3/2 primary antibody was used at a 1:50– 1:100 dilution. In the experiment to determine the effect of phosphatase treatment on 3F3/2 staining, cells were first fixed in the presence of 100 nM microcystin and then treated with 100 units/ml phosphatase for 30 min at 37°C, either in the presence or absence of 5 μM microcystin. In cases where MAD2, CENPC, and tubulin were detected simultaneously , MAD2 and CENPC were direct-labeled (see below), and tubulin was detected using CY5-conjugated anti– mouse antibodies (115-175-146; Jackson ImmunoResearch Laboratories). Chromosomes were stained with DAPI at 0.1 μg/ml. To visualize MAD2 and CENPC simultaneously (antibodies to both were generated in rabbits), fluorescent dyes were directly coupled to primary antibodies using the Alexa 546 and Alexa 488 Protein Labeling Kits (Molecular Probes). For the data in Fig. 3 A, affinity-purified anti-CENPC and affinity-purified anti-MAD2 antibodies were used for direct labeling. The efficiency of labeling for the CENPC antibody was poor at ∼1 mol dye/mol protein whereas the efficiency of labeling for the MAD2 antibody was excellent at ∼12 mol dye/mol protein. The poor labeling of the CENPC made it difficult to obtain high-contrast images. Therefore the CENPC antibody labeling was repeated on the proteins derived from a 40% ammonium sulfate precipitation of the crude antiserum . When the labeled protein preparation was used to detect CENPC in double-labeling experiments with the directly labeled anti-MAD2, the CENPC staining was much brighter and qualitatively indiscernible from the results obtained when directly labeled affinity-purified CENPC antibodies were used. The labeled ammonium sulfate precipitate was used in most of the experiments where quantitative data was collected and for Figs. 3 B, 6, and 7. Since the affinity-purified MAD2 antibodies were well-labeled with Alexa 546, ammonium sulfate precipitates were not used to detect MAD2. Chromosomes were stained with DAPI at 0.1 μg/ml. Kinetochores were not detected either by the crude rabbit CENPC or MAD2 preimmune sera, or by purified IgGs derived from these sera (data not shown). Data from both living and fixed cells were collected using an Applied Precision, Inc. SA3.1 multidimensional light microscope system . Optical sections were taken using a 60× Nikon objective at 0.2 μm to 0.4 μm intervals either with or without a 1.5× Optivar (pixel size = 0.065 μm or 0.097 μm, respectively). The data from living cells were binned, resulting in an effective pixel size of 0.196 μm . The three dimensional data sets were mathematically deconvolved, to remove the out-of-focus information, with software supplied with the DeltaVision system. The images were scaled to optimize contrast but not enhanced further. The intensity of MAD2 staining at kinetochores (see Results on MAD2 staining in mitosis) was measured by identifying optical sections with the most intense MAD2 staining, and averaging the grey level values from 9 pixels in a 3 × 3 square. The kinetochore-kinetochore distances were measured using software supplied with the DeltaVision system. Image color was modified using the GraphicConverter program (Lemke Software) and printed using a Techtronix Phaser IIsdx dye sublimation printer. A full-length cDNA homologous to Saccharomyces cerevisiae Mad2 was identified in a maize EST data base compiled by Pioneer Hi-bred. Complete sequencing revealed that the Mad2 gene encodes a polypeptide of 208 amino acids with a predicted molecular mass of 24 kD. BLAST alignments revealed 42% identity (64% similarity) to yeast Mad2, 45% identity (65% similarity) to human MAD2 and 46% identity (70% similarity) to Xenopus XMAD2. The sequence alignment is shown in Fig. 1 . Using the full-length maize Mad2 cDNA, a thioredoxin-MAD2 fusion protein was generated, purified, and injected into rabbits. The resulting antibodies were affinity-purified against the recombinant MAD2 protein and used on protein blots. As shown in Fig. 2 , the purified antibody preparation recognized a single 24-kD protein in tassel, ear, and root tissue. The results are consistent with the predicted molecular mass of maize MAD2 as well as previous animal studies where 24-kD MAD2 proteins have been consistently observed . To determine the precise subcellular localization of MAD2, we made use of a recently generated antibody to maize CENPC . CENPC is a kinetochore structural component in yeast, mammals and maize, where it appears to localize close to the centromeric DNA . Because the MAD2 and CENPC antisera were both prepared in rabbits, the affinity-purified antisera were differentiated from each other by direct labeling using differing fluorochromes. Fig. 3 A shows a triple-labeled prometaphase I cell, where DNA, MAD2, and CENPC were each detected using different wavelengths on a three-dimensional light microscope workstation. The data demonstrate that MAD2 and CENPC occupy essentially nonoverlapping domains in the maize kinetochore: CENPC occupies an inner domain close to the chromosome while MAD2 occupies an outer domain. As shown in Fig. 3 B, similar results were obtained in prometaphase II cells. The affinity-purified MAD2 antiserum also recognized mitotic kinetochores from maize root tip cells (see below). Because the directly labeled anti-CENPC antibodies only weakly recognized these kinetochores, however, we were unable to determine whether mitotic kinetochores have a substructure similar to meiotic kinetochores. In mitotic root tip cells, we observed a cell cycle–specific pattern of MAD2 staining that is essentially the same as that reported for mammalian cultured cells and newt lung cells . The important features of the MAD2 localization patterns are shown in Fig. 4 . The interphase and prophase stages can be identified by the state of chromatin condensation and the characteristic perinuclear array of microtubules that mark the presence of the nuclear envelope . In prometaphase, the nuclear envelope and perinuclear array are broken down and the chromosomes begin to interact directly with microtubules. Using indirect immunofluorescence we were unable to detect MAD2 staining at the interphase and prophase stages , but detected intense kinetochore staining in prometaphase . All 20 kinetochores were visible in some prometaphase cells, with most chromosomes containing paired spots representing sister kinetochores. The identity of the MAD2-positive regions as kinetochores was further verified in several cases by combined direct immunofluorescence of MAD2 and CENPC. MAD2 staining became undetectable in metaphase , and remained undetectable in anaphase and telophase (data not shown). Further inspection of prometaphase cells revealed that the MAD2 staining on sister kinetochores was frequently unequal. Bright MAD2 staining was correlated with weak staining of associated microtubules (known as kinetochore fibers, or K-fibers) and weak MAD2 staining was correlated with bright staining of the associated K-fibers . A quantitative analysis of 10 chromosomes with a single attached kinetochore fiber indicated that, on average, microtubule attachment caused an ∼5.7-fold reduction in the intensity of MAD2 staining (SD = 4.0, with a low of 2.8 and a high of 14.4). Based on these data, we expected that artificial depolymerization of microtubules would result in bright MAD2 staining at all kinetochores, as had been established in previous studies . A variety of microtubule destabilizing agents is available for plants, many of which are used as herbicides. One particularly effective microtubule destabilizing agent is oryzalin . As shown in Fig. 4 E, a 4-h treatment of oryzalin disrupted mitotic spindles and arrested the cells at a prometaphase-like stage. Nearly all the microtubules were depolymerized with only short K-fibers remaining at the kinetochores of some chromosomes . MAD2 staining was relatively bright at kinetochores with thin or no K-fibers attached and weak or absent on kinetochores with short K-fibers. An 8-h oryzalin treatment depolymerized all the microtubules, including the short K-fibers visible in four-hour treated cells. Consistent with expectations, all kinetochores stained brightly with the anti-MAD2 antibodies in the 8-h arrested cells . These data lend support to the argument that microtubule attachment is sufficient to cause the dissociation of MAD2 and the inactivation of the spindle checkpoint . To further test the idea that microtubule attachment is associated with a loss of MAD2 staining, we extended our studies to the more specialized meiotic cell divisions. We began our analysis of meiosis I with living meiocytes to determine the timing and characteristic chromosome movements of this cell division. Using the cell culture and chromosome staining techniques that were developed in an earlier study of meiosis II , we successfully recorded the prometaphase-metaphase transition in six living meiosis I cells. Time-lapse data from one of these cells is illustrated in Fig. 5 . The data indicate that during meiotic prometaphase I the chromosomes accumulate at the spindle midzone relatively quickly, but can take up to 60 min to form a definite metaphase plate. The metaphase I plate was characterized by a continuous gap that appeared to separate the homologues (the chiasmata were apparently stretched to the point that they were difficult to see). Once such a plate was formed, anaphase onset occurred within 30 min. Immunocytochemical analysis of fixed cells indicated that MAD2 was not detectable during the prophase stages of meiosis I. This is illustrated by the cell in Fig. 6 A, which at a late stage of prophase I (diplotene-diakinesis) lacks any evidence of MAD2 staining on the chromosomes. It is unlikely that the absence of prophase staining was a consequence of poor antibody penetration, because when the cells were counterstained with anti-CENPC antibodies the kinetochores were clearly visible . MAD2 was readily detectable on congressing chromosomes during prometaphase I . Unlike in mitosis, however, the association of microtubules with the kinetochores did not appear to dim the intensity of MAD2 staining . The result was that the paired homologous kinetochores appeared to stain brightly and with nearly equal intensity well into late prometaphase. A hallmark of late prometaphase-metaphase I transition is that the sister kinetochores begin to separate from each other ; the fact that we were able to detect sister kinetochore separation using MAD2 antibodies is one indication that the chromosomes are MAD2-positive until immediately before metaphase. The dissociation of MAD2 appeared to occur gradually on a chromosome-by-chromosome basis, such that during the transition from prometaphase to metaphase some chromosomes had MAD2 staining while others lacked MAD2 staining (discussed below with respect to 3F3/2 staining). In cells that fit our strict definition of metaphase I (a gap between homologues) MAD2 was undetectable. The patterns of MAD2 staining in meiosis II were similar in most respects to the staining in meiosis I. As with the meiosis I analysis, we determined the stages of the fixed meiosis II cells by comparing the images to data derived from live studies . MAD2 staining was not observed until prometaphase II, at which point the kinetochores stained brightly for MAD2 even though they were clearly attached to microtubules. Fig. 7 , A and B illustrate a cell in mid-prometaphase II. By viewing this cell in stereo, we were able to identify 10 pairs of kinetochores by MAD2 staining alone . The images in Fig. 7 , D–F further illustrate that MAD2 was lost on a chromosome-by-chromosome basis as the cells approached metaphase II. Once the chromosomes had fully aligned at the metaphase plate, MAD2 staining was no longer detectable and remained undetectable on chromosomes throughout anaphase and telophase (not shown). The data from both meiosis I and II appear to be at odds with the idea that microtubule attachment results in the dissociation of MAD2. An alternative proposal for the mechanism of MAD2 action involves the idea that kinetochores can in some way sense the tension that is applied to kinetochores by the attached microtubules . Since chromatin is elastic , one measure of the tension applied to kinetochores during metaphase is the distance between sister kinetochores . By analyzing fixed meiocytes in a variety of stages, we could demonstrate that the kinetochore-to-kinetochore distance varied by a factor of two in both meiosis I and II. Further, as shown in Fig. 8 , the variation in kinetochore-to-kinetochore distance was correlated with MAD2 staining. The kinetochores stained positive for MAD2 staining when the distance was beneath a specific threshold (∼5.6 μm in meiosis I and ∼1.7 μm in meiosis II), and stained negative when the distance was above the threshold. Thus, while the meiotic data are not consistent with the hypothesis that microtubule attachment is sufficient for the dissociation of MAD2, they are consistent with the hypothesis that a tension threshold must be reached before MAD2 is released or destroyed. The 3F3/2 antibody recognizes a kinetochore phosphoepitope that disappears when tension is applied to the kinetochore . Previously, 3F3/2 staining had only been demonstrated in animal systems. We were able to obtain reproducible staining of the 3F3/2 epitope in maize, but only in meiotic cells (I and II), and only with an ∼10-fold higher concentration of antibody than is normally used in animal cells. When cells were treated with phosphatase, weak or no staining was observed, whereas staining was preserved when the phosphatase treatment was accompanied by the phosphatase inhibitor microcystin (data not shown). Strong staining with the 3F3/2 antibody was observed at prometaphase kinetochores in both meiosis I and II. Double labeling for CENPC and the 3F3/2 antigen revealed the same kinetochore substructure observed when CENPC and MAD2 were observed together . When cells were double-labeled for MAD2 and the 3F3/2 antigen, the two signals almost perfectly colocalized . We also observed 3F3/2 staining in the vicinity of chiasmata . Not all of the maize bivalents stained at presumed chiasmata, and frequently only one arm of a bivalent demonstrated staining, even though the other appeared to have crossed over. The staining patterns may indicate that tension at meiosis I is registered not only at kinetochores but at the chiasmata as well. We are pursuing this idea with further studies involving meiotic mutants. Like MAD2, the 3F3/2 staining disappeared from kinetochores as the cell exited prometaphase and was undetectable when all the chromosomes had properly aligned at the plate. We observed that kinetochores lost MAD2 and 3F3/2 staining concomitantly, such that the majority of kinetochores at prometaphase-metaphase were either both positively stained or both negatively stained. This is shown in Fig. 9 (G–I), which shows a cell with two pairs of homologous kinetochores that stained positively for both antibodies, and eight pairs of homologous kinetochores that failed to stain for either antibody. Among 240 kinetochores from 12 similar cells, only 1.3% stained singly for MAD2 and 7.1% stained singly for 3F3/2 (28.3% did not stain for either and 63.3% stained for both MAD2 and 3F3/2). In cases where only one or the other protein were detectable, the staining was usually very weak (data not shown). These observations suggest that dissociation of MAD2 from the meiotic kinetochore occurs contemporaneously with the dephosphorylation of the 3F3/2 antigen. Assuming that the 3F3/2 epitope reports tension in maize as it does in animals, these data support the idea that MAD2 dissociation occurs in response to tension applied at the meiotic kinetochore. MAD2 plays a key role in the evolutionarily conserved process of spindle checkpoint activation . As a member of a group of kinetochore proteins that senses the presence of unaligned chromosomes, MAD2 relays a stop anaphase signal through the action of CDC20 and the APC . Once metaphase is achieved, MAD2 is degraded or released from kinetochores and anaphase is allowed to proceed. Here we report the identification of the maize homologue of MAD2, show that it is an outer kinetochore protein, and demonstrate differing localization patterns in the mitotic and meiotic cell cycles. Plant kinetochores have an unorganized ball-shaped appearance when viewed under the electron microscope . In contrast, animal kinetochores have an highly ordered trilamellar construction, composed of an inner, middle, and outer plate . A major focus of mammalian kinetochore research has been to ascribe functions to these conspicuous domains. A case in point is CENPC, which is thought to be involved in the early stages of kinetochore assembly and is a component of the inner kinetochore plate . In a separate report we demonstrate that the maize homologue of CENPC is localized to an inner domain of the maize kinetochore close to the centromeric DNA . We show here that maize MAD2 and 3F3/2 antigen localize to a domain of the kinetochore that lies outside of the region containing CENPC. These data provide encouraging evidence of a functional homology among eukaryotic kinetochores and of a domain structure within the plant kinetochore that can be observed using appropriate antisera. The first studies of MAD2 in higher eukaryotes clearly demonstrated that its presence at kinetochores was limited to prometaphase when the chromosomes were aligning on the metaphase plate . Waters et al. subsequently demonstrated that the dissociation of MAD2 was not immediate but occurred over a period of minutes, with the intensity of MAD2 staining decreasing over time. Since the initial interaction of kinetochores with the animal spindle is a stochastic process, this often resulted in a distinct difference in the intensity of MAD2 staining between the two kinetochores of a chromosome. The authors went on to use the microtubule stabilizing drug taxol to release the tension applied during chromosome alignment, and did not observe an effect on MAD2 staining. Based on these data, they argued that microtubule attachment, not tension, is responsible for disappearance of MAD2 staining in metaphase . The results of MAD2 immunolocalization in maize mitotic cells are consistent with the idea that microtubule attachment is an important factor in the dissociation of MAD2 at kinetochores. The disappearance of MAD2 staining at prometaphase was correlated with the interaction of kinetochores with microtubules. Those kinetochores that lacked an associated bundle of microtubules (K-fiber) had intense MAD2 staining, whereas in the presence of a K-fiber the MAD2 staining was reduced or absent . A 4 h incubation of the mitotic cells with oryzalin destabilized the microtubules and produced short K-fiber remnants at the kinetochores . Significantly, even these short K-fibers were sufficient to reduce or abolish MAD2 staining. The fact that MAD2 staining was negatively correlated with the presence of K-fibers even in the absence of an intact spindle apparatus suggests that microtubule attachment has a major role in the dissociation of MAD2 during mitosis. Tension, applied to the kinetochore by the attached kinetochore fiber, is an important component of the spindle checkpoint in animal meiotic cells . In an elegant study, Li and Nicklas demonstrated that if a fine needle was used to apply tension to a mal-oriented chromosome the cell could be induced to proceed from an arrested metaphase state into anaphase. The mechanism for tension-sensing is not known. Recent studies indicate that at least one kinetochore protein, recognized by the 3F3/2 antibody, becomes dephosphorylated in response to tension . The as yet unidentified antigen recognized by the 3F3/2 antibody appears to be phosphorylated in animals by the mitogen-activated protein (MAP) kinase pathway . Our experiments provide evidence that tension is also correlated with a loss of MAD2 staining in maize meiosis. Unlike in mitosis, immunolocalization of meiotic cells revealed uniformly MAD2-stained kinetochores interacting with thick K-fibers throughout early to mid prometaphase I and II . Despite the fact that kinetochores interacted with microtubules from the earliest stages of prometaphase, there was no noticeable reduction of MAD2 staining until late prometaphase when opposing kinetochores began to visibly separate from each other. The loss of MAD2 was positively correlated with the kinetochore-kinetochore distance: beneath a threshold value, MAD2 was readily detectable, whereas above the threshold MAD2 was undetectable . These observations, that MAD2 was detected throughout prometaphase regardless of microtubule attachment, and that MAD2 only became undetectable after poleward forces were sufficient to separate opposing kinetochores, suggest that tension is involved in the dissociation of MAD2 from meiotic kinetochores. In addition, we show that MAD2 and the 3F3/2 epitope are colocalized on the meiotic kinetochore both spatially and temporally . This correlation provides further evidence that MAD2 and the 3F3/2 antigen are involved in a common process and that tension is a prerequisite for the timely initiation of anaphase. The distinct differences in MAD2 staining between mitotic and meiotic cells led us to consider the differences in spindle formation between the two cell types . A comparison of the mitotic and meiotic spindle formation in maize is presented here for reference only , since similar data have been illustrated and discussed in previous reports . All higher plant spindles lack centrosomes , but in mitosis a fusiform spindle apparatus is nevertheless apparent before nuclear envelope breakdown . In animals, where a similar pattern of spindle morphogenesis is mediated by centrosomes, microtubules originate from poles and search for kinetochores in a random fashion . Upon encountering a kinetochore, the inherently unstable microtubules are captured and stabilized. This stable interaction of microtubules with kinetochores, in conjunction with microtubule-based motor proteins, is thought to generate chromosome congression . In the mitotic search and capture type of spindle assembly, many correct chromosome-spindle interactions are likely to occur early in prometaphase. In contrast, maize and many other animal meiotic spindles appear to form after nuclear envelope breakdown by an inside-out mechanism . The spindles assemble around the mass of chromosomes and initially appear as poorly organized, often multipolar structures . Bipolar meiotic spindles emerge at mid-prometaphase both in meiosis I and II . The progressive self-organization of the focused bipolar spindle probably occurs through the combined effects of microtubule bundling and specific motor activities . Since in meiosis spindle assembly begins at chromatin/kinetochores, initial microtubule attachment is a poor indicator of correct spindle formation; and tension is likely to have an important role. Therefore, while we believe that attachment and tension are both factors in the mitotic and meiotic spindle checkpoints, differences in the timing and/or relative contributions of the two factors may exist to accommodate basic differences in spindle assembly. How a single protein or signal transduction pathway can detect both attachment and tension remains an interesting question. The answer may lie in the observation that in vivo, tension is required to stabilize the attachment of kinetochores to the spindle. Ault and Nicklas carried out an ultrastructural study of mal-oriented chromosomes that were undergoing reorientation. They consistently observed that reorienting kinetochores (not under tension) lost microtubule attachments at both the pole and the kinetochore . Since unattached microtubules are highly unstable, the loss of attachment is expected to result in the rapid loss of the microtubules . Under this view, MAD2 and its associated checkpoint proteins are dissociated by stable microtubule attachment , and the role of tension in the spindle checkpoint is to increase the stability or number of microtubule attachments. Supporting this proposition is the fact that a destabilization of kinetochore microtubules by vinblastine delays anaphase onset and the fact that the microtubule stabilizing agent taxol results in a near-complete loss of MAD2 staining from kinetochores . An additional prediction, which should be testable by current electron microscopic imaging techniques, is that there is a reciprocal relationship between MAD2 and attached microtubules in both mitosis and meiosis. | Study | biomedical | en | 0.999997 |
10225946 | The phyB-5 mutant of Arabidopsis thaliana (ecotype, Landsberg er ) was used as the host for transformation. Arabidopsis thaliana (ecotype Landsberg er ) and the phyB - 5 mutant were used as controls for physiological, immunochemical, and microscopic experiments. A full-length PHYB cDNA clone was isolated from an Arabidopsis (ecotype Columbia) cDNA library. Cloned PHYB cDNA was almost identical to a previously reported sequence except that a C to T substitution at the base position 971, which does not cause amino acid difference, was detected. To construct the PHYB - GFP fusion sequence, PHYB translational termination codon (TAG) was replaced with an oligonucleotide sequence (GGAGGTGGAGGTATCGAT) by PCR. This oligonucleotide introduces a unique ClaI restriction site at its 3′ terminus. The GFP clone (blue-sGFP-TYG-nos KS) was a kind gift from Dr. J. Sheen (Massachusetts General Hospital, Boston, MA). This clone contains a unique ClaI restriction site that shortly precedes the ATG start codon of the GFP gene. The PHYB and GFP clones were ligated at the ClaI restriction site to generate PHYB - GFP translational fusion. As the result, an oligoamino acid sequence (GGGGIDKLDP) was inserted between the phyB and GFP amino acid sequences . This PHYB - GFP chimeric cassette was inserted between the constitutive cauliflower mosaic virus 35S promoter and the Nos terminator of an Agrobacterium transformation vector pBI-Hyg/35S-NosT, which is derived from another transformation vector pBI101-Hm (a gift from Dr. Kenzo Nakamura, Nagoya University, Japan) by removing its uidA gene (Nakamura, M., unpublished observation). The resulting vector was designated pBI-Hyg/35S-PHYB-sGFP-NosT . Arabidopsis phyB mutant was transformed using Agrobacterium -mediated in planta transformation . Transformed plants were selected on the medium containing 25 mg ml −1 hygromycin B ( Boehringer Mannheim ) and 166 mg ml −1 claforan (Hoechst). The transgenic lines PBG-5 and PBG-7 were selected from the drug-resistant lines by phyB immunoblotting and GFP epifluorescence microscopy. For growth of plants, seeds were sown on 0.6% agar plates containing the Murashige-Skoog medium with 2% (wt/vol) sucrose and grown under continuous white light from fluorescent tubes (FLR40SW/M-B; Hitachi). The plants were then transplanted to pots containing vermiculite and grown to maturity under continuous white light from fluorescent tubes. For the immunochemical detection of the fusion protein, rosette leaves were harvested from 3-wk-old plants. For the hypocotyl assay and microscopic observation, seeds were sown on agar plates containing Murashige-Skoog salt mixture without sucrose. The plates were placed at 4°C for 12 h and then irradiated with continuous white light for 12 h at 23°C to induce germination. For the hypocotyl assay, seedlings were grown for 5 d under continuous red light (6.0 W m −2 ) from red fluorescent tubes (FL20S/R-F; National) or in darkness. For microscopic observation, seedlings were grown for 5 d under continuous white light (15 W m −2 ) from fluorescent tubes (FLR40SW/M-B; Hitachi) or in darkness. To detect the phyB-GFP fusion protein and the authentic phyB, ∼0.1 g of rosette leaves was glass homogenized in the presence of 0.1 ml of the phytochrome extraction buffer (100 mM Tris-HCl, 2 mM DTT, 5 mM EDTA, pH 8.3) containing proteinase inhibitor cocktails for general use and for fungal and yeast extracts at the concentrations recommended by the manufacturer. Debris was removed by centrifugation. Proteins were concentrated from the crude homogenate by ammonium sulfate precipitation. The precipitated protein was dissolved in the SDS-PAGE sample buffer and subjected to immunoblot analysis . Antibodies used were an anti-phyB mAb, mBA2 , and an anti-GFP mAb ( Clontech ). Molecular weight markers (prestained SDS molecular weight standard mixture) were from Sigma Chemical Co. Arabidopsis seedlings were soaked in 2 μg ml −1 Hoechst No. 33342 ( Sigma Chemical Co. ) solution made in H 2 O for visualization of the nucleus in some experiments. Epidermal layers including cortex were peeled from the hypocotyls and placed on glass slides. For the other parts of seedlings, whole organs were placed on glass slides and pressed gently. The specimens were observed using an Olympus BX60 microscope equipped with ×20, ×40, and ×100 objectives, differential interference contrast (DIC) optics, and a 100-W mercury arc light source. Fluorescence was filtered using UV (U-MWU) or FITC (U-MNIBA) filter sets ( Olympus ). For confocal microscopy, trichomes were removed from the surface of cotyledons with a razor blade and placed on glass slides. Root tips were placed on glass slides without any pretreatment. The specimens were observed using an inverted laser scan microscope (LSM410 invert; Carl Zeiss Jena) equipped with ×40 and ×63 objectives. The laser scan images were obtained with a combination of 488 nm laser excitation and 515 nm longpass emission filter (LP515; Carl Zeiss Jena). Sequential images from different focus planes were recorded automatically. To examine biological activity and intracellular localization of the phyB-GFP fusion protein, the phyB-5 mutant of Arabidopsis was transformed with a vector harboring the 35S::PHYB-GFP construct. The resulting transgenic lines, PBG-5 and PBG-7, exhibited an overall dwarfing of mature plants under continuous white light . They flowered a few days later than the wild-type under the conditions tested. Similar phenotypes, which are opposite to those of the phyB -deficient mutants , have been reported in phyB overaccumulating plants . Hence, the phyB-GFP fusion protein is likely to be fully functional. It is known that inhibition of hypocotyl elongation by continuous red light is mediated primarily by phyB . To confirm the biological activity of phyB-GFP further, heterozygous progeny of the PBG-5 plant was examined for this response. The seedlings were grown under continuous red light for 5 d and hypocotyl lengths were determined. As shown in Fig. 2 , a short population segregated from a longer one at about a 3:1 ratio. Hypocotyl lengths in the longer population matched well with those in the parental phyB mutant. In contrast, the shorter seedlings were significantly shorter than the wild-type seedlings, which is consistent with the phyB overexpression phenotypes reported by other groups . Cosegregation of the short phenotype with the expression of phyB-GFP was then examined. As expected, all the short seedlings exhibited GFP fluorescence whereas no fluorescence was observed in the longer seedlings . The seedling phenotype was examined in darkness as well. As is the case with the phyB overexpressing plants , no clear segregation of shorter seedlings was observed in the PBG-5 heterozygous progeny . The average hypocotyl lengths in the fluorescent and nonfluorescent populations were indistinguishable. Hence, phyB-GFP was suggested to be not only biologically but also photochemically active. To examine the accumulation of phyB-GFP fusion protein in the transgenic plants, immunoblot analysis was performed. Proteins were extracted from rosette leaves of the PBG-5 plants and probed with anti-phyB and anti-GFP antibodies . The anti-phyB mAb detected a major band of ∼143 kD in the PBG-5 extracts . The size was consistent with the expected mass of the phyB-GFP fusion protein. A band at the same size was detected with the anti-GFP antibody , confirming that the band represented the phyB-GFP fusion protein. The higher intensity of the phyB-GFP band compared with that of the authentic phyB indicated that the phyB-GFP was overaccumulated in the transgenic plants. A similar result was obtained for the other transgenic line, PBG-7 (data not shown). In addition to the major 143-kD band, a weak band of ∼123 kD was detected in the PBG-5 plants . The intensity of the band was comparable to that of authentic phyB. To confirm that the fragment is larger than the authentic phyB (117 kD on the blot), extracts from the PBG-5 and the wild-type plants were mixed and probed with the anti-phyB antibody. As expected, the two bands were separated on the blot. Since the fragment was not detected with anti-GFP antibody, it is speculated that proteolysis of phyB-GFP within the GFP portion yielded this fragment. In accordance with this, minor bands around 20 kD were detected on the anti-GFP blot. In the absence of the protease inhibitor cocktails, fragmentation was much more severe (data not shown). Hence, the 123-kD fragment is likely to be produced by the residual proteolytic activity in the extract during the extraction procedure. Intracellular localization of the phyB-GFP fusion protein in the PBG-5 seedlings was examined. Epidermal layers including cortex were peeled from the light-grown seedlings and observed under a fluorescence microscope. At lower magnification, bright green spots of GFP fluorescence were observed . Positions of the spots matched well with those of the nuclei revealed by the Hoechst staining. Similar fluorescence images were obtained for another transgenic line, PBG-7 (data not shown). Interestingly, observation at higher magnification revealed that the phyB-GFP fluorescence was speckled within the nuclear region . The apparent size of each speckle appeared to be <1 μm. Although speckles were observed in all of the nuclei, the number per nucleus varied. In most cases, one nucleus contained 5–10 speckles. The intracellular localization of phyB-GFP was then examined in other parts of the seedling. As shown in Fig. 5 , nuclear fluorescence was confirmed in leaf , root , and root hair cells . Furthermore, the speckles were observed in all of the cell types examined. To determine the spatial distribution pattern of the speckles within the nucleus, optical sectioning of the cell with a confocal microscope was performed . For this purpose, trichomes were chosen for observation because of the large size of their nuclei. As shown in Fig. 6 , the speckles appeared to be distributed more or less evenly in the nucleus. In this particular case, at least 24 spots were recognized. This is probably due to the large size of the trichome nucleus. The images clearly demonstrated that the size of each speckle varied substantially even within one nucleus. A previous study suggested that the nuclear localization of phyB is light dependent . In accordance with this, weak fluorescence was observed throughout the cell in PBG-5 dark-grown seedlings . Since the intensity of fluorescence was low, it was difficult to determine the intracellular localization in detail. However, higher intensity in the peripheries of the cells indicated that phyB-GFP was distributed in the cytoplasm. Those cells were highly vacuolated and the cytoplasm was observed mostly in the peripheral region, as observed by DIC microscopy . In some cells, fluorescence was observed not only in the peripheries but also in the nuclear region . However, it was difficult to conclude that the phyB-GFP exists inside the nucleus even by confocal observation (data not shown). Intracellular distribution of phyB-GFP in the light and darkness was compared in root tip cells with a confocal microscope. As shown in Fig. 7 j, the speckles of fluorescence were observed in light-grown seedlings. In contrast, relatively uniform fluorescence was observed in the peripheries of the cells in dark-grown seedlings , which provided further evidence that phyB-GFP was distributed outside the nucleus and throughout the cell in darkness. The time course of nuclear accumulation of phyB-GFP during the dark to light transition was followed. The PBG-5 dark-grown seedlings were transferred under continuous red light. As shown in Fig. 8 , nuclear fluorescence was not clear at time 0 . After 2 h in red light, the intensity of the nuclear GFP signal was increased . However, fluorescence remained detectable in the periphery of the cells. Speckles in the nucleus were rarely observed at this time point, although a few tiny spots were detected in some cases. After 4 h in red light, many small speckles were observed . Fluorescence in the cell periphery was greatly reduced. After 6 h in red light, the speckles became larger but the number per nucleus was reduced . Hence, translocation of phyB-GFP to the nucleus appeared to be completed within 4–6 h in hypocotyl cells under continuous red light. In the course of these experiments, we noticed that the translocation took longer in root cells, although the reason for this was not clear (data not shown). It is known that phytochromes overexpressed in transgenic plants are biologically active. Transgenic Arabidopsis expressing exogenous phyB exhibits increased sensitivity to red light . In this study, we have demonstrated that the plants expressing phyB-GFP show similar light-dependent phenotypes . Since the expression level of phyB-GFP was comparable to those reported for the phyB overexpressing plants , it is concluded that phyB-GFP is as active as authentic phyB. Furthermore, the fusion protein was expressed in the phyB -deficient background in the present study, confirming that the presence of endogenous phyB is not required for correct functioning of the phyB-GFP fusion protein. In the PBG-5 seedlings, a proteolytic fragment of the fusion protein was detected . However, its level was as low as the authentic phyB. Furthermore, the fragmentation might have occurred during the extraction. Even if the fragment existed in vivo, it would not contribute to the fluorescence. The immunoblot analysis suggests that the fragment resulted from proteolysis within the GFP portion, which would cause the loss of fluorescence. Likewise, it is unlikely that the fragment alone caused the phyB overexpression phenotypes, although it might have contributed to the phenotype to some extent. Although associations of phyA with various organelles have been reported, only a small portion of the total cellular phyA was recovered in those cases . It is also known that the Pfr form of phyA tends to associate with particulate material under certain cell extraction conditions . Thus, it had remained obscure whether phytochrome indeed resides within organelles in vivo. More recently, we have shown that COOH-terminal fragments of phyB fused to GUS are localized to the nucleus . Furthermore, a substantial amount of endogenous phyB has been detected in isolated nuclei. On the basis of these findings, we had tentatively proposed that phyB translocates to the nucleus to mediate the light responses . However, this observation could have been due to a cryptic nuclear localization signal that is exposed only in the context of the fusion protein. It is also difficult to exclude the possibility that phyB detected in the isolated nuclei might be due to contamination during specimen preparation and staining. This study provides more dramatic evidence for phyB nuclear localization. Localization of the phyB-GFP fusion protein in the nucleus has been observed in intact live cells without any pretreatment . The optical sectioning by a confocal microscope clearly indicates that the fluorescence is distributed inside the nucleus . Furthermore, the fusion protein appears to be fully functional as a photoreceptor (see above), suggesting that the structure of phyB is preserved in the phyB-GFP fusion context. However, it should be noted here that phyB-GFP is overexpressed under control of the 35S promoter. Hence, there remains the possibility that ectopic expression contributes somewhat to the observed intracellular distribution. To address this question, we are now screening for transgenic lines with lower accumulation levels. In the light, fluorescence of the phyB-GFP protein was observed mainly in the nucleus in all the cell types examined. Hence, the nucleus is likely to be the major site of the phyB action, although it remains possible that a minor fraction of phyB-GFP is present in other compartments of the cell and might contribute to the overall response. All the phenotypes observed in this study, such as the shorter hypocotyls, overall dwarfing in mature plants, and late flowering, can ultimately be explained by alteration in gene expression patterns. Hence, it is an intriguing possibility that phyB-GFP translocates to the nucleus to affect the transcription of target genes. Although phytochrome appears to have neither DNA-binding nor transactivation domains, it could interact with other factors that directly regulate transcription. PIF3, a nuclear-localized basic helix-loop-helix protein that binds to the COOH-terminal domain of phytochrome , is a potential candidate for such a factor. Interestingly, the phyB-GFP fusion protein forms speckles in the nucleus. The sizes of the speckles are mostly <1 μm. However, the size varies even within one nucleus under continuous light . The number of speckles per nucleus also varies. Interestingly, the size of the speckle gradually increases during the dark to light transition . Conversely, the number of speckles decreases. Speckled structures similar to the one observed in this study have been reported in animal cells . Factors involved in the processing and transcription of RNA are found in those speckled structures in the nucleus. The promyelocytic leukemia (PML) nuclear body is another example of such a structure. However, the biological relevance of those structures remains unclear, although they may function as a repressor of transcription . In plant cells, COP1, which is a negative regulator of plant photomorphogenesis or light responses , has been shown recently to form speckles in the nucleus . Hence, those speckles observed in plant cells might represent the site where the photoreceptor and other nuclear factors such as COP1 and PIF3 interact with each other to mediate light signals. Identification of proteins present in the phyB speckles is awaited. It should be noted here that the phyB-GFP speckles could be due to an artifact caused by overaccumulation of the fusion protein at nonphysiological concentration. It is known that phytochrome in general tends to form aggregates in vitro . Hence, as with all studies using GFP fusion proteins, it is difficult at present to exclude this possibility. However, the light-dependent nature of the nuclear translocation supports the view that this distribution is indeed relevant to phytochrome function. Detailed analysis of transgenic lines that accumulate the fusion protein at lower levels is awaited. A search for mutations that abolish the speckles would greatly help to answer the question. For example, it has been shown in onion cells that GFP-HY5 fails to form speckles in the absence of COP1 expression . It will be of interest to investigate the effect of removing known light transduction components upon the subnuclear distribution of phyB. In dark-grown seedlings, the phyB-GFP fusion protein appeared to be distributed evenly throughout the cytoplasm . This is consistent with the previous observation that phyB is not detected in the nuclei isolated from the dark-adapted rosette leaves . In addition, a similar result has been obtained in dark-grown pea seedlings (Nagatani, A., unpublished observations). However, the detailed distribution of phyB-GFP could not be determined because of the resolution limit within the small Arabidopsis cells. At present, it is not clear whether the fusion protein is excluded completely from the nucleus in darkness. Although fluorescence was observed in the nuclear region, this could be due to fluorescence from the cytoplasm surrounding the nucleus. It would be helpful to isolate intact cells or protoplasts to determine the localization pattern in greater resolution. To observe GFP fluorescence, the seedlings received relatively intense actinic blue light. In theory, this could alter the localization pattern of phyB-GFP. The apparent nuclear fluorescence observed in the dark-grown seedlings might be due to this effect. However, the distribution of phyB-GFP did not change significantly during the observation period (10–20 min). This is consistent with the relatively slow kinetics of phyB-GFP accumulation in the nucleus induced by continuous red light . In addition, no change was observed in light-grown seedlings during the observation. Hence, the actinic blue light does not appear to disturb the distribution pattern at least during the observation period of 10–20 min. Continuous red light induced accumulation of phyB-GFP in the nucleus . Relatively slow kinetics of this process is consistent with the observation made in pea seedlings (Nagatani, A., unpublished observations). phyB was not detected in the nuclei isolated from dark-grown pea seedlings. However, treatment of the seedlings with continuous red light induced nuclear localization of phyB. The level of nuclear phyB reached a plateau ∼4 h after the onset of light treatment. Phytochrome is known to regulate expression of various genes, of which CAB is the best characterized . In Arabidopsis , the time course of the CAB gene induction by a red light pulse has been examined at high time resolution by using luciferase as a reporter . The results indicate that the induction is multiphasic. An acute response occurs in Arabidopsis with the peak at 2 h after the pulse treatment. Subsequently, the expression oscillates under control of the biological clock. Namely, the level falls to a trough at 6.5–8 h and peaks again at 15.5 h. Analysis of the phyB mutant has demonstrated that phyB contributes to both the acute and clock-dependent responses . As shown in Fig. 8 , it takes ∼4 h for phyB-GFP to complete the translocation from the cytoplasm to the nucleus, which is significantly slower than the acute response of the CAB gene expression. This may indicate that the level of nuclear phyB attained 2 h after the onset of light might be sufficient to induce the acute response. It is also possible that phyB-GFP migrates more slowly than the authentic phyB. Alternatively, the signal transduction from phyB to the downstream components may take place in the cytoplasm in the early phase of the response. Interestingly, it seems here that the speckle formation is not required for the acute response of the CAB gene expression. Almost no speckles could be observed 2 h after the onset of red light treatment . It is more probable that the clock-dependent induction of the CAB gene expression is under control of nuclear-localized phyB. The extent of clock-dependent expression is maximal under continuous light. In such a condition, phyB-GFP is localized to the nucleus almost exclusively . In addition, we have confirmed in pea seedlings that a pulse of red light can induce long-lasting accumulation of phyB in the nucleus (Nagatani, A., unpublished observations). It has been proposed that the circadian clock confines the ability of light to induce CAB expression . Hence, it is an intriguing possibility that phyB and components of the biological clock directly interact with each other within the nucleus. Together with the previous report , the present results provide compelling evidence that the nucleus is at least one of the sites of phyB action. In animal and yeast cells, many signal transduction factors are known to translocate to the nucleus upon receipt of the signal . For example, steroid hormone receptors are targeted to the nucleus upon binding of the hormonal ligands . Therefore, it is not surprising that phyB translocates to the nucleus upon light stimulation. The det , cop , and fus mutants of Arabidopsis exhibit the constitutive photomorphogenic phenotypes in darkness . The DET1, COP1, COP9, and FUS6 proteins can be localized to the nucleus. It is especially interesting that the nuclear localization of COP1 is light dependent . Conversely, the hy5 mutant is impaired in the light signal transduction. The HY5 gene has been cloned recently . The gene encodes a putative transcription factor which is constitutively localized to the nucleus . The HY5 protein binds to the light-responsive promoters of the CAB and CHS genes . More recently, a putative transcription factor, PIF3, has been identified as a phytochrome-interacting protein . Hence, it is an attractive possibility that phyB interacts with those proteins in the nucleus to modify transcription of the target genes. The biological relevance of the phyB-GFP speckles in the nucleus is unknown as discussed above. The time course analysis in this study suggests that speckles are not required for the acute response of the CAB gene expression . However, the speckled structure may contribute to long-term effects of phyB. It is intriguing here that GFP-COP1 fusion protein forms speckles in the nucleus . Furthermore, GFP-HY5 is recruited to the speckles if it is coexpressed with COP1. Hence, the speckles of COP1 may represent the site where nuclear factors interact with each other to mediate light signals. In this connection, it would be particularly interesting to know whether phyB colocalizes with other factors in the nucleus. As discussed above, the present results suggest that phyB translocates to the nucleus upon light stimulation. In the nucleus, phyB may interact with other nuclear factors to transduce the light signal to alter transcription of target genes. However, the kinetics of the light-induced accumulation of phyB-GFP in the nucleus is clearly too slow to explain rapid phytochrome responses such as light-induced changes in intracellular Ca 2+ levels . Hence, phyB might be functioning not only in the nucleus but also in the cytoplasm. Alternatively, different molecular species of phytochromes may function at different sites within the cell. In this connection, it will be important to know whether other molecular species are localized to the nucleus. | Study | biomedical | en | 0.999996 |
10225947 | GST pull-down assays were performed as described . Bacterially expressed GST-ASF/SF2 (100 μg) was phosphorylated in vitro using baculovirus-expressed GST-SRPK1 for 6 h at 30°C with or without 1 mM ATP. Proteins were desalted on G-50 columns ( Pharmacia ) and rebound to glutathione beads before binding to TNT ( Promega ) translated U1-70K or U2AF35. Empty beads or GST-loaded beads were used as controls. Two-hybrid assays were performed as described using the LexA system . Strain EGY48 was deleted of the SKY1 gene by recombination with a kanamycin resistance expression unit flanked by SKY1 genomic sequences . The deletion encompassed 316 bp 5′ of the initiation codon to 541 bp 5′ of the termination codon, and was confirmed by PCR. Bait and prey plasmids were gifts from J. Wu or were constructed in pEG202 and pJG4-5 with modified polylinkers . Expressed proteins were verified by Western blotting with a monoclonal anti-LexA antibody ( Clontech ) using the enhanced chemiluminescent detection system (Pierce) in conjunction with HRP-labeled goat anti–mouse antibodies. Kinase expression plasmids were constructed in p415GAL1 for selecting Leu + phenotype . Deletions of the RS domains in U2AF65 (residues 1–65) and U2AF35 (residues 191– 240) were made by PCR. β-Galactosidase activity is expressed as Miller units: OD420 × 1,000/OD600 × time (min) × culture volume assayed (ml), determined at 30°C. Freshly streaked yeast clones bearing prey plasmids (HA tagged) were inoculated in 5 ml YPGal/Raff and grown for 3 h at 30°C. Yeast were fixed with 0.7 ml 37% formaldehyde and processed for immunofluorescence as described , using the 12CA5 antibody at 1:100. Humanized GFP (pGreenLantern-1; Life Technologies) was fused in frame to the NH 2 terminus of ASF/SF2 wild-type cDNA or the KS substitution mutant . The fusion protein was expressed from pcDNA3 (Invitrogen) in HeLa cells for 48 h before visualization of live cells. Whole cell extracts (200 μg) were treated with 500 U CIP and subjected to Western analysis using an anti-GFP antibody (Chemicon). Both the parent EGY48 and corresponding sky1 Δ strains were grown on 5-FOA plates to eliminate the pSH18-24 two-hybrid reporter (lacZ) plasmid. The resulting strains were cotransformed with pEG202-based bait plasmids and pJK101, a transcriptional repression reporter , then assayed for β-galactosidase activity. Maximal transcription was determined using a modified bait plasmid lacking LexA. Phosphorylation of the SR protein ASF/SF2 by SRPK1, SRPK2, or Clk/Sty has been shown previously to enhance its interaction in vitro with the U1 snRNP-specific 70-kD protein (U1-70K) in a GST pull-down assay . This enhancement may result from the neutralization of charges, allowing RS domains to interact efficiently . To test the generality of this principle, we examined the effect of SRPK1-mediated phosphorylation on ASF/SF2 binding to another RS domain–containing splicing factor, U2AF35, an interaction proposed for pairing the 5′ and 3′ splice sites . Unexpectedly, binding of unphosphorylated ASF/SF2 to U2AF35 was readily detectable, and this interaction was largely unaffected by SRPK1-mediated phosphorylation . These results indicate that phosphorylation differentially modulates the affinity between RS domain–containing splicing factors, depending on the pairs examined. A similar observation was also made independently by Xiao and Manley . To examine the role of phosphorylation in protein–protein interactions under physiological conditions, we took advantage of the single conserved SR protein–specific kinase in S . cerevisiae to investigate RS domain interactions in the presence or absence of phosphorylation. Mammalian SR proteins expressed in both wild-type and SKY1 deletion ( sky1 Δ) yeast strains were soluble, but differed in their phosphorylation states, as determined by mobility shift on SDS-PAGE as well as Western blotting with mAb 104, a monoclonal antibody specific for the phosphoepitope present in all SR proteins . As shown in Fig. 2 a, LexA-ASF/SF2 and LexA-SC35 expressed in wild-type yeast migrated more slowly than those from sky1 Δ yeast (left four lanes), indicative of a change in phosphorylation states . Reexpression of Sky1p, but not an ATP binding site mutant (Sky1p K187M), restored the phosphorylated form of ASF/SF2 (right two lanes). Therefore, Sky1p appears to be the only endogenous kinase capable of efficiently phosphorylating mammalian SR proteins expressed in yeast. This system offers a unique opportunity to test the phosphorylation dependence of RS domain interactions in vivo using the two-hybrid strategy. The results of two-hybrid assays are shown in Fig. 2 b. As a control, the interaction between the nuclear receptor transcription factor T3R and its corepressor NCoR, which are not SRPK1 substrates (data not shown), was unaffected by the SKY1 deletion. In contrast, the interactions between RS domain–containing splicing factors, all SRPK substrates, were largely eliminated in sky1 Δ yeast. Growth assays of these interactions agreed with the quantitative assays (data not shown). In particular, RS domain–mediated interactions between U1-70K and either ASF/SF2 or SC35 were completely abolished in sky1 Δ yeast, consistent with the in vitro binding data. As expected, the interaction between ASF/SF2 or SC35 with U2AF35 was detectable in wild-type yeast, but we never detected any interaction between these three RS domain–containing splicing factors in parallel experiments in sky1 Δ yeast. Surprisingly, the two-hybrid interaction between U2AF65 and U2AF35 was also severely affected by the SKY1 deletion, despite the fact that their interaction takes place outside their RS domains . These results clearly indicate that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro, reflecting additional phosphorylation-dependent events for the interaction between RS domain proteins in cells. To further demonstrate that Sky1p-mediated phosphorylation is critical for RS domain–containing proteins to interact with one another, we conducted kinase rescue experiments. As shown in Table I , Sky1p, but not its ATP binding site mutant, was able to restore the two-hybrid interactions examined, proving that Sky1p kinase activity is required. Because mammalian cells express more than one kinase family for SR proteins, the yeast model system provides an opportunity to evaluate functional similarities between different mammalian SR protein–specific kinases. Therefore, we extended the rescue experiments to SRPK1 and Clk/Sty from mammalian cells. As shown in Table I , both SRPK1 and Clk/Sty, but not corresponding ATP binding site mutants, were able to restore the interactions of U1-70K with both ASF/SF2 and SC35. In contrast, an unrelated kinase, the catalytic subunit of protein kinase A, did not rescue these interactions. Therefore, kinases from both the SRPK and Clk/Sty families appear to be functionally equivalent in this assay. The restoration of these two-hybrid interactions by different SR protein kinases strongly argues against the possibility that the lack of RS domain–mediated interactions in sky1 Δ yeast is due to a general defect resulting from the SKY1 deletion. Furthermore, the ability of Clk/Sty to restore the two-hybrid interactions in sky1 Δ yeast implies that there may be no endogenous kinase with the same activity and specificity as Clk/ Sty in S . cerevisiae , whereas mammalian cells have evolved multiple SR protein kinases to regulate RS domain–containing splicing factors. The absolute dependence of RS domain–mediated interactions in vivo on phosphorylation is in sharp contrast to the affinity changes modulated by phosphorylation in vitro. This difference raises the possibility that other aspects of SR protein function may be affected by phosphorylation in vivo and, therefore, offers an opportunity to investigate additional phosphorylation-dependent events. For instance, hypophosphorylated SR proteins may be sequestered in the nucleus due to nonspecific binding to other proteins or nucleic acids, as suggested by in vitro RNA binding studies . Therefore, we examined the localization of expressed SR proteins in yeast by immunohistochemistry. As shown in Fig. 3 , SC35 expressed from the prey vector was largely localized in the nucleus in wild-type yeast, but dispersed uniformly in both the cytoplasm and the nucleus in sky1 Δ yeast. In other experiments using a prey vector lacking an exogenous nuclear localization signal, SC35 was uniformly distributed in wild-type cells, but excluded from the nucleus in sky1 Δ yeast, leaving a “hole” in the immunofluorescence image (data not shown). These unanticipated findings suggest that Sky1p-mediated phosphorylation may regulate nuclear import of SC35 expressed in yeast. However, the localization of ASF/SF2, U1-70K, and U2AF35 seemed mostly unaffected by the SKY1 deletion as they were generally uniformly distributed in both the nucleus and cytoplasm of wild-type and sky1 Δ yeast . These observations suggest for the first time that SRPK-mediated phosphorylation plays an important role in nuclear import of SR proteins, although not all SR proteins are affected in the same way. To obtain further evidence that SRPK-mediated phosphorylation plays a role in nuclear localization of RS domain proteins in mammalian cells, we took advantage of the KS substitution mutant of ASF/SF2 in which arginines in the RS domain are replaced by lysines . We have shown previously that ASF/SF2-KS is not a substrate for the SRPK family of kinases, but can be phosphorylated by Clk/Sty . Consistent with the observation that there appears to be no Clk/Sty-like kinase activity in yeast, ASF/SF2-KS did not interact with U1-70K in the two-hybrid assay even in wild-type yeast (data not shown). We expressed wild-type ASF/SF2 and ASF/SF2-KS as GFP fusion proteins in HeLa cells, and expressed proteins were examined for phosphorylation-dependent mobility shifts on SDS-PAGE . Treatment with alkaline phosphatase caused an increase in the mobility of wild-type ASF/SF2, but had no effect on that of the KS mutant, suggesting that GFP-ASF/ SF2-KS expressed in mammalian cells is not phosphorylated. The localization of these proteins in HeLa cells is shown in Fig. 4 b. Wild-type GFP-ASF/SF2 localized in the nucleus in a speckled pattern characteristic of endogenous splicing factors. In contrast, GFP-ASF/SF2-KS showed significant accumulation in the cytoplasm. ASF/ SF2-KS was also localized in the nucleus in enlarged speckles, which is consistent with a role of SRPKs in dissociating splicing factors from nuclear speckles, as suggested earlier . These observations reinforce the idea that SRPK-mediated phosphorylation plays an important role in both nuclear import and intranuclear localization of SR proteins. Although inefficient nuclear localization may be a contributing factor for impaired protein–protein interactions involving SC35, it cannot explain the absolute phosphorylation dependence of all the RS domain–mediated interactions we tested in sky1 Δ yeast. Furthermore, the two-hybrid interaction between U2AF65 and U2AF35 in sky1 Δ yeast was also substantially reduced despite the fact that this interaction is not RS domain–mediated . When the RS domains of U2AF65 and U2AF35 were deleted, their two-hybrid interaction became highly efficient in both wild-type and sky1 Δ yeast, even surpassing the interaction between full-length proteins in wild-type yeast (Table II ). These observations suggest that the phosphorylation state of their RS domains may influence the U2AF65-35 interaction in eukaryotic cells, even though this interaction can take place in bacteria . In an attempt to correlate inefficient two-hybrid interactions with a defect in heterodimer formation between U2AF65 and U2AF35, we conducted gel filtration and coimmunoprecipitation experiments and detected similar amounts of U2AF65 and 35 in large protein complexes from both wild-type and sky1 Δ yeast (data not shown). Because these complexes may contain additional proteins, we cannot determine whether phosphorylation directly affects the efficiency of heterodimer formation. Alternatively, Sky1p may indirectly affect the outcome of the two-hybrid interaction assay by regulating molecular targeting of its substrates to each other and/or to the reporter gene in the nucleus. To demonstrate that SRPKs play a direct role in assisting RS domain–containing proteins to locate their specific protein or RNA targets, we adapted the following transcriptional repression assay , which allowed a quantitative measure of phosphorylation-dependent molecular targeting of RS domain proteins in vivo. As diagrammed in Fig. 5 , LexA operators were inserted between a GAL1 upstream activating sequence (UAS) and the transcriptional start site for lacZ such that LexA or LexA fusion proteins bind to the operators and repress lacZ expression. In both wild-type and sky1 Δ yeast, lacZ expression was constitutive in the absence of LexA, and repressed in its presence. The ability of a control LexA-T3R fusion protein to repress lacZ expression was not affected by the SKY1 deletion. In contrast, the targeting of LexA-SR fusion proteins was dramatically, although not equally, affected by the SKY1 deletion in this assay . In particular, the LexA-SC35 fusion protein fully repressed lacZ expression in wild-type yeast, but showed little repression in sky1 Δ yeast, probably reflecting the significant impact of Sky1p-mediated phosphorylation on the fusion protein at both nuclear localization and intranuclear targeting steps. A similar SKY1 -dependent effect was also seen, although less pronounced, with ASF/ SF2, U1-70K, and U2AF65, but was not evident with U2AF35. These observations provide an explanation for the decreased interaction between U2AF65 and U2AF35 in sky1 Δ yeast in the two-hybrid assay. The U2AF65 fusion protein expressed from the bait vector in sky1 Δ yeast could not efficiently locate its target and activate transcription of the reporter gene, even though targeting of U2AF35 was unaffected by phosphorylation. Based on these results, we conclude that phosphorylation plays a direct role in the molecular targeting of RS domain–containing proteins within the nucleus. In this report, we have taken advantage of the fact that apparently only one SR protein–specific kinase is conserved in the relatively simple eukaryote S . cerevisiae . Therefore, we were able to address the functional requirements of SR protein–specific kinases in mediating SR protein–protein interactions in vivo. An equivalent kinase deletion experiment cannot be done in mammalian cells due to the presence of at least four identified kinase activities capable of phosphorylating RS domain–containing proteins . The yeast model system is especially attractive due to the fact that many aspects of RNA metabolism and fundamental mechanisms of nuclear import/export are well conserved between mammalian cells and yeast. For example, this model organism was used successfully to address HIV rev function in mediating specific RNA transport in yeast, although yeast is not a natural host for this virus . Furthermore, the two-hybrid protein–protein interaction and transcriptional repression assays have been used widely to address the function of mammalian proteins in yeast, and often accurately reflect interactions that occur normally in mammalian cells. Our current findings illustrate multiple steps at which the SR superfamily of splicing factors may be regulated by phosphorylation and our data are consistent with published observations on the effects of phosphorylation and dephosphorylation on SR proteins in mammalian cells (see below). Although a phosphorylation-dependent nuclear localization defect was observed using mammalian SC35 expressed in yeast, a similar phenomenon also occurs with an endogenous Sky1p substrate, Npl3p (Yun, C.Y., and X.-D. Fu, manuscript submitted for publication), indicating that conserved regulatory mechanisms may operate in both yeast and mammalian cells. Most advantageously, the use of yeast has made it possible to provide quantitative measures of intracellular trafficking of SR proteins, which have been largely descriptive in previous studies. Our localization studies showed increased cytoplasmic accumulation of SR proteins in the absence of SRPK-mediated phosphorylation. The nuclear localization signal for SC35 appears to be confined to its RS domain , and, therefore, the effect of SRPK-mediated phosphorylation on SC35 localization may be readily detectable. In contrast, sequences both inside and outside of the RS domain of ASF/SF2 seem to be required for the protein to properly localize in the nucleus , and, as a result, nuclear import of ASF/SF2 may be less efficient but not abolished without SRPK-mediated phosphorylation. This possibility is consistent with the observations that ASF/SF2 mutants, including ASF/SF2-KS and RS domain–deleted ASF/SF2 , were localized in both the cytoplasm and the nucleus of transfected HeLa cells. Together, these results suggest that the localization of different SR proteins may be differentially facilitated by phosphorylation, which may reflect distinct mechanisms for their nuclear import. Increased cytoplasmic localization of hypophosphorylated SR proteins may be due to inefficient nuclear import or accelerated export. Recently, it was reported that overexpression of Clk/Sty or SRPK2 in mammalian cells increases cytoplasmic localization of ASF/SF2 , indicating that SR protein–specific kinases may play an active role in facilitating nuclear export of SR proteins. Alternatively, the dissolution of nuclear speckles by these kinases elevates the nucleoplasmic pool of SR proteins, which may indirectly lead to an increase in detectable cytoplasmic protein levels, as previously suggested . In either case, the observation that over-phosphorylation appears to stimulate SR protein export is incompatible with the idea that the lack of phosphorylation also accelerates nuclear export of SC35 in sky1 Δ yeast. Therefore, we favor the interpretation that SKY1 -mediated phosphorylation functions at the nuclear import step, which provides a rationale for our earlier findings that all SRPK family members from yeast to humans are largely, but not exclusively, localized in the cytoplasm . Together, these observations suggest that SRPKs may catalyze phosphorylation of their substrates in the cytoplasm and facilitate their nuclear import. In yeast, we observed that unphosphorylated SR proteins appear to distribute uniformly in the nucleoplasm, yet their efficient targeting to appropriate molecular targets (in this case, the LexA binding site) requires phosphorylation mediated by Sky1p. These data are consistent with the recent report on phosphorylation-dependent recruitment of SR proteins to nascent transcripts . In addition, the transcriptional repression assay provided a quantitative measure of recruitment, and, therefore, allowed the comparison of different RS domain–containing splicing factors. For example, we observed that targeting of the SC35 fusion protein is more dependent on phosphorylation than that of ASF/SF2 and other RS domain–containing splicing factors, which may result from the phosphorylation dependence of SC35 for both efficient nuclear import and intranuclear movement. The mechanism for phosphorylation-dependent targeting is not entirely clear, but may be explained by the possibility that an unphosphorylated RS domain may interact with other nuclear constituents. For example, it was suggested, based on in vitro experiments, that phosphorylation is necessary for sequence-specific binding of SR proteins to their RNA targets . In addition to increasing protein–nucleic acid binding specificity, phosphorylation can modulate protein–protein interactions among RS domains in vitro . Our finding that phosphorylation increases ASF/SF2-U1-70K interactions, but has little effect on ASF/SF2-U2AF35 interactions, suggests that phosphorylation can differentially modulate protein–protein interactions depending on the protein pairs. Therefore, phosphorylation may regulate molecular targeting of RS domain–containing proteins to their appropriate protein partners after being recruited to actively transcribing regions in the nucleus. This phosphorylation-dependent selectivity may be crucial for an orderly assembly of splicing factors on specific transcripts at specific stages of the splicing reaction. In mammalian cells, splicing factors are believed to be “stored” in nuclear speckles and recruited to nascent transcripts to carry out the splicing reaction. The exchange of SR proteins between the nucleoplasm and nuclear speckles is highly dynamic, as illustrated by monitoring the movement of GFP-ASF/SF2 in living cells . Earlier studies have shown that increased phosphorylation releases SR proteins to the nucleoplasm , making them generally available to be recruited to the sites of transcription and splicing, whereas dephosphorylation appears to be essential for the resolution of spliceosomes , and as a result, contributes to the accumulation of SR proteins in nuclear speckles . In the current study, SR proteins in sky1 Δ yeast remained soluble, suggesting that unphosphorylated SR proteins may not form nonspecific aggregates resembling inclusion bodies in yeast, yet they do not interact with each other efficiently. Interestingly, we observed that removal of the RS domains from both U2AF65 and U2AF35 allowed them to interact very efficiently in both wild-type and sky1 Δ yeast, even better than the interaction between the full-length proteins in wild-type yeast. This result implies that even in wild-type cells, some proteins may not be sufficiently phosphorylated for efficient interaction. Because we detect U2AF65/35 in large complexes in both wild-type and sky1 Δ yeast, it is possible that phosphorylated and unphosphorylated proteins may interact with different sets of proteins. In fact, interactions involving hypophosphorylated RS domain–containing splicing factors may resemble those in nuclear speckles where a series of rearranged protein–protein and protein–RNA interactions have been triggered by phosphatases during splicing. To some extent, these dephosphorylation-induced rearrangements may cause the coalescence of splicing complexes on nascent transcripts to give rise to the appearance of speckles in the nucleus. Therefore, one might imagine that nuclear speckles are postsplicing structures for recycling of splicing factors, rather than storage sites where splicing factors are randomly accumulated. This scenario may best explain the observations that nuclear speckles contain essentially all splicing factors as well as poly (A) + mRNA , but are not sites for the majority of nascent transcripts detected by BrUTP pulse labeling . A synthesis of our results with published reports is presented in a model , depicting the dependence of SR protein trafficking on phosphorylation. In this model, nuclear import of newly translated or shuttling RS domain–containing proteins is facilitated by phosphorylation. Newly imported SR proteins are then partitioned between speckles and the nucleoplasm based in part on the extent of phosphorylation. A large body of evidence suggests that mRNA splicing takes place cotranscriptionally in the nucleoplasm whereas nuclear speckles have been described as “storage sites” for splicing factors. Release of SR proteins from speckles is facilitated by phosphorylation , which is a prerequisite for their subsequent recruitment to nascent transcripts . We show in the current study that specific targeting of nucleoplasmic SR proteins to their appropriate RNA and protein partners are also affected by phosphorylation. Once assembled on nascent transcripts, the resolution of splicing complexes during the splicing reaction appears to require dephosphorylation of SR proteins , which may accompany a series of rearrangements involving both RNAs and proteins in the spliceosome. As a result, hypophosphorylated SR proteins may be engaged in interactions with a distinct set of proteins, causing them to remain in speckles, as mature transcripts proceed to nuclear export. In summary, the novel system developed in this study enabled us to dissect this complex process in a relatively simple genetic background. The data presented in this report illustrate that SR protein kinases can affect translocation of RS domain–containing proteins from the cytoplasm to the nucleus as well as molecular targeting within the nucleus, in addition to modulation of RS domain affinities observed in vitro. Defects in combinations of these processes provide a probable explanation for the critical dependence of RS domain interactions on SRPK-mediated phosphorylation in cells. These phosphorylation-dependent steps provide multiple avenues for pre-mRNA splicing regulation in response to internal or external signaling. | Study | biomedical | en | 0.999997 |
10225948 | Tissue culture reagents and plastics were from Gibco Ltd. and other chemicals were from Sigma Chemical Co. , unless indicated otherwise. The human T cell line SupT1 and Jurkat cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin (pen/strep). The mutant Jurkat cell line JCam.1 was provided by A. Weiss (University of California, San Francisco, CA). These cells express low levels of a shorter mutant form of Lck, but not wild-type (wt) Lck . NIH-3T3 cells stably transfected with human Lck were cultured in DMEM, 10% FCS, pen/strep, 1 mg/ml G418. Two polyclonal rabbit anti-Lck sera, here designated LckN and LckC, were used. LckN was provided by J. Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and used in immunoprecipitation experiments. This serum was raised against an NH 2 -terminal Lck peptide comprising residues 39–58. In addition to Lck, LckN also immunoprecipitates and immunoblots an unidentified protein with a slightly higher molecular weight than Lck . LckC , provided by S. Ley (National Institute for Medical Research, Mill Hill, United Kingdom), was used in Western blotting. This antibody was raised against a synthetic Lck peptide comprising residues 478–509. For immunoprecipitation of CD4, a mixture of two mouse mAbs, #4 and #19, provided by J. Hoxie (University of Philadelphia, Philadelphia, PA) was used. The anti-CD4 mouse mAb Q4120 was used for immunoblotting. Peroxidase-conjugated goat anti–rabbit antibodies were from Pierce and Warriner. Cells were washed once and incubated for 45 min in methionine- and cysteine-free DME medium (ICN Biomedicals Ltd.). For pulse-labeling of suspension cells (SupT1 and Jurkat cells), typically 1 mCi [ 35 S]methionine/ cysteine (Express [1,175 Ci/mmol]; DuPont ) was used per 10 8 cells in 1 ml methionine- and cysteine-free DME medium, 10% FCS. When chase times did not exceed 15 min, incorporation of label was terminated by the addition of nonradioactive methionine and cysteine to a final concentration of 1 mM each. For experiments with chase times >15 min, cells were transferred after the pulse to 10 ml warm (37°C) DMEM with methionine and cysteine at a final concentration of 1 mM. Where indicated, BFA was present at a final concentration of 10 μg/ml (added from a 2.5 mg/ml stock in EtOH) before and during the pulse, and at 2 μg/ml during the chase. Samples taken at indicated chase times were kept in 10 ml ice-cold DMEM until the final time point, pelleted by centrifugation (5 min at 1,500 rpm, 4°C), and further processed as described for individual experiments. For the adherent NIH-3T3 cells, per time point one 10-cm dish of cells (∼7 × 10 6 ) was labeled with 0.75 mCi [ 35 S]methionine/cysteine in 1 ml methionine- and cysteine-free DME medium without FCS. The chase was started by replacing the radiolabel with prewarmed DMEM containing cysteine and methionine at 1 mM each. At the end of the chase this medium was replaced by ice-cold DMEM. The cells were put on ice, gently scraped, and recovered by centrifugation at 1,500 rpm for 5 min at 4°C. Unless indicated otherwise, cells were lysed in NP-40 buffer (2% NP-40 [Pierce and Warriner], 20 mM Tris, pH 7.8, 150 mM NaCl, 2 mM MgCl 2 , 1 mM EDTA) containing the protease inhibitors PMSF (at 1 mM) and CLAP (5 μg/ml each of chymostatin, pepstatin A, antipain hydrochloride, and 10 μg/ml leupeptin hemisulphate). After removal of nuclei and cell debris by centrifugation at 13,000 g for 5 min at 4°C, the lysates were cleared of nonspecifically binding proteins by three rounds of incubations with normal rabbit serum (3 μl) and 20 μl packed protein A–Sepharose beads ( Pharmacia Biotech AB ) for 30 min at 4°C. For specific immunoprecipitations, samples were incubated on ice for 45 min with relevant antibodies: 1 μl of the polyclonal rabbit serum LckN for immunoprecipitation of Lck or a mixture of mAbs #4 (1.7 μg) and #19 (0.5 μg) for CD4. Immune complexes were recovered by incubation with protein A–Sepharose (25 μl packed beads) for 45 min at 4°C and washed five times in NP-40 lysis buffer. For Endo H digestion, washed immunoprecipitates were incubated for 1 h at 37°C with 1 mU Endo H ( Boehringer Mann-heim) in 50 mM sodium citrate (pH 5.5), 0.02% SDS. Immune complexes were eluted by addition of nonreducing SDS sample buffer, incubated for 5 min at 95°C, and loaded on 8% SDS-polyacrylamide gels. After electrophoresis, gels were enhanced in salicylic acid (16% wt/vol in 30% methanol), dried, and exposed to Kodak X-Omat AR film ( Eastman Kodak Co. ) for 1–9 d. After gel electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher and Schuell). The blots were incubated in blocking buffer (10% skimmed milk, 0.1% Tween 20 in PBS) for 1 h at room temperature. Incubations with primary and secondary antibodies were in blocking buffer for 1 h each at room temperature. To detect Lck, the rabbit antiserum LckC (1:1,000) and HRP-conjugated goat anti–rabbit antibodies (1:2,000) were used. Q4120 (1.6 μg/ml) and HRP-conjugated goat anti–mouse antibodies (1:2,000) were used to detect CD4. Blots were developed using enhanced chemiluminescence ( Amersham International plc ) and visualized with autoradiography film (Fuji Photo Film Co. Ltd.). After pulse–chase labeling, cells were incubated in 1 ml hypotonic buffer (20 mM Tris, pH 7.8, 2 mM MgCl 2 , 1 mM EDTA, 1 mM PMSF, CLAP as above) on ice for 12 min and homogenized by 15 strokes in a Dounce homogenizer (Wheaton Scientific). To remove nuclei, cell homogenates were centrifuged for 5 min at 1,500 rpm, 4°C. The postnuclear supernatant was centrifuged in an Optima TL Ultracentrifuge ( Beckman Instruments ) for 45 min at 100,000 g , 4°C, to recover total cellular membranes. The pellet (membrane fraction) was resuspended in hypotonic buffer, Dounce homogenized (20 strokes), and adjusted to 2% NP-40, 150 mM NaCl, 1 mM PMSF, and CLAP. Similarly, the soluble fractions were adjusted to 2% NP-40 and 150 mM NaCl. The final volume of both fractions was equivalent. Samples (2% of total volume) were analyzed by immunoblotting to check the efficiency of membrane separation and the remainder was subjected to immunoprecipitation. In control experiments, to examine the presence of Lck in the nuclear fraction, nuclei were resuspended in an equal volume of the same buffer as the membrane and soluble fractions and analyzed for Lck by immunoprecipitation and immunoblotting. The amount of Lck detected in this fraction was negligible . After pulse–chase labeling, SupT1 cells (2.5 × 10 7 for each time point) were washed once with ice-cold DMEM and incubated with the anti-CD4 antibodies #4 (1.7 μg/ml) and #19 (0.5 μg/ml) for 1 h at 4°C to absorb cell surface CD4. During incubation, the cells were kept in suspension by rotation. The cells were washed three times with 10 ml ice-cold DMEM to remove unbound antibody. Next, the cells were lysed in NP-40 buffer (see above) to which soluble nonlabeled CD4 (150 ng/ml, diluted from a 500 μg/ml stock; American Biotechnologies) was added to prevent binding of intracellular labeled CD4 to free antigen-binding sites. Nuclei and cell debris were removed by centrifugation (5 min at 13,000 g , 4°C) and cell surface CD4 was recovered by incubation with 25 μl packed protein A–Sepharose beads. To recover the remaining intracellular CD4, the lysate was precleared twice with normal rabbit serum and protein A–Sepharose and subsequently incubated with antibodies #4 and #19 and protein A–Sepharose again. Autoradiograms were digitized using Sony XC-77CE CCD video camera and NIH Image, stored as TIFF files and imported into the Bio-Rad Molecular Analyst program for analysis. The relative amount of membrane-associated Lck was determined as follows: [ M − BG1 /( M − BG1 ) + ( S − BG2 )] × 100%, where M is the density of the membrane-associated Lck band, BG1 the background in the membrane lane, S the density of the soluble Lck band, and BG2 the background in the soluble fraction. The relative amount of CD4-associated Lck was determined as follows: [ CD4 − BG1 /( CD4 − BG1 ) + ( Lck − BG2 )] × 100%, with CD4 being the density of the Lck band in the anti-CD4 immunoprecipitation, and BG1 the background in the anti-CD4 immunoprecipitation; Lck the density of the Lck band in the anti-Lck immunoprecipitation, and BG2 the background in the anti-Lck immunoprecipitation. The relative amount of Lck associated with cell surface CD4 was determined as follows [ Lck ( CS ) − BG1 /{ Lck ( CS ) − BG1 } + { Lck ( IC ) − BG2 }] × 100%, where Lck(CS) is the density of the Lck band in the immunoprecipitation of cell surface CD4, Lck(IC) the density of the Lck band in the immunoprecipitation of intracellular CD4, and BG1 and BG2 the background in cell surface and intracellular immunoprecipitation, respectively. The relative amount of CD4 at the plasma membrane was determined in the same way for the CD4 bands. We found that quantitation of autoradiograms leads to an overestimation of signals with low intensity, as a result of which curves do not reach the 0 and 100% as would be expected based on the autoradiograms. Similar curves were obtained when the gels were analyzed using a PhosphorImager. We used the human leukemia T cell line SupT1 to study the mechanism by which newly synthesized Lck reaches the plasma membrane. In these cells, the majority of Lck is detected at the cytosolic side of the plasma membrane by immunofluorescence . In the study described here, we used pulse–chase labeling and immunoprecipitation to follow newly synthesized Lck. The rabbit antiserum used for immunoprecipitations recognizes Lck and, in addition, a protein with a slightly higher molecular weight. Lck was identified by comparing Jurkat cells that express wt Lck, with JCam.1 cells, a Jurkat derivative that expresses very low levels of a shorter, mutant Lck . The background band was detected in both cell lysates whereas wt Lck was only detected in Jurkat lysates (the shorter mutant Lck in JCam.1 was only visible after long exposures of the autoradiogram, not shown). The background band (marked with ★) was also detected in a variety of Lck-negative cell lines including HeLa and NIH-3T3 (not shown), indicating that this protein is not associated with, nor related to, Lck. Lck was further identified by its association with CD4 in SupT1 cells. Lck and CD4 have similar molecular weights; however, in SDS-PAGE, under nonreducing conditions, CD4 migrates slightly faster than Lck and the two proteins can be clearly resolved. Immunoprecipitation with anti-CD4 antibodies resulted in coimmunoprecipitation of Lck, but not of the background band . Vice versa, immunoprecipitation with anti-Lck antibodies coimmunoprecipitated CD4. As expected, CD4 only coimmunoprecipitated from a membrane, but not from a soluble fraction . To establish the kinetics with which newly synthesized Lck becomes membrane-associated, SupT1 cells were pulse-labeled for 5 min with [ 35 S]methionine/cysteine and chased for various times. The cells were then broken in hypotonic buffer, the nuclei removed by centrifugation, and total cellular membranes recovered by centrifugation at 100,000 g . By immunoblotting, total cellular Lck was detected exclusively in the membrane fraction , indicating that virtually all Lck is membrane-associated at steady state. To study newly synthesized labeled molecules, Lck was immunoprecipitated from the membrane and soluble fractions and analyzed by SDS-PAGE. After 5 min of pulse-labeling only a small amount of Lck (∼15%) was found at membranes . This amount increased with time, and membrane association was complete after 30–45 min of chase . Newly synthesized CD4 coimmunoprecipitated with Lck already at early chase times (5 min), and, as expected, was only detected in membrane fractions . This coimmunoprecipitation was easier to detect when cells were directly lysed in NP-40 buffer . Quantitation showed that the kinetics of Lck membrane association were consistent between experiments and occurred with a calculated half time of 9 min. In SupT1 cells, the majority of Lck (at least 70%) is associated with CD4, as determined by coimmunoprecipitation experiments (not shown). However, this interaction is not essential for membrane binding of Lck, since also in CD4/ CD8-negative T cells, Lck is completely membrane-associated at steady state . Nevertheless, CD4 or CD8 could influence the rate of membrane binding of newly synthesized Lck. To examine this, we studied Lck membrane binding in BC7 cells, a CD4-negative derivative of SupT1. The kinetics of membrane association were identical to those seen in SupT1 cells (not shown), indicating that membrane recruitment of Lck is not dependent on the presence of CD4. However, BC7 and SupT1 cells do express CD8, and therefore a role for this protein could not be excluded, although an interaction with CD8 was not observed by immunoprecipitation. Therefore, we also determined Lck membrane-binding kinetics in the CD8-negative Jurkat cells. Three different Jurkat clones were screened by FACS ® analysis for CD4 expression. Two were found to express very little if any CD4, whereas one expressed considerable amounts of CD4 . This was confirmed by immunoblotting . Again, we did not find a difference in membrane association of newly synthesized Lck between CD4-positive and -negative cells . However, membrane-binding kinetics of Lck were slower in Jurkat cells than in SupT1 cells ( t 1/2 21 min in Jurkat vs. 9 min in SupT1), indicating that cell type–specific differences can influence these kinetics. The membrane-binding kinetics measured here for Lck differ from those reported recently for another member of the Src-family, Fyn , which is also myristoylated and palmitoylated. Membrane binding of Fyn was studied in transfected NIH-3T3 fibroblasts and COS cells and found to be complete within 5 min of synthesis. To investigate whether this apparent difference between Lck and Fyn was due to the different cellular backgrounds, we also analyzed Lck in stably transfected NIH-3T3 cells. Previously, we established that at steady state all the Lck is membrane bound in these cells . We found little Lck on membranes after 5 min of labeling, whereas membrane association of newly synthesized Lck proceeded at rates similar to those seen in SupT1 cells, with 50% membrane-associated after 10 min . Thus, T cell–specific proteins do not enhance membrane binding of newly synthesized Lck and Lck differs markedly from Fyn in its rate of membrane association. In mammalian cells, nonpalmitoylated soluble Lck does not interact with CD4 , suggesting that membrane binding of Lck is necessary for stable association with CD4. To establish the kinetics of CD4 interaction, SupT1 cells were pulse-labeled and chased, and cell lysates were then subjected to sequential immunoprecipitations, first with anti-CD4 antibodies and then with anti-Lck antibodies. With the anti-CD4 antibodies we recovered CD4 and Lck and, in addition, a background band with a molecular weight similar to the one seen in Lck immunoprecipitates . The latter band (★) was also present in the normal rabbit serum control , showing that it is precipitated nonspecifically. In contrast to the anti-Lck immunoprecipitates, the background band is not consistently found in anti-CD4 immunoprecipitates . Directly after 5 min of pulse-labeling, some Lck coimmunoprecipitated with CD4, while at 15 min chase ∼50% interacted with CD4 . Previously, we determined that in SupT1 cells, at least 70% of the total Lck is associated with CD4 at steady state. This situation is apparently reached 60 min after synthesis . The kinetics of the interaction with CD4 are similar to those seen for membrane association of Lck, suggesting that the two processes are closely linked. We next labeled the cells for only 2 min to detect the earliest membrane and CD4 association of Lck. On long exposures of autoradiograms, a small amount of Lck can be seen associated with membranes directly after the 2-min pulse . Association of Lck with CD4, by contrast, can be detected only after a 3-min chase , with a further increase in both the amounts of membrane- and CD4-associated Lck after a 6-min chase. Thus, membrane association of Lck starts soon after synthesis and is rapidly followed by CD4 association. Note that in these experiments, all the CD4 is immunoprecipitated and therefore no distinction can be made between Lck binding to newly or previously synthesized CD4. To determine at which time after synthesis CD4 associates with Lck, we followed the coimmunoprecipitation of labeled CD4 with anti-Lck antibodies. CD4 was detected in Lck immunoprecipitates already at 5-min chase after 5 min of pulse-labeling, and possibly even at 0-min chase . This is similar to the CD4 coimmunoprecipitation with Lck in membrane fractions shown in Fig. 2 B. Human CD4 carries two N-linked oligosaccharides , one of which becomes resistant to Endo H, an enzyme that selectively removes high mannose N-linked oligosaccharides . Endo H resistance is acquired when the protein is delivered to the cis-Golgi where trimming of the high mannose carbohydrate occurs. Directly after pulse-labeling and at 5 min of chase, the majority of CD4 is Endo H–sensitive, indicating that the protein has not yet reached the cis-Golgi . Endo H–resistant CD4 first appeared at 10 and 15 min of chase and coincided with a decrease in the amount of Endo H–sensitive CD4 . We also detected small amounts of Endo H–sensitive CD4 in Lck immunoprecipitates at 5 min of chase (not shown). Together, the data show that CD4 interacts with Lck early after synthesis, possibly on the ER or intermediate compartment. The previous experiment indicates that CD4 associates with Lck early in the exocytic pathway; however, this experiment does not discriminate between association of CD4 with newly or previously synthesized Lck. To determine the route through which Lck traffics to the plasma membrane, it is necessary to follow the pool of newly synthesized Lck exclusively. Therefore, we investigated the arrival of Lck at the plasma membrane at various times after pulse-labeling by selectively immunoprecipitating cell surface CD4. For this purpose, intact SupT1 cells were incubated with anti-CD4 antibodies on ice, thereby ensuring that only cell surface CD4 was complexed with antibody. Next the cells were lysed in the presence of soluble unlabeled CD4 to prevent binding of intracellular labeled CD4 to antibodies during lysis. Cell surface CD4 was then isolated by incubation with protein A–Sepharose. Intracellular CD4 was subsequently recovered by a second round of immunoprecipitation with anti-CD4 antibodies. In agreement with reported transport rates of CD4 , we observed that at 0, 10, and 20 min of chase, the majority of newly synthesized CD4 was found inside the cell . Approximately 50% was at the cell surface after 40 min of chase and at 90 min the majority was at the cell surface. At 0 min of chase, CD4 is completely Endo H–sensitive and has therefore not yet reached the cis-Golgi. Therefore, the small amount of labeled CD4 in the cell surface immunoprecipitation at this time most likely reflects an incomplete block of free antibody-binding sites during cell lysis and, consequently, the recovery of some intracellular CD4. A similar explanation accounts for the small amount of labeled CD4 in the cell surface immunoprecipitation at 10 and 20 min of chase. Like CD4, newly synthesized Lck can be seen to move from the intracellular to the plasma membrane fraction with time. Lck predominantly associated with intracellular CD4 at early time points (0, 10, and 20 min of chase), while the majority was associated with cell surface CD4 at 40 and 90 min of chase . Thus, it appears that the initial association between newly synthesized Lck and CD4 occurs on intracellular membranes and that Lck is subsequently transported to the plasma membrane. Little if any newly synthesized Lck associates directly with CD4 at the plasma membrane although >95% of the total amount of CD4 is located at this site. Given our observation that binding of Lck to CD4 occurs very rapidly after membrane binding , the initial membrane association of Lck most likely occurs at the same intracellular site as CD4 binding. The transfer of newly synthesized Lck from intracellular membranes to the plasma membrane with time suggests that CD4-associated Lck is transported via the exocytic pathway. To further investigate this, we studied transport of Lck in the presence and absence of the drug BFA. BFA causes disruption of the Golgi apparatus and blocks transport from the ER and Golgi complex, but not from the TGN to the plasma membrane . The drug was added 2 min before pulse-labeling and remained present during the chase. We again followed transport of Lck by its association with cell surface versus intracellular CD4. In the absence of BFA, the amount of cell surface CD4 increased with time , while in its presence all CD4 remained intracellular, consistent with an inhibition of transport via the secretory pathway. Similarly, the relative amount of cell surface–bound Lck at 30 and 60 min of chase was significantly reduced in the presence of BFA , suggesting that the newly synthesized Lck was associated with early compartments of the exocytic pathway and its transport to the plasma membrane inhibited by BFA. The block in transport caused by BFA was not as complete for Lck as for CD4: some Lck associated with nonlabeled cell surface CD4 at 30 and 60 min chase . This minor fraction of Lck might be targeted from its site of synthesis to the plasma membrane directly. CD4 synthesized in the presence of BFA coimmunoprecipitated with anti-Lck antibodies albeit to a lower extent than in the absence of BFA . This is consistent with the notion that CD4 associates with Lck on an early exocytic compartment. The palmitoylation of the cytosolic proteins SNAP-25 and GAP43, but not of the alpha subunits of heterotrimeric G proteins, was shown to be inhibited in the presence of BFA . Our data suggest that palmitoylation of Lck is not affected by BFA since its association with CD4 occurs normally . We also studied membrane binding of Lck in the presence of BFA and found no difference in rates compared with untreated cells , suggesting that Lck palmitoylation indeed occurred normally. Furthermore, incorporation of [ 3 H]palmitic acid into Lck was not decreased in the presence of BFA (not shown). Thus, the reduction in the amount of Lck associated with cell surface CD4 in the presence of BFA is most likely caused only by the inhibition of transport through the exocytic pathway. To understand the mechanism(s) through which acylated cytosolic proteins are targeted to their functional locations in cells, we have studied the early events in the membrane binding and cellular localization of the Src-related tyrosine kinase Lck. We have shown previously that Lck contains intrinsic targeting signals in its NH 2 -terminal unique domain that direct the protein to the cytosolic leaflet of the plasma membrane . In the present study we have used pulse–chase analysis to investigate how newly synthesized Lck is delivered to this location. Our data indicate that in the lymphoid T cell line SupT1, a large proportion of Lck is initially targeted to an intracellular compartment, most likely an early station of the exocytic pathway, where it becomes stably membrane-bound and can interact with CD4, before it is transported to the cell surface. Membrane binding of Lck is a relatively slow process; it starts soon after synthesis but is not complete until 30–45 min later. An Lck mutant that cannot be palmitoylated is unable to associate with membranes , indicating that palmitoylation is required for stable membrane interaction of Lck. Membrane association has similar kinetics in the presence or absence of CD4 and CD8 . Thus, the rate of membrane association likely reflects the rate of Lck palmitoylation and not its interaction with these transmembrane proteins. Membrane binding has been studied for only a few acylated proteins and at present it remains unclear what elements are important for the kinetics of this process. Between different T cell lines, we did observe slight differences in membrane-binding rates. However, all our kinetics for Lck differ significantly from those measured by others for another Src-kinase, Fyn, which is completely membrane-bound within 5 min of synthesis . The rapid membrane association of Fyn was shown to require myristoylation at the NH 2 -terminal glycine and palmitoylation at cysteine 3 . Significantly, both Lck and Fyn are myristoylated and can be palmitoylated at cysteine 3 . In addition, Lck can also be palmitoylated at cysteine 5 and Fyn at cysteine 6. Cysteine 3 was found to be the major palmitoylation site for both proteins , although a conflicting report exists for Lck . Ostensibly, the modifications appear similar for the two proteins, suggesting that the acylations alone do not determine the rate of membrane association and that other features of the unique domains might be important. One difference between the unique domains of Lck and Fyn is the presence of two cysteines in Lck (Cys 20 and 23) that are required for the interaction with the cytoplasmic domains of CD4 and CD8. However, we excluded a role for this interaction in membrane-binding kinetics of Lck, since the kinetics are similar in the presence and absence of CD4/CD8. Another difference between the unique domains of Lck and Fyn is the absence of positively charged amino acids in the NH 2 -terminal 30 residues of Lck, while five positively charged amino acids are present in the corresponding region of Fyn. Given that Src requires positively charged amino acids for membrane association , it is possible that these charges in Fyn also facilitate membrane binding. It is noteworthy that a mutant Fyn which is myristoylated but not palmitoylated can associate with membranes to some extent , whereas the corresponding Lck mutant does not . This implies that, in contrast to Lck, newly synthesized, myristoylated Fyn might have some affinity for membranes and as a result may translocate faster to membranes than Lck. van't Hof and Resh showed that NH 2 -terminal amino acid substitutions, apart from the myristoylation and palmitoylation sites at Gly 2 and Cys 3, respectively, did not change membrane-binding rates for Fyn. However, all their reported mutants contained at least one positively charged amino acid in the NH 2 -terminal 10 residues. The interaction of newly synthesized Lck with CD4 occurred with similar kinetics to its interaction with membranes . CD4 binding must succeed membrane association since nonpalmitoylated Lck is unable to associate with CD4 . Consistent with this, we observed the earliest CD4 association of Lck shortly after the earliest detectable membrane association . This suggests that CD4 binding follows rapidly upon membrane binding, and therefore upon palmitoylation of Lck. Thus, it is likely that all three events (palmitoylation, membrane binding, and CD4 binding) occur at the same location in the cell. While we have no direct data concerning the site where palmitoylation or initial membrane association occurs, several results suggest that the initial interaction with CD4 takes place on an early compartment of the exocytic pathway. First, the association of newly synthesized CD4 with Lck starts within 10 min of CD4 synthesis , at a time when CD4 has not yet reached the plasma membrane and is located in the exocytic pathway . Secondly, newly synthesized Lck associates initially with intracellular CD4 and only at later times (after 40 min of chase) with cell surface CD4 . Thirdly, in the presence of BFA, transport of CD4-associated Lck to the plasma membrane was inhibited . Previously, Crise and Rose showed that Lck can interact with CD4 retained in the ER. Interaction at the ER was also found in cells overexpressing both Lck and a chimera containing the VSV G protein ectodomain and the cytoplasmic tail of CD4 . However, in these latter experiments the CD4 construct resides in the ER for a long time, at least 60 min after synthesis . The pulse–chase experiments described here show that the interaction of CD4 with Lck takes place early in the exocytic pathway irrespective of expression levels or mislocalizations of CD4 and/or Lck. Our previous immunofluorescence experiments with transfected HeLa cells indicate that CD4 and Lck can interact at the Golgi complex and travel to the plasma membrane together . Thus, the normal route for the bulk of newly synthesized Lck is initial association with intracellular CD4 followed by delivery to the plasma membrane. The BFA experiments support this conclusion and further indicate that newly synthesized Lck associates with CD4 at a compartment located before the TGN . The ability of newly synthesized CD4 to associate with Lck in the presence of BFA and the observation that Endo H–sensitive CD4 coimmunoprecipitates with Lck (not shown) suggest that Lck can associate with membranes of the ER or intermediate compartment. Since palmitoylation is required for stable membrane association of Lck, our results imply that palmitoylation of newly synthesized Lck occurs on intracellular membranes of the early exocytic pathway. To date, palmitoyl transferase activity has been detected at the plasma membrane , intermediate compartment , Golgi complex , and mitochondria . Palmitoyl transferase activities have been partially purified , but the number of different palmitoyl transferases in the cell, their specificities, and subcellular distributions are unknown. Several transmembrane proteins are palmitoylated early after synthesis in the ER or intermediate compartment and the cytosolic GAD65 requires targeting to the Golgi for palmitoylation. Possibly, newly synthesized Lck uses the same palmitoyl transferase(s) as these proteins. Palmitoylation of two cytosolic proteins, SNAP25 and GAP43, is inhibited by BFA, suggesting that an intact secretory pathway is required for the palmitoylation of these proteins . On the other hand, palmitoylation of several viral transmembrane proteins is not affected by BFA . Also for Lck, palmitoylation in the presence of BFA is apparently normal since membrane-binding kinetics and palmitic acid incorporation (not shown) were unaffected. Currently, there are no direct data concerning the cellular sites of palmitoylation of Src-family proteins. It has been suggested that this modification can occur at the plasma membrane since a short fluorescent peptide containing the first three amino acids of Lck (myrGlyCysGly) is palmitoylated and incorporates into the plasma membrane when added to intact cells . Palmitoylation of Lck is reversible , suggesting that the protein undergoes cycles of de-palmitoylation and re-palmitoylation. The re-palmitoylation might well occur at the plasma membrane where most steady-state Lck is located. However, the experiments described here indicate that newly synthesized Lck is palmitoylated on early membranes of the exocytic pathway. The delivery of Lck from an intracellular membrane compartment to the plasma membrane in a BFA-sensitive manner presents a novel transport route for an acylated, cytosolic protein. The routes of only two other acylated proteins have been studied: Fyn which is myristoylated and palmitoylated, and the Gag protein of Moloney murine leukemia virus, which is myristoylated only. In contrast to Lck, both of these proteins were found to be targeted to the plasma membrane directly . Fyn resembles Lck in the nature of its acylations, its expression in T lymphocytes, and its role in signaling through the TcR. However, while Lck interacts with CD4 and/or CD8 , no high stoichiometry interactions with transmembrane proteins have been identified for Fyn. Although expression of CD4 and CD8 does not affect steady-state localization nor membrane-binding kinetics of Lck, these proteins could still influence the transport route of Lck. If, for instance, newly synthesized CD4/CD8 is the only pool capable of interacting with Lck, targeting of Lck to early exocytic compartments would be required to facilitate efficient assembly of the coreceptor–Lck complexes. Possibly, Lck contains intrinsic signals for targeting to an intracellular membrane compartment. Alternatively, the presence of available CD4/CD8 might recruit newly synthesized Lck to early exocytic compartments and thereby favor palmitoylation and membrane association at these sites. Newly synthesized, nonpalmitoylated Lck is located primarily in the cytosol and shows no stable interaction with CD4. However, a weak interaction might occur since soluble Lck associates with CD4 in the yeast two-hybrid system and when coexpressed in Escherichia coli . Determining the precise site(s) of the initial Lck interaction with membranes and the influence of CD4/CD8 in defining this site will require further analysis. Nevertheless, the experiments reported here indicate that palmitoylation of Lck occurs on intracellular membranes and that Lck is transported to the plasma membrane via the exocytic pathway. Further characterization of the cellular and molecular mechanisms that underlie this transport will be essential for understanding the properties and functions of acylated proteins. | Other | biomedical | en | 0.999996 |
10225949 | Degenerate PCR was performed on a mouse retinal cDNA (oligo-dT primed) library using primers to conserved motor domain amino acids, MGKTY/FTM (EcoRI · GGG/A/T/CAAA/GACG/A/T/CTA/TT/ CACG/A/T/CATG) and DLAGSE (BamHI · TCA/GCTG/A/T/CCCG/ A/T/CGCC/TAAA/GTC). PCR products were cloned into pBluescript (Stratagene) using the EcoRI and BamHI sites from the oligos and sequenced. One novel cDNA (KIF21A) was identified and used to screen the retinal cDNA library from which it was isolated. Five overlapping clones containing the entire coding sequence of KIF21A were identified from the dT primed retinal library, as well as a random primed retinal library (B6 strain mouse retina), and sequenced. A second gene, KIF21B, was isolated due to its cross hybridization to a KIF21A cDNA probe. The entire KIF21B coding sequence is contained on two overlapping clones. The GCG sequence analysis software package was used to align the KIF21A and KIF21B amino acids sequences (gap program) as well as produce a dendrogram comparing the motor domain core amino acids of many KLPs (from IFAY to LAGSE). The murine chromosomal locations of KIF21A and KIF21B genes were determined using an interspecific backcross DNA panel obtained from The Jackson Laboratories . The panel consists of 94 F2 progeny from a (C57BL/6J × SPRET/Ei) F1 female mated to a SPRET/Ei male and DNA from parental C57BL/6J and Mus spretus . ScaI polymorphisms between SPRET/Ei and C57BL/6J mouse strains were identified by Southern blotting for KIF21A and KIF21B . The distribution of the C57BL/6J allele among the F1 progeny was used to estimate gene locus position and linkage distance by The Jackson Laboratories . Human chromosomal locations that are syntenic to the mouse regions were determined by using the map generated by DeBry and Seldon . Total RNA was isolated from various mouse tissues using the guanidinium isothiocyanate extraction method as previously described . RNA amount and purity was determined by absorbance at 260 nm and the 260/280 nm ratio using a spectrometer. 30 μg of total RNA was separated on a 1% formaldehyde agarose gel, transferred to GeneScreen Plus membrane ( New England Nuclear ), and fixed by baking. The blots were probed with random primed DNA and with α[ 32 P]dATP incorporation in a buffer of 0.5 M NaPO 4 and 7% SDS at 65°C for 16 h. Blots were washed in 0.1× SSC/1% SDS at 65°C, twice for 30 min. Washed blots were exposed to Biomax MS film ( Kodak ). A KIF21A-HIS fusion protein construct which contained amino acids 1124–1355 (KLCG to QINQ) was generated in pET-23b (Novagen, Inc.). A comparable KIF21B-HIS fusion protein containing amino acids 1135– 1419 (KFKG to QINQ) was also generated in pET-23b. These fusion proteins were grown in pLysS BL21(DE3) bacteria, induced with 0.5 mM IPTG, and purified using Ni-NTA–agarose (Qiagen, Inc.). Each fusion protein was further purified by SDS-PAGE. Gel slices (containing 300 μg of fusion protein) were injected into three rabbits to produce polyclonal sera against KIF21A or KIF21B. To generate antisera that recognize only KIF21A or KIF21B, each antiserum was incubated with the conflicting Affigel (Bio-Rad Laboratories) bound fusion protein to immunodeplete antibodies that recognize both KIF21 proteins. Immunodepletion was confirmed by Western immunoblot analysis of a dilution series of known amounts of KIF21A and KIF21B fusion proteins. Affinity-purified antibody was generated by incubating each antiserum with its Affigel-bound antigen used in its generation. The antibody was then released from the column by lowering the pH. Tissue was homogenized in 25 mM sodium phosphate, 5 mM EDTA, 1% SDS, pH 7.5, buffer using a polytron. Protein concentrations were determined using the Bio-Rad D c protein assay kit. Protein samples were separated on a SDS-PAGE gel using standard Laemmli method and then transferred to PVDF membrane (Bio-Rad), dried, and blocked in 5% dry milk in 1× TBS/0.05% Tween for 1 h. Primary antibodies were incubated 1 h in the same solution at room temperature, washed, incubated with an HRP-conjugated secondary antibody, and then washed again. ECL (Nycomed- Amersham Inc. ) was used for detecting the antibodies by exposing the blots to X-OMAT-XAR5 ( Kodak ) film. Antibody concentrations used: polyclonals α-KIF21A and α-KIF21B whole serum, 1:1,000; α-KIF3A, 1:5,000; nKHC, 1:2,000 (a gift from Dr. Ron Vale, UCSF); monoclonals α-synaptotagmin, 1:500; Stressgen α-SV2, 1:50 (Hybridoma Bank, University of Iowa). A mouse brain was homogenized in 3 ml microtubule binding buffer (0.1 M Pipes, 0.9 M glycerol, 5 mM EGTA, 0.5 mM EDTA, 2.5 mM MgSO 4 , pH 6.4) using a glass dounce homogenizer (Kontes). The homogenate was centrifuged at 25,000 rpm and 25°C in a Sorvall 1270 rotor for 20 min. The supernatant was recentrifuged for 30 min to remove vesicles and endogenous microtubules. 90 μl of supernatant was supplemented with 10 μl of purified taxol stabilized mouse microtubules (3 mg/ml). To this, 10 μl of either 0.1 M Mg · ATP, or 1 μl 0.5 M Mg · AMP-PNP was added for 15 min at room temperature. Then the samples were layered onto 100 μl of a 40% sucrose cushion in the same buffer and centrifuged for 30 min at 25,000 rpm and 25°C in a Sorvall 42.2 TI rotor. Taxol was added to 10 mM in all the buffers. Pellets were resuspended in 1× SDS-PAGE sample buffer (10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 700 mM β-mercaptoethanol) and analyzed by Western blotting. A mouse brain was homogenized in 4 ml cell fractionation buffer (20 mM Hepes, 100 mM sodium aspartate, 40 mM KCl, 5 mM EGTA, 5 mM MgCl 2 , 2 mM Mg-ATP, 1 mM DTT, pH 7.2) supplemented with protease inhibitors (to 1 mM PMSF, 0.7 ng/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor; final concentrations). The homogenate was centrifuged twice at 8,800 rpm 10 min each in a Sorvall SS34 rotor. 1.2 ml of lysate was preabsorbed with 100 μl of protein A–Sepharose beads ( Pharmacia Biotech, Inc. ). 5 μl of immunodepleted KIF21A or KIF21B whole serum was added to the lysate and incubated 1.5 h at 4°C on a rotator. 20 μl of protein A beads were added to lysate for 2 h at 4°C on a rotator. Proteins were eluted by boiling 5 min with 75 μl of 1× SDS-PAGE sample buffer without β-mercaptoethanol and spun in microfuge. To the supernatant, 25 μl of 4× buffer + β-mercaptoethanol was added and boiled for 5 min more. 15 μl of each sample was analyzed by Western immunoblotting. Cell fractionation was performed using a modified version of the protocol described by Okada et al. . In brief, one mouse brain was homogenized on ice with a glass dounce homogenizer in 3 ml of CF buffer (see Immunoprecipitations section). The homogenate was spun at 3,000 g avg for 5 min, 9,000 g avg for 10 min, then centrifuged in a Sorvall 1270 rotor at 100,000 g avg for 1 h. 50 μg of total protein from each fraction, as determined using Bio-Rad D c protein assay kit, was separated on 7.5% polyacrylamide gels and transferred to PVDF membrane (Bio-Rad Laboratories) for Western immunoblotting. P3 pellets were extracted by homogenization with a dounce homogenizer and recentrifuged at 100,000 g avg for 1 h. The pellet was resuspended in the starting volume and equal volumes of the pellet and supernatant were analyzed by Western immunoblotting. A KIF21B motor construct (amino acids 1–750) was generated by PCR with the following primers that contained either a NdeI or XhoI restriction enzyme site (5′-CTG GTG CCG GAG CAT ATG GCT GGC CAG GGC, and 3′-CGC TTG TAG CTT CTC GAG CTC CCT TTC ATA). The PCR product was cloned into the NdeI and XhoI sites of pET-23b (Novagen Inc.). The construct was introduced into BL21 (DE3) bacteria and cells were grown at 37°C until an OD 600 ∼1.5 and then induced with 0.5 mM IPTG overnight at room temperature. Cells were harvested by centrifugation and resuspended in lysis buffer (300 mM NaCl, 50 mM sodium phosphate, 0.5 mM MgCl 2 , 0.01% NP-40, 10 μg/ml soybean trypsin inhibitor, 0.7 μl/ml β-ME, 1 mM PMSF, 0.1 μM ATP, pH 7.4) at 1 g/5 ml. Cells were lysed three times with a French press and then spun for 45 min at 30,000 rpm in a 647.5 Sorvall rotor at 4°C. KIF21B-HIS protein was isolated by incubating the high speed supernatant with 0.5 ml of Ni-NTA– agarose beads (Qiagen Inc.) for 2 h. The beads were washed three times with lysis buffer supplemented with 25 mM imidazole and 1 μM ATP, and protein was eluted with lysis buffer + 200 mM imidazole and 1 μM ATP. Protein was concentrated by centrifuging the protein in a Millipore -4 concentrator. Polarity marked microtubules were generated by the method described in Howard and Hyman , using unlabeled bovine tubulin, rhodamine-labeled tubulin (Cytoskeleton), and N -ethyl maleimide tubulin . 5 μl of 0.5 mg/ml KIF21B motor protein was absorbed onto a coverslip for 3 min and then washed with PEM40 buffer (40 mM Pipes, pH 6.9, 1 mM EGTA, 1 MgCl 2 ) for 1 min. The motility assay was performed by adding the following components: 1:1,000 polarity marked microtubules; 1 mM MgATP; 10 μM taxol; and an oxygen scavenging system to PEM40 buffer. Images were gathered using a Zeiss Axioplan fluorescence microscope, a Princeton Instruments cooled CCD, and the MetaMorph software package ( Universal Imaging Corp. ). Animals were killed by suffocation with carbon dioxide and their circulatory system flushed by intracardial perfusion of 1× PBS, followed by fixation with 4% paraformaldehyde in 0.1 M PO 4 . Tissue was dissected and cryoprotected by overnight incubation in 20% sucrose/1× PBS at 4°C. Tissue was then embedded in OCT compound and 10–15 μm sections were cut on a cryostat. Sections were dried 1 h at room temperature and then blocked with 1× PBS/0.1% Triton X-100/1% BSA for 1 h. Tissue was incubated in 1° antibody for 1 h at room temperature in incubation buffer (1× PBS/0.1% Triton X-100/1% BSA), washed, incubated with the appropriate 2° antibody for 1 h, washed, and then mounted with Vectashield (Vector Labs, Inc.). Tissue was observed using a Bio-Rad MRC-1000 scanning confocal imaging system. Antibody concentrations used: affinity-purified KIF21A and KIF21B, 1 μg/ml; SMI-31, 1:1,000 (Sternberger and Sternberger); MAP2, 1:400 ( Boehringer Mannheim ); goat α–rabbit Cy5 1:200 (Jackson ImmunoResearch Laboratories); and goat α–mouse Texas red, 1:200 (Jackson ImmunoResearch Laboratories). Adult BALB/C mice were killed by cervical dislocation and isolated brains were frozen using Tissue-Tek OCT (Miles Inc.) and Histofreeze ( Fisher Scientific Co. ). Parasagittal cryostat sections (20 μm) were cut, thaw-mounted onto charged microscope slides (Superfrost Plus; Fisher Scientific Co. ), fixed, and processed as previously described . Digoxigenin-labeled riboprobes were transcribed in the sense and antisense orientations from linearized plasmids containing: KIF21A, nucleotide (nt) 2720 (BamHI) to nt 4260 (EcoO109); KIF21B, nt 3020 (HindIII) to nt 4430 (EcoRV); and est W46113 for MAP2 cDNAs using standard protocols ( Boehringer Mannheim Corp. ). Hybridization and color reaction were carried out as previously described . To identify novel KLPs that may be involved in axonal and/or dendritic transport, a degenerate PCR based screen was performed using a retinal mouse cDNA library. Two novel KLPs, which we named KIF21A and KIF21B, as well as many previously reported KLPs sequences were identified. Based on the cDNA sequences, KIF21A protein is predicted to contain 1,573 amino acids, while KIF21B protein is predicted to contain 1,668 amino acids . To determine whether known mouse mutants or human diseases mapped to the same chromosomal regions as the KIF21A and KIF21B genes, the chromosomal location of each KIF21 gene was established using The Jackson Laboratories SBS backcross panel (see Materials and Methods). The KIF21A gene maps to 39.7 on mouse chromosome 15 (syntenic to human chromosome 8 at ∼8q24), and KIF21B maps to 64.7 on chromosome 1 (syntenic to human chromosome 2 at ∼2cen-q21). The unique chromosome locations establish KIF21A and KIF21B as independent genes, but no known mouse mutants or human diseases map close to these chromosomal locations. Members of a protein family often share a high degree of amino acid similarity, as well as common protein motifs. A comparison of the core amino acids of the KIF21A and KIF21B motor domains to previously identified KLPs reveals that KIF21A and KIF21B are most similar to each other and a Caenorhabditis elegans KLP sequence (CET01G1) identified during the C . elegans genome sequencing project . KIF21A and KIF21B proteins share 61% amino acid sequence identity along their entire length with the highest identity in the NH 2 -terminal 25% and COOH-terminal 25% of the proteins. Like true kinesin, KIF21A and KIF21B proteins are comprised of three functional domains: an NH 2 -terminal head motor domain (1–400), a predicted coiled-coil stalk (data not shown; 400–1,000), and COOH tail . Both proteins have a cluster of negatively charged amino acids of unknown function within their stalk domain and seven consensus WD-40 repeats in their tails . WD-40 repeats were first identified in β-transducin , and subsequently have been found in numerous, functionally unrelated proteins and are believed to be involved in protein–protein interactions . Thus, the KIF21 family of KLPs may mediate interactions with their cargoes through these WD-40 domains. To determine which tissues KIF21A and KIF21B proteins function in, Northern and Western analyses were performed using neural and nonneural tissues. A 6.2-kb KIF21A transcript was identified in all tissues examined except the spleen, and a 6.0-kb KIF21B transcript was identified in the brain, eye, and spleen . Protein immunoblotting for KIF21A primarily identifies an ∼180-kD polypeptide in brain extract, with more moderate levels in the kidney, liver, and testes, and low levels in all other tissues except the spleen, where no protein is observed . KIF21B antibody recognizes an ∼178-kD band in the brain and spleen with lower levels in testes . The ∼6-kb mRNAs and ∼180-kD proteins sizes observed with mRNA and protein blots are consistent with sizes predicted from the isolated KIF21A and KIF21B cDNA sequences. To consider potential neuronal functions of KIF21A and KIF21B, the distribution of these proteins in various neural regions was examined by protein immunoblotting. KIF21A and KIF21B proteins were present in all regions of the murine nervous system examined, except the sciatic nerve, where expression varied between the two proteins . In this axon-rich tissue, KIF21A protein is readily detected while KIF21B protein is barely detectable. This low expression of KIF21B in the sciatic nerve is not expected for a protein predicted to be involved in axonal transport. Like KIF21A, KIF3A and nKHC, two plus end-directed KLPs believed to participate in axonal transport , are expressed at similar levels in the sciatic nerve compared with the rest of the nervous system . To determine if KIF21A and KIF21B share similar functional characteristics, the ability to bind microtubules, to form heterodimers, and to interact with different cellular components were examined. Like most KLPs, KIF21A and KIF21B bind strongly to microtubules in the presence of the nonhydrolyzable ATP analogue AMP-PNP . Although KIF21A and KIF21B proteins share amino acid identity along their lengths, they do not form heterodimers with each other, since neither antibody was able to immunoprecipitate both proteins . To determine whether KIF21A and KIF21B proteins associate with vesicles or protein complexes, differential centrifugation was performed. KIF21A and KIF21B, like previously characterized neuronally expressed KLPs, KIF3A and nKHC, were observed in soluble (S3) and insoluble (P1, P2, and P3) pools, while synaptotagmin, an integral membrane protein , was found only in the insoluble pool . The KLP/cargo interaction was examined directly by either extracting the P3 pellet with buffer only or with buffer plus detergent and then recentrifuging. Much less KIF21B protein was released from the P3 pellet than KIF21A, nKHC, or KIF3A, suggesting that KIF21B has a higher affinity for its cargo than axonally localized kinesins . Extraction of the P3 pellet with Triton X-100, which solubilizes membranes , was performed to determine whether the insoluble fraction of KLPs is associated with membranous vesicles. Triton X-100 did not increase further the dissociation of any of these KLPs from the P3 pellet, suggesting that the KLP pools isolated in the P3 pellet are not associated with membranous vesicles. To directly determine whether the KIF21 family members are plus end-directed microtubule motors, the direction of movement of polarity marked microtubules by a KIF21B truncated motor fusion protein was determined. We found that all microtubules for which we could unambiguously identify a single bright seed moved with the bright seed leading, indicating movement by a plus end-directed microtubule motor . In this assay, microtubules moved at a rate between 0.1 and 0.3 μm/s, independent of the length of the microtubules. Sciatic nerve ligations were also performed and stained for the presence of KIF21A and KIF21B protein (data not shown). Previous work demonstrates that plus end-directed motors accumulate only on the proximal side of a ligation, while minus end-directed motors accumulate on both sides of a ligation . KIF21A and KIF21B accumulate only on the proximal side of the ligation, consistent with both proteins being plus end-directed motors. However, the amount of signal detected for KIF21B was greatly reduced compared with KIF21A and required 10-fold more laser power to see comparable levels of staining as KIF21A, which is consistent with the low protein level observed by protein immunoblotting . To determine in which neurons, and where within neurons KIF21A and KIF21B function, an immunofluorescent survey of the mouse nervous system was performed . Protein immunoblotting indicated that KIF21A and KIF21B are present throughout the nervous system. Although KIF21A and KIF21B are sometimes expressed in the same cells , their intracellular staining patterns are markedly different from each other. Strong KIF21A protein staining was observed in cell bodies and processes extending from it . KIF21B signal, on the other hand, much lower in the cell bodies (arrows) than in the processes . To determine whether KIF21A or KIF21B protein is present in axons and/or dendrites, the spinal cord was double immunostained for each KIF21 protein and either phosphorylated neurofilament H (pNF-H), an axonal marker , or MAP2, a dendrite specific marker . KIF21A protein colocalized with pNF-H and MAP2 positive processes, indicating KIF21A presence in dendrites and axons . KIF21B protein only colocalized with MAP2 positive processes , but not with most pNF-H positive processes , indicating that KIF21B protein is primarily a somatodendritic protein that is highly enriched within dendrites. This localization pattern is consistent with the protein level observed by protein immunoblotting , and the low levels of accumulation observed in a sciatic nerve ligation experiment (data not shown). In situ hybridization analysis was performed on mouse hippocampus to determine if KIF21B protein enrichment within dendrites might result from its mRNA being localized to the dendrites. KIF21A and KIF21B mRNA signal was detected only in the cell bodies of the CA pyramidal neurons . When this same tissue was probed for MAP2 mRNA, which localizes partially to dendrites, signal was detected mainly in the cell bodies and in the molecular layer where the pyramidal neuron dendrites reside , consistent with previous reports . The restriction of the majority, if not all of the KIF21B mRNA to the cell body suggests that KIF21B protein is enriched within dendrites via a protein sorting mechanism rather than by local dendritic synthesis following mRNA localization. We report here the identification and initial characterization of two KLPs, KIF21A and KIF21B. These proteins share extensive amino acid and motif similarity to each other throughout their lengths, but not to any previously characterized kinesins outside of the motor domain, suggesting that they are the founding members of a new kinesin family. Not only do KIF21A and KIF21B share amino acid sequence similarity, but each also contains seven analogous WD-40 repeats. WD-40 repeat domains have been shown in other proteins to interact with pleckstrin homology and tetratrico peptide repeats (TPR) domains . Modular protein–protein interaction domains have also been identified in other kinesins, for example, TPR domains in kinesin light chain and PH domains in KIF1A . Data from detergent extraction of high speed cell fractionation pellets suggest that the KIF21 proteins may interact with the insoluble cytoskeleton, a large protein complex, or the detergent insoluble vesicle cytoskeleton, possibly through their WD-40 repeat domains. Northern and Western data indicate that KIF21A and KIF21B mRNAs and proteins are not expressed in the same tissues except for the nervous system. Differential tissue expression among family members is not uncommon and may represent specialized tissue or unique intracellular functions for each member. While KIF21A protein was found throughout the neuron in a pattern similar to other plus end-directed kinesins that are believed to function in axonal transport , KIF21B protein was found to localize primarily to the somatodendritic compartment of neurons, being highly enriched in the dendritic processes compared with the cell body. KIF21B protein distribution is unique from the ubiquitous localization observed for plus end-directed motors and distinct from the somatodendritic distribution observed for the putative minus end-directed kinesin, KIFC2 . Current proposals to explain how cargoes are differentially targeted to either a dendrite or the axon have focused on the differences in microtubule polarity between the processes and the directionality of the motors that move along the microtubules . It has been suggested that plus end-directed motors may perform most of the axon specific anterograde transport, while minus end-directed motors may perform most of the dendrite specific transport. This model is based on the premise that orientation of axonal microtubules would not allow minus end-directed motors to enter the axon from the cell body under their own power. The mixed microtubule polarity in the proximal portion of dendrites should allow all kinesin motors to cycle between the cell body and the dendrite. Since active minus end-directed motors should only enter the dendrite and not the axon, they are ideal for sorting dendrite specific cargo to the dendrites. Plus end-directed kinesins should eventually enter the axon from the cell body and then unidirectionally translocate down the axon where they should then be capable of releasing their cargo. So far, the intracellular distribution observed for all characterized neuronally expressed kinesins, including KIF21A, conform to the prediction made by this model. The recent identification of the first putative minus end-directed neuronal kinesin, KIFC2, and its predominantly somatodendritic localization provided further support for this kinesin-dependent sorting mechanism. Our identification of a plus end-directed kinesin that exhibits somatodendritic localization and enrichment within dendrites, KIF21B, is inconsistent with the current kinesin sorting model, indicating that this model is too simple to account for all aspects of neuronal process transport. We have shown that KIF21B mRNA is confined almost exclusively to the cell body and that KIF21B protein levels are very low in the cell body and axon, compared with the dendrites. Several different mechanisms could account for the dendritic enrichment observed for KIF21B protein within neurons. There is some evidence that differences in protein stability between the axonal and dendritic processes can account for enrichment of protein within one of the processes . It is possible that active KIF21B moves into both axons and dendrites, but becomes stabilized only within the dendrite while it is actively degraded within the axon. Another way to enrich KIF21B protein within the dendrite would be to inactivate the motor function within the cell body and then use a minus end motor, such as KIFC2, to transport KIF21B specifically into dendrites where it then can be sequestered. Pellet extraction data showed minimal KIF21B release from the pellet, suggesting that there may be a strong interaction between KIF21B protein and an insoluble cargo. This insoluble cargo may be the dendritic cytoskeleton and is being used by the neuron to sequester inactive KIF21B motor until it is needed. What function might KIF21B perform within the dendrites? It has been shown that the mixture of microtubule polarity in the dendrites of cultured hippocampal neurons is not constant along its proximal to distal length . In the proximal portion of the dendrite, there is a 50/50 mixture of oppositely oriented microtubules. However, in the most distal portion of the dendrites, as many as 95% of the plus ends of microtubules are oriented towards the synapse. It is unclear whether the distal portion of dendrites in vivo possess similar microtubule distributions as cultured neurons. If this distribution does occur in vivo, it produces an environment that would favor movement of a plus end-directed kinesin toward the synapse. KIF21B may function within distal dendrites to deliver cargoes to the distal regions of dendrites. To create and maintain enrichment within the dendrite when KIF21B becomes active, it must be continuously returned to the dendrite from the cell body. KIF21B may be targeted to dendrites via a dendritic sorting signal sequence that is possessed by KIF21B, but not KIF21A. Recently, it has been shown that membrane proteins, which target to the basolateral surface within polarized epithelial cells (e.g., transferrin receptor), also target to dendrites when they are expressed in neurons. When the targeting signal is disrupted the protein is no longer restricted to the somatodendritic compartment, but becomes dispersed throughout the neuron . Future generation of KIF21A/KIF21B chimeric proteins will be useful in separating the different functional domains within each protein, and identifying the sequence differences that produce the markedly different localizations and characteristics observed for the two proteins. | Study | biomedical | en | 0.999996 |
10225950 | Plasmids pCMVNot6.2 and pCMVNot6.2-ΔF containing expressible human CFTR cDNAs were the generous gift of Dr. Johanna Rommens (The Hospital For Sick Children, Toronto). Two anti–human (COOH terminus) CFTR antibodies were used: mouse monoclonal antibody 24-1 from Genzyme Diagnostics and rabbit polyclonal antibody R3194 . Mouse monoclonal anti–rat Grp78 (BiP) was obtained from StressGen Biotechnologies Corp. Monoclonal anti-ubiquitin and anti–γ-tubulin antibodies and rhodamine-conjugated wheat germ agglutinin were purchased from Sigma Chemical Co. Mouse monoclonal anti-Hsp70 and anti-Hsp90 antibodies were from Affinity Bioreagents , Inc. Goat polyclonal anti-aldolase was from Biodesign International. Mouse monoclonal anti– lamin B 1 antibody was from Zymed Laboratories, Inc. Mouse monoclonal anti–β-cop antibodies contained in media from secreting hybridoma cells were the generous gift of Dr. George Bloom (Department of Cell Biology, University of Texas Southwestern Medical Center). Fluorescein-conjugated concanavalin A was from Molecular Probes, Inc. Fluorescently labeled secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Lactacystin was purchased from Calbiochem . All other materials were of the highest quality commercially available. Polyclonal anti-PA28 , anti–PA28-α , and anti-20S proteasome were generated as described. Polyclonal antibodies were prepared in chickens against highly purified bovine PA700. Chicken IgY was purified from egg yolks of immunized birds. The antibodies specifically recognized multiple subunits of the PA700 complex, including p112, p97, p58, p56, and p45, upon Western blot analysis of crude cell lysates and could specifically immunoprecipitate PA700 from solution using an agarose bound anti-IgY antibody. Polyclonal antibodies were prepared in rabbits against an HPLC-purified peptide representing the 16 COOH-terminal amino acids of human p31 (Nin1p), a subunit of PA700 . Oligonucleotide-directed mutagenesis as described was used to generate the mutant CFTR from the parent expression vector pCMVNot6.2. In brief, mutants were selected based upon the incorporation of a second-site mutation in β-lactamase which alters its substrate specificity leading to resistance of transformed bacteria to cefotaxime and ceftriaxone in addition to ampicillin. The sequence of the mutagenic primer used to create P205S was 5′-CGTGTGGATCGCTTCTTTGCAAGTGGC-3′. Incorporation of the mutation was verified by DNA sequencing. Transfection-quality plasmid DNA was prepared using reagents supplied by Qiagen Inc. Wild-type and mutant CFTR cDNAs were transfected into human embryonic kidney (HEK) 293 or HeLa cells (American Type Culture Collection) using the Fugene Mammalian Transfection Reagent ( Boehringer Mannheim ). Cell lines were maintained in DME supplemented with 10% FCS, 50 μg/ml streptomycin, and 50 units/ml penicillin. Centrosomes were isolated from HEK 293 and HeLa cells by discontinuous gradient ultracentrifugation according to the method of Moudjou and Bornens . In brief, cells in the exponential phase of growth were treated with 1 μg/ml cytochalasin D and 0.2 μM nocodazole for 1 h. Cells were collected by trypsinization and centrifugation and the resulting pellet was washed in TBS followed by 0.1× TBS/8% sucrose. Cells were resuspended in 2 ml of 0.1× TBS/8% sucrose followed by addition of 8 ml lysis buffer (1 mM Hepes, pH 7.2, 0.5% NP-40, 0.5 mM MgCl 2 , 0.1% β-mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, and 1 mM PMSF). The suspension was gently shaken and passed five times through a 10-ml narrow-mouth serological pipette to lyse the cells. The lysate was spun at 2,500 g for 10 min to remove swollen nuclei, chromatin aggregates, and unlysed cells. The resulting supernatant was filtered through a nylon membrane followed by addition of Hepes buffer and DNase 1 to a final concentration of 10 mM and 1 μg/ml, respectively, and incubated on ice for 30 min. The mixture was gently underlaid with 1 ml of 60% sucrose solution (10 mM Pipes pH 7.2, 0.1% Triton X-100, and 0.1% β-mercaptoethanol containing 60% [wt/wt] sucrose) and spun at 10,000 g for 30 min to sediment centrosomes onto the cushion. The upper 8 ml of the supernatant was removed and the remainder, including the cushion, containing the concentrated centrosomes was gently vortexed and loaded onto a discontinuous sucrose gradient consisting of 70, 50, and 40% solutions from the bottom, respectively, and spun at 120,000 g for 1 h. Fractions were collected and stored at –70°C before further analysis. Density gradient fractions were diluted into 1 ml of 10 mM Pipes, pH 7.2, and centrosomes were sedimented at 14,000 rpm in a microfuge for 15 min at 4°C. Centrosome pellets were resuspended in Laemmli sample buffer containing 5% β-mercaptoethanol and electrophoresed on 10% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes in the presence of Towbin transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.3). Membranes were blocked in TBS-T (Tris-buffered saline/0.1% Tween-20) containing 10% nonfat dry milk for 1 h and then incubated in fresh blocking buffer containing primary antibody at the desired concentration. Membranes were washed several times in TBS-T followed by incubation with the appropriate horseradish peroxidase–conjugated secondary antibody. Immunoreactive proteins were visualized with enhanced chemiluminescence. The proteolytic activity of the endogenous proteasomal pool was inhibited by the addition of the potent fungal product lactacystin. The inhibitor was added to 293 cells 48–72 h after transfection at a concentration of 10 μM. Treatment of cells was carried out for 2 to 12 h and was immediately followed by several washes with PBS before fixation for immunocytochemistry. For immunofluorescent subcellular localization of CFTR , transiently transfected cells attached to glass coverslips were rinsed three times with PBS followed by fixation and permeabilization with 1 ml of ice-cold methanol for 10 min at –20°C. Upon removal of methanol, cells were again rinsed three times with PBS and incubated 10 min in 1 ml of PBS supplemented with 50 mM glycine (this and all subsequent manipulations were carried out at room temperature). This buffer was then removed and nonspecific sites were blocked with 0.1 ml of blocking medium (PBS supplemented with 5% goat serum, 1% BSA, and 0.1% gelatin) for 1 h in a humidified chamber. After blocking, the medium was replaced with 0.1 ml of blocking medium containing a 1/100 dilution of the respective primary antibody for 1 h. Cells were next washed three times with blocking medium and incubated for 1 h with blocking medium containing a 1/100 dilution of the appropriate fluorescently labeled secondary antibody. For double-labeling experiments, these primary and secondary incubations were repeated with antibodies against the second protein of interest. For lectin affinity staining involving either fluorescently tagged wheat germ agglutinin or concanavalin A, these reagents were added at either a 1/250 dilution with the secondary antibody, or at 1 μg/ml in PBS for 30 min before the initial fixation and permeabilization, respectively. Fluorescent images were obtained using a Bio-Rad MRC 1024 confocal microscope. Expression levels of cells expressing CFTR were divided into two categories, those which required a gain of 1,150 or higher to visualize (low expressers) and those which required a gain setting of 950 or less (high expressers). In each case the Iris was held constant at 3.5. CMV-driven CFTR expression levels can be categorized as either low or high. At the high expression level, some cells form CFTR inclusions without lactacystin treatment. This is observed for the folding mutants as well as for wild-type CFTR. This is likely due to an overload of the cellular folding machinery and may account, in part, for the low (∼30%) maturation efficiency observed for wild-type CFTR overexpressed in cultured cells . We have therefore restricted our study to cells expressing low levels of CFTR. Due to periodic irregularities in the shape of the perinuclear structure, morphometric analysis describing their apparent diameter, as delineated by confocal imaging of immunostained HEK 293 cells, was limited to cells containing reasonably symmetric centrosomes. Measurements of diameter were performed using software supplied by the manufacturer. Three fields (×400) of each experiment were randomly selected and the diameters were measured from all the cells in each field. In the case of elliptically shaped structures, the geometric mean of the diameters were used. Data presented are reported as the mean ± SEM. 2.5 × 10 5 HeLa cells per 3-cm dish were either mock-transfected or transfected with P205S mutant CFTR expression plasmid. 48 h post-transfection, one of each was treated with 10 μM lactacystin for 12 h. Cells were washed with PBS, collected by trypsinization, and pelleted in a microfuge at 4°C. Each pellet was washed twice with PBS and resuspended in 100 μl PBS supplemented with Complete protease inhibitor cocktail ( Boehringer Mannheim ). After lysis by passage 10–20 times through a 27-gauge needle, lysates were spun at 14,000 g for 1 h at 4°C. The supernatants were collected and the pellets were washed in supplemented PBS followed by centrifugation at 14,000 g for 30 min. After aspiration of the wash solution, the pellets were resuspended in 100 μl supplemented PBS. Both pellets and supernatants were stored at –70°C until analysis. Equal volumes of each pellet and supernatant were analyzed by SDS PAGE through 4–20% gels followed by Western immunoblotting versus the indicated antibodies as described above. A central question with respect to proteasome-mediated degradation of misfolded integral membrane proteins is where in the cell does proteolysis occur. To address this question, we employed immunocytochemistry to delineate the subcellular distribution of several key components of the proteasome pathway including: the 20S proteasome, the PA700 and PA28 activator complexes, ubiquitin, and Hsp70. In HEK 293 cells, antibodies directed against each of these proteasome components identified multiple distinct subcellular pools. The 20S proteasome , the PA700 complex , and ubiquitin are each clearly discernible in both a nuclear pool and a cytoplasmic pool. In contrast, PA28-associated immunofluorescence was identified mainly in the nucleus , and Hsp70 was primarily found in a reticular pattern in the cytosol . In addition to these general distributions, staining with each of these specific antibodies revealed the existence of a unique perinuclear site in which all of the studied components concentrate . The structure is surrounded by but does not colocalize with staining against the ER lumenal chaperone BiP , and is proximal to the Golgi apparatus as illustrated by staining with the lectin, WGA . These same subcellular distributions were also observed in HeLa, COS, and CHO cells suggesting generality of the observation (data not shown). Further investigation of this perinuclear structure using double-labeled immunofluorescence revealed exact colocalization of the 20S proteasome, PA700, PA28, ubiquitin, and Hsp70 with γ-tubulin, an established centrosomal marker . The majority of γ-tubulin staining was restricted to the centrosome, although an expected faint diffuse cytosolic staining was also observed corresponding to the soluble fraction of this protein. These data implicate the centrosome as a unique site for the colocalization and concentration of the proteasomal machinery and certain cell stress chaperones under basal conditions, suggesting a novel function for the centrosome. The specificity of these proteasomal antibodies for immunocytochemistry is supported by several control experiments. First, using indirect immunofluorescence and confocal microscopy, staining with non- and preimmune rabbit sera or secondary antibody alone revealed virtually no detectable signal (data not shown). Second, preabsorption of chicken anti-PA700 antiserum against purified antigen completely eliminated immunocytochemical fluorescence . Likewise, a similar preabsorption of rabbit anti-PA28 and Western blot analysis demonstrated the specificity of this antisera. And third, staining with anti-p31, a rabbit polyclonal antibody directed against a single subunit of PA700, yielded a staining pattern that is indistinguishable from that generated with the chicken anti-PA700 complex antibodies. Similarly, staining with polyclonal antisera raised against a peptide from the α subunit of PA28 was identical to that obtained with whole anti-PA28 (data not shown). To confirm our immunocytochemical observations, centrosomes were purified from nocodazole/cytochalasin D–treated HEK 293 and HeLa cells by sucrose gradient ultracentrifugation and subjected to Western blotting . Analysis of the final purification step revealed a peak of γ-tubulin immunoreactivity in fractions 3 through 6 corresponding to sucrose densities of between 50 and 60%. In agreement with the immunocytochemical data, 20S proteasome, p31 (PA700), PA28, and Hsp70 were observed in fractions containing γ-tubulin, indicating their copurification with the centrosome in the absence of an intact cytoskeleton. In addition, Hsp90 also copurified with γ-tubulin. Interestingly, Hsp70, Hsp90, and to a lesser extent PA28, were also observed in lighter gradient fractions, where faint γ-tubulin immunoreactivity was detected. This observation may be explained either by a heterogeneous population of centrosomes or lower-affinity binding of the chaperones and PA28 to the centrosome relative to that of 20S and PA700. Sucrose fractions were devoid of other subcellular markers, including BiP (ER), aldolase (cytosol), Lamin B 1 (nucleus), and β-cop (Golgi), establishing the purity of the centrosome preparations. Treatment of cells with lactacystin, a potent and specific inhibitor of the proteasome resulted in a significant increase in size of the centrosome. Representative images of cells treated and untreated with lactacystin, were stained for PA28 and the lectin Con A . Similar results were obtained for cells stained for 20S proteasome, Ub, PA700, Hsp70, and γ-tubulin. These results were quantified by morphometric analysis of centrosome diameters (identified by fluorescent staining for γ-tubulin or perinuclear proteasomal components). Comparison of lactacystin-treated and untreated HEK 293 cells revealed a twofold increase in mean diameter in response to the proteasome inhibition . These data suggest that the centrosome is a dynamic structure, capable of expansion in response to inhibition of the proteasome. This expansion may be due to a build up of misfolded proteins, which would otherwise be degraded by the proteasome concentrated at the centrosome. To further test this hypothesis, we expressed in HEK 293 cells wild-type CFTR and two variants known to misfold, namely ΔF508 and P205S, and examined their effect on the centrosome and the proteasome machinery. Consistent with previous reports , immunolocalization of CFTR in transiently transfected HEK 293 cells clearly differentiates the wild-type CFTR subcellular locale from that of the folding mutants. Antiserum directed against the COOH terminus of CFTR recognized wild-type CFTR at the plasma membrane of transfected cells and at an early stage of maturation that colocalized with the ER-resident chaperone BiP . The ER is the site of initial membrane translocation and integration of nascent polytopic membrane proteins. In sharp contrast, ΔF508 and P205S mutant CFTR are detected predominantly in the ER of transfected cells as illustrated by the ER pattern of CFTR staining and the complete colocalization with BiP staining . Cells expressing low levels of the wild-type CFTR do not significantly perturb either the PA28 distribution or size of the centrosome, although in highly expressing cells a fraction of CFTR colocalizes within this structure as seen in Fig. 5 A. Similar results were obtained with other proteasome components studied (data not shown). In striking contrast, expression of P205S or ΔF508 (data not shown) expands the centrosome in a manner similar to that observed when cells are treated with lactacystin alone . This observation provides further support for the accumulation of misfolded proteins at the site of the centrosome even when the proteasome is functional. In cells overexpressing mutant CFTR (P205S) and treated with lactacystin, we observed the formation of large, perinuclear aggregates of misfolded CFTR which appear to arise from the centrosome as indicated by colocalization with γ-tubulin . Identical results were observed with ΔF508 expressing cells (data not shown). In addition, a remarkable and comprehensive recruitment of the cytosolic pools of the 20S proteasome , PA700 (B), ubiquitin (D), and Hsp70 (E) to the site of CFTR aggregate formation was observed. This recruitment is coincident with a depletion of the cytosolic pools implicating this as the source. PA28 is also recruited to the inclusion in response to the build up of misfolded CFTR . Morphometric analysis of the CFTR aggregate formed in lactacystin treated cells is presented in Fig. 7 F. A composite of data from Fig. 3 E describing centrosome expansion due to proteasome inhibition alone relative to nontreated cells is included for comparison. The diameter of the aggregate formed in lactacystin-treated cells expands to four to six times the size of the centrosome from which it is apparently derived. Next, to further investigate the observed redistribution of the proteasomal machinery and Hsp70 in response to the formation of lactacystin-induced intracellular inclusions of misfolded CFTR, we separated cell lysates into soluble and insoluble cellular fractions. In mock-transfected cells, γ-tubulin was observed primarily in the soluble fraction . However, in cells expressing P205S mutant CFTR and treated with lactacystin, γ-tubulin was observed distributed between the soluble and insoluble fractions. PA28, PA700, and Hsp70 redistributed in a manner parallel to γ-tubulin . An intermediate degree of redistribution was observed for mock-transfected cells treated with lactacystin and untreated mutant transfected cells (data not shown). The work reported here describes the immunocytochemical and biochemical identification of the centrosome as a unique subcellular location in which, under basal conditions, components of the proteasome proteolytic pathway and certain relevant heat shock chaperones concentrate. This localization was observed under normal growth conditions in HEK 293, HeLa, COS, and CHO cells. Moreover, the association of the proteasome with the centrosome does not require an intact F-actin nor microtubular network as indicated by their presence in the purified centrosomal fractions obtained from nocodazole/ cytochalasin D–treated cells , indicating that their localization at this site is not simply a result of clustering at the minus-ends of microtubules. This does not, however, exclude the possibility that the cytoskeleton may be required for trafficking to and from this location. When the cellular level of misfolded protein is high, either due to the overexpression of a misfolded mutant protein (such as ΔF508 or P205S CFTR) or the inhibition of the proteasome, the cell responds by expanding the diameter of the centrosome up to twofold . Assuming a spherical, three-dimensional shape, this would translate to more than a fourfold increase in its volume. Under high loads of misfolded substrate and/or insufficient proteasome activity, the centrosome, Hsp70, and the proteolytic machinery undergo correspondent redistribution to a sedimentable fraction . The presence of the perinuclear proteasome concentration in control cells argues that the simplest explanation for this expansion involves the targeting of misfolded proteins to this centralized locale for rapid and efficient degradation. Consistent with this hypothesis, the centrosomal-associated proteasomal machinery is active (Fabunmi, R.P., W.C. Wigley, P.J. Thomas, and G.N. DeMartino, unpublished observations). When degradation is insufficient the misfolded proteins accumulate at and proximal to this site eventually forming a large inclusion. In light of this finding and the proximity of the centrosome to the Golgi and lysosomes, care should be taken when interpreting subcellular localization of overexpressed proteins. It is particularly interesting that under the highest loads of misfolded protein, when the proteasome is inhibited and a mutant protein is overexpressed, the cell responds by extensively recruiting additional proteasomal machinery from the cytosolic pools to the centrosome-associated inclusion . The function of the centrosome in nucleating and organizing microtubules suggests the involvement of microtubule-based motors in the recruitment process. Consistent with this notion is the observation that the nuclear pools of proteasomal component remain unperturbed . Further experiments will be required to determine if the recruitment is an active process or simply due to diffusion although the latter possibility is unlikely because molecules the size of the 26S proteasome are too large to simply diffuse through the cytosol . Similar questions exist as to the targeting of the substrates to this site. While this manuscript was under review, an interesting and complementary study reported the formation of CFTR aggregates at the centrosomes . Formation of the aggregates required an intact microtubular system implicating a motor-based translocation of substrate to the centrosome, analogous to the potential mechanism of proteasome recruitment suggested here. Interestingly, fibrillar extensions, as observed by CFTR and proteasomal staining , radiate from the centrosomally localized aggregates. These fibrils are sensitive to treatment with nocodazole suggesting their microtubular origin. However, the centrosomal localization of the aggregates and associated proteasomal components persist in lactacystin treated cells after nocodazole treatment. The mechanisms employed for assembly and, potentially, disassembly of these structures deserves further study. Regardless of the means of assembly, it is clear that this structure concentrates and recruits proteins that would be expected to perform a censor function by monitoring and perhaps controlling the balance between folding, degradation, and aggregation of nascent membrane proteins in the cell. Understanding the composition and assembly of the proteasome-enriched centrosomes should provide new insight into the mechanisms of quality control employed by eukaryotic cells. For example, it is unclear if PA700, free or in complex with the 20S proteasome, participates directly in the recognition of the misfolded CFTR, altered localization, or the formation of the inclusions. However, it is possible that free PA700 uses its poly-Ub binding domains and other cues to identify substrates not only for the proteasome, but also for transport to the centrosome. Subsequently or coincidentally, ATP-dependent association with the 20S proteasome, PA700-mediated Ub isopeptidase activity, and other likely activities such as active unfolding would then serve to reexamine the substrate and perhaps, as an alternative to degradation, allow a second attempt at folding before degradation. Such iterative steps are known to occur in the Hsp60 class of chaperones and the Clp family of proteases . The accumulation of overexpressed mutant CFTR at the centrosome upon inhibition of proteolysis suggests that it may serve as a terminal point in the pathway of misfolded polypeptides suggesting that they either assemble or nucleate at this location. It is interesting to note that similar inclusions have been previously observed in heat-stressed , protease-inhibited cells , and a growing family of pathologies related to protein misfolding such as Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and type-1 spinocerebellar ataxia (SCA1) . The detailed structural and functional relationships among these inclusions are unknown and warrants further investigation. The centrosomal localization of the proteolytic machinery under basal conditions described in this study may play an important role in the degradation of proteins involved in progression through the cell cycle . In addition, this position also places the proteasome and chaperones proximal to the cellular organelles directly involved in the production, maturation, and trafficking of membrane proteins, a strategic locale for machinery involved in the recognition and processing of mutant proteins. Interestingly, recent studies in yeast, which lack the resolution of the current work, place the 26S proteasome at either the nuclear envelope-endoplasmic reticulum or, in fission yeast, at the nuclear periphery at rest and in a nuclear-associated spot during meiosis . Although the ER is generally considered the quality control organelle, based on BiP, WGA, and Con A staining, the current higher resolution studies indicate that in mammalian cells the proteasome concentrates in a compartment probably post-ER, lacking the complex glycoproteins of the Golgi. Dissecting the biochemical function of the centrosome should reveal if it is the site of proteasome-mediated degradation of misfolded and mutant membrane proteins. We are actively addressing these issues. | Other | biomedical | en | 0.999995 |
10225951 | Wild-type C . elegans strain N2 was obtained from the Caenorhabditis Genetics stock center. unc-60 mutants were obtained from various colleagues and their origins were described previously . All mutant animals used in this study were homozygous for each allele. A motility assay was performed as described by Epstein and Thomson . In brief, adult nematodes carrying eggs in a single row were placed in a droplet (10 μl) of M9 buffer. One beat was counted when a worm swung its head to either right or left. The total number of beats in 1 min was recorded. The data were taken only when the worms kept beating for 1 min. Genomic DNA for unc-60A (1 kb) and unc-60B (2.5 kb) were amplified from two adult worms from each strain with Pfu DNA polymerase (Stratagene Inc.) and all of the exons were sequenced with an automated sequencer (ABI373). Sequencing was performed on at least two independent PCR products to insure that there were no PCR-induced errors. After we identified the mutation sites in e677 , m35 , and r398 , we focused only on unc-60B because a fine structure map showed that the other mutations were located between e677 and m35 . Site-directed mutagenesis was performed on pET-UNC-60B , a bacterial expression construct for wild type (that expresses UNC-60B without any additional sequences), with a mutagenesis kit (Quick-change; Stratagene). Recombinant proteins except UNC-60B ( m35 ) were expressed and purified as described previously for wild-type UNC-60B . Because a large portion of UNC-60B ( m35 ) was insoluble after induction of expression, the insoluble proteins were solubilized by 6 M urea. After urea was removed by dialysis, the protein was purified with DEAE-cellulose (DE-52; Whatman), followed by gel filtration (Sephacryl S-200; Pharmacia Biotech ). The concentrations of UNC-60B proteins were spectrophotometrically determined using a calculated extinction coefficient of 8,040 M −1 cm −1 at 280 nm. Actin was purified from wild-type C . elegans as described previously . This actin preparation probably contains both muscle and nonmuscle actins. Nonetheless, we expect that most of this actin preparation consists of the muscle isoforms because of extensive extraction of nematodes in the early steps of the purification . C . elegans actin was used in this study because UNC-60B interacts with C . elegans actin differently than rabbit muscle actin . Copelleting assays of wild-type and mutant UNC-60B proteins with C . elegans F-actin were performed as described previously in a buffer containing 0.1 M KCl, 2 mM MgCl 2 , 1 mM DTT, 20 mM Hepes-NaOH, pH 7.5. Ultracentrifugation was performed with a Beckman Airfuge at 28 psi for 20 min. Control experiments were performed by replacing actin with 0.4 mg/ml BSA and nonspecific sedimentations of wild-type and mutant UNC-60B proteins were determined and subtracted from the data of the assays with actin. For the actin polymerization assays, C . elegans G-actin at 5 μM was incubated with UNC-60B proteins for 2 min in a buffer containing 2 mM Tris-HCl, 0.2 mM CaCl 2 , 0.2 mM ATP, 0.2 mM DTT, pH 8.0, and polymerization was initiated at time 0 by adding salts at final concentrations of 0.1 M KCl, 2 mM MgCl 2 , 0.2 mM EGTA, 20 mM Hepes-NaOH, pH 7.5. The kinetics of actin polymerization were monitored as changes in turbidity at a wavelength of 310 nm at 20°C. To determine the effects of UNC-60B proteins on the extent of actin polymerization, C . elegans G-actin at 5 μM was polymerized in the presence of UNC-60B proteins in a buffer containing 0.1 M KCl, 2 mM MgCl 2 , 0.2 mM EGTA, 0.2 mM ATP, 0.2 mM DTT, 20 mM Hepes-NaOH, pH 7.5, for 4 h and the concentrations of the remaining G-actin were quantified with a DNase I inhibition assay as described . It was established previously that a DNase I inhibition assay is a valid measurement of C . elegans G-actin concentration. To determine whether wild-type and mutant UNC-60B proteins sever actin filaments and increase the filament ends, effects of UNC-60B proteins on the activity of F-actin to nucleate actin polymerization were examined as described . C . elegans F-actin at 10 μM was mixed for 30 s with 0–10 μM of wild-type or mutant UNC-60B in a buffer containing 0.1 M KCl, 2 mM MgCl 2 , 0.1 mM ATP, 1 mM DTT, 20 mM Hepes-NaOH, pH 7.5. These mixtures were added as nuclei at 1.25 μM actin to 2.5 μM pyrene-labeled G-actin and the changes in fluorescence of excitation at 366 nm and emission at 384 nm were monitored over time. The rates of nucleation for actin polymerization were determined as the initial rates of the increase in fluorescence intensity. The nucleation rates in the presence of UNC-60B proteins were divided by the rate of F-actin alone and expressed as the relative nucleation rates in Fig. 5 . Pyrene-actin was prepared by labeling rabbit muscle actin with N -(1-pyrene) iodoacetamide (Molecular Probes Inc.) by the method of Kouyama and Mihashi and finally purified by gel filtration with Sephacryl S-300. The labeled G-actin was mixed with nonlabeled G-actin to make 10% pyrene-labeled G-actin and converted to MgATP–actin by incubating 5 μM of G-actin with 15 μM MgCl 2 and 0.2 mM EGTA for 3 min before the assay. Synthetic peptides that correspond to 15 amino acids from the COOH terminus of UNC-60A and UNC-60B and cysteine at the NH 2 terminus were synthesized and coupled to keyhole limpet hemocyanin by the Microchemical Facility at Emory University. The rabbit antisera against these conjugates were raised at Spring Valley Laboratories, Inc., and affinity-purified with Sepharose-6B columns to which the synthetic peptides had been immobilized. Total SDS-soluble protein extracts from each strain (mostly adults) were prepared as described in Benian et al. . Protein concentrations were determined by a filter paper dye-binding assay . 10 μg of each lysate was separated on a 15% SDS–polyacrylamide gel and transferred onto polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp. ). The membranes were blocked in 5% nonfat milk in PBS containing 0.1% Tween 20 and incubated for 1 h with anti– UNC-60A, anti–UNC-60B, or antiactin (clone C4; ICN Pharmaceuticals, Inc.) antibodies followed by treatment with peroxidase-labeled donkey anti–rabbit or mouse IgG ( Amersham Life Science). The reactivities were detected with a chemiluminescence reagent (Renaissance; NEN). Immunofluorescent staining of whole adult nematodes was performed essentially as described . Immunofluorescent staining of embryos was performed by two methods. For double staining of myosin and UNC-60B, embryos were obtained by cutting gravid adults on polylysine-coated slides, freeze-cracked as described , fixed with−20°C methanol for 5 min, washed with PBS, and incubated with antibodies. The staining of actin was performed using the method of Barstead and Waterston with slight modifications. In brief, embryos were collected by a hypochlorite treatment of gravid adults, fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with −20°C methanol for 5 min, washed with PBS and 0.5% Triton X-100, and incubated with antibodies. In addition to anti–UNC-60B, we used antimyosin A mAb (clone 5.6) and antiactin mAb (clone C4; ICN Pharmaceuticals, Inc.). To understand the function of a particular gene, availability of several different alleles allows us to correlate the properties of the gene products and the phenotypes. Viable unc-60 alleles were previously described and the homozygous mutant animals show a wide range of phenotypic severity based on mobility . Based upon our quantitative motility assay, they are from strongest to weakest alleles: e677 , m35 , s1309 , r398 , and s1307 . All of them are recessive and hypomorphic because they exhibit more severe phenotypes when placed over a deficiency , suggesting that the mutations cause partial defects in the function of the unc-60 gene product. Sequence analyses of the genomic DNA from the unc-60 mutants showed that mutations in the unc-60B but not in the unc-60A coding region cause the Unc-60 phenotypes . s1309 , m35 , and s1307 have missense mutations within a region homologous in sequence to the α-helix that contains the putative actin-binding surface of the members of the ADF/cofilin family for which structures have been solved . e723 , s1310 , and s1331 have the same mutation as m35 . The missense mutation in e677 lies close to the NH 2 terminus. r398 has a premature stop codon that truncates three amino acids from the COOH terminus. Both the NH 2 terminus and the COOH terminus of ADF/ cofilin have been implicated in binding to actin . Thus, these sequence alterations suggest that the actin-binding activity of UNC-60B is changed in the unc-60 mutants, but these alterations do not allow us to correlate the properties of the mutant proteins with the phenotypes. Therefore, we produced recombinant mutant UNC-60B proteins and examined their activities in vitro. Mutant UNC-60B proteins showed various alterations in their effects on F-actin as compared with wild type. Wild-type UNC-60B partially depolymerizes and also binds to C . elegans F-actin . The extent of depolymerization of F-actin was consistent regardless of the concentration of UNC-60B and binding of UNC-60B to F-actin was saturated at a 1:1 molar ratio. e677 , m35 , and s1309 showed weaker activities in both partial depolymerization and F-actin binding than wild type . Maximum depolymerization was achieved by 5 μM of wild type , but e677 and s1309 required 10 and 20 μM to reach the same level of depolymerization . m35 did not cause the equivalent level of depolymerization even at an extremely high concentration . These weak actin-depolymerization phenotypes probably indicate weak affinities of these mutants with ADP–G-actin. Weak binding of these mutants to F-actin was obvious because the amounts of these mutants that cosedimented with F-actin were lower than wild type at any equivalent concentrations examined here. Binding of m35 and s1309 to F-actin was so weak that saturation was not reached until 50 to 80 μM . Both r398 and s1307 showed stronger actin-depolymerizing and weaker F-actin binding than wild type . When the concentrations of these mutants were increased, more F-actin was depolymerized . In contrast, these mutants were poorly cosedimented with F-actin. However, a marked difference between these mutants was detected in the extent of depolymerization when their concentrations were substoichiometric to actin. At 5 μM of the mutants to 10 μM of F-actin, s1307 showed greater actin-depolymerizing activity than r398 . Wild-type UNC-60B strongly accelerated the rate of spontaneous polymerization of C . elegans actin . However, all the mutant proteins were less potent in this effect than wild type to different extents . In the presence of wild-type UNC-60B, the elongation phase of actin polymerization (200–1,000 s) was accelerated and the kinetics reached a plateau much earlier than actin alone . This effect was obvious when UNC-60B was present at a concentration as low as 20% of actin in a molar ratio. The increases in turbidity in the later phase (after 1,000 s) were caused by binding of UNC-60B to F-actin that resulted in an increase in the mass of actin filaments . In contrast, m35 and s1309 showed only minor effects on the kinetics when these mutants were present at 1:1 molar ratio to actin . Interestingly, r398 had an inhibitory effect on the polymerization kinetics . Although e677 and s1307 accelerated polymerization kinetics, both of them did not cause strong effects when they were present at 20% of actin, at which wild type can accelerate polymerization . In addition, acceleration by e677 was less steep than wild type at the equivalent concentrations . Acceleration by s1307 was comparable to the effect of wild type, whereas the timing when the acceleration began was later for s1307 (500–600 s) than for wild type (200 s) . Effects of wild-type and mutant UNC-60B proteins on the concentration of unassembled actin at steady state were determined quantitatively by a DNase I inhibition assay. In the presence of wild-type UNC-60B, the concentration of G-actin was increased . However, all the mutant proteins at 1 μM did not increase the concentration of G-actin in 5 μM of total actin . m35 and s1309 did not affect the level of G-actin at any concentrations examined in this study. e677 and r398 increased the G-actin level to lesser extents than wild type only when they were present at 1:1 molar ratio to actin . Therefore, m35 , s1309 , e677 , and r398 are less active than wild type in this regard. However, s1307 increased the G-actin concentration greater than wild type when s1307 was present at >50% of actin . Therefore, s1307 is less active than wild type at low concentrations, but at high concentrations it shows hyperactivity in increasing the level of unpolymerized actin. Acceleration of actin polymerization by ADF/cofilin has been interpreted as its severing activity that increases filament ends . Therefore, we tested whether wild-type and mutant UNC-60B sever actin filaments. As shown in Fig. 5 inset (closed circles), the elongation rate of actin polymerization from F-actin nuclei was increased by preincubating the nuclei with wild-type UNC-60B. This effect can be explained by fragmentation of the F-actin nuclei by UNC-60B that increased the number of filament ends . The rate was increased by 30% at a 1:1 molar ratio of UNC-60B to actin subunits, suggesting that UNC-60B weakly severs actin filaments. In contrast, the s1307 mutant remarkably increased the elongation rate to a much greater extent than wild type . There was a threefold increase in the elongation rate at a 1:1 molar ratio of s1307 to actin subunits, indicating that this mutant has much stronger actin-severing activity than wild type. The other mutants, e677 , m35 , s1309 , and r398 did not increase the elongation rates . It should be noted that r398 decreased the elongation rate , suggesting that some actin filaments were depolymerized without being severed. These data provide supportive evidence for the turbidity measurements in Fig. 3 that wild type and s1307 enhanced actin polymerization by severing actin filaments, whereas e677 , m35 , and s1309 increased the turbidity by decorating actin filaments. However, a four- to fivefold increase in the polymerization rate in the presence of wild-type UNC-60B cannot be fully explained by a weak severing activity (a maximum of 30% increase in filament ends). Therefore, wild-type UNC-60B may also enhance association of actin monomers to barbed ends of actin filaments as reported for Arabidopsis ADF1 . Our observations and interpretations are inconsistent with the report by Du and Frieden that fragmentation of actin filaments by yeast cofilin is sufficient to explain the enhanced rate of actin polymerization with the assumption that cofilin tightly binds ATP-G-actin and prevents polymerization. This inconsistency needs to be clarified by considering differences in experimental conditions, methods of analysis, and possible variances in the biochemical properties of ADF/cofilin isoforms. As tools to identify UNC-60 proteins, we produced specific antibodies against UNC-60A and B using synthetic peptides as antigens . Western blot analyses using these antibodies showed that the levels of UNC-60A were consistent among the homozygous mutant animals, whereas those of UNC-60B were variable . The mutants, m35 and s1307 , contained moderately increased levels of UNC-60B as compared with wild type . However, r398 and s1309 contained three- and fourfold larger amounts of UNC-60B as compared with wild type . In contrast, e677 had <10% (the lower limit of quantification of this Western blot) of UNC-60B as wild type . Although the band of UNC-60B in e677 is not visible in Fig. 6 B, it appeared after a longer exposure of the blot. UNC-60B was expressed in embryonic body wall muscle cells, supporting its function as an essential regulator of myofibril assembly during development. Although weak maternal expression of UNC-60B was observed (data not shown), its expression was remarkably upregulated in developing body wall muscle cells from the 1.5-fold stage , when nascent myosin filaments were formed before obvious striations of myofibrils were established . As myofibrils are assembled beneath the hypodermis in two- to threefold stages , UNC-60B was localized both in diffuse cytoplasm and striated myofibrils . The spindle-shaped outline of the muscle cells were visible by the UNC-60B staining, but the nuclei were devoid of staining . In contrast, myosin staining was limited to the myofibrils. Because of the bright UNC-60B staining of diffuse cytoplasm, localization of UNC-60B in myofibrils was visible only in some areas of the image . These data indicate that most of UNC-60B is diffusely located in the cytoplasm of embryonic muscle cells, suggesting that UNC-60B functions as a soluble regulator of actin polymerization in the cytoplasm of developing muscle cells. In wild-type threefold stage embryos, actin was localized in myofibrils and the striation patterns were evenly distributed throughout the arrays of body wall muscle cells . The staining of UNC-60B was found in body wall muscle cells in the same embryos, but it was diffuse and wider than the staining of actin, suggesting that UNC-60B was diffusely localized in cytoplasm, whereas actin is mostly assembled into myofibrils. We have noticed that the formaldehyde-methanol fixation optimized for actin staining gives less sharp staining of UNC-60B than the freeze-crack–methanol fixation . Especially, striated staining of UNC-60B and outlines of nuclei that were often clear by the freeze-crack–methanol fixation were not preserved in the formaldehyde– methanol-fixed embryos . Disorganization of myofibrils was observed in embryonic muscle cells of the unc-60 mutants . In the unc-60 mutant embryos of the threefold stage, actin was found in discrete thick bundles that were not in the normal positions of thin filaments . Although e677 shows a much more severe phenotype as adult worms than r398 , they exhibited similar degrees of disorganization of actin in embryonic stages. The weakest mutant, s1307 , also showed a high degree of disorganization of myofibrils in embryos . The aggregates of actin in s1307 were slightly larger than e677 and r398 , whereas the distribution of actin was discontinuous and was not found as organized myofibrils. In e677 , only a trace level of UNC-60B protein was detected by the antibody staining , which is consistent with the result of Western blot . However, in r398 and s1307 , the immunostaining of UNC-60B was indistinguishable from that of wild type although the staining was not quantitative. These results suggest that the proper function of UNC-60B is critical for the process of actin filament assembly into myofibrils during embryonic stages. In adult animals carrying unc-60 mutations, myofibrils of body wall muscle were highly disorganized, but exhibited a variety of disorganization depending upon the mutant alleles. In wild-type animals, myosin and actin were organized into striated sarcomeres . Some UNC-60B was localized in a striated pattern and colocalized with actin by confocal microscopy (data not shown, see Discussion for details). In contrast, in unc-60 mutant animals, large aggregates of actin were found at the ends of body wall muscle cells and myosin was also disorganized . In the most severe mutant, e677 , most of the actin was associated with the aggregates and myosin failed to form clear striations . The staining of UNC–60B was only found in small dots in the muscle cells , which was consistent with the low level of UNC-60B protein as shown by the Western blot . In the second most severe mutant, m35 , myosin was relatively aligned in striations , although it was not as tight as in wild type . However, most actin in m35 was included in the large aggregates with UNC-60B and the occurrence of actin in striations was rare . In the intermediate mutants, s1309 and r398 , actin was found in the aggregates with UNC-60B, whereas some actin formed a striated pattern . Myosin was disorganized to similar extents as m35 . The weakest mutant, s1307 , showed relatively clear striations of myosin , although the width of the striations were often greater than in wild type. The striated pattern of actin was clearer than in any of the other mutants . Aggregates of actin in s1307 were smaller than in the other mutants . However, the mutant UNC-60B was associated preferentially with the aggregates rather than the thin filaments . Thus, there was correlation between the severities in the slowness of movement and the extents of disorganization of myofibrils . However, it is likely that these various phenotypes in adult muscles were caused by effects in the growth and/or the maintenance of myofibrils, because these mutants showed similar phenotypes in embryonic stages. Here, we described an essential role of a particular isoform of ADF/cofilin for proper assembly of actin into myofibrils. This was concluded from three major findings: mutations in UNC-60B, one of the two ADF/cofilin isoforms encoded by the unc-60 gene, were sufficient to cause disorganization of myofibrils; these mutations resulted in abnormal actin-regulating activities of UNC-60B; and UNC-60B was primarily expressed in body wall muscle cells during embryonic stages. We detected three major in vitro functions for UNC-60B as an actin binding protein: F-actin binding activity, partial F-actin depolymerizing activity to maintain a high concentration of unassembled actin, and weak F-actin severing activity to accelerate actin polymerization. The combinations of biochemical and phenotypic consequences of each unc-60 mutation as summarized in Table I suggests that UNC-60B is required to maintain a dynamic state of actin filaments. The most severe phenotype of e677 is probably caused by its low level of UNC-60B protein rather than its reduced activity. The m35 mutation also caused a severe phenotype, yet UNC-60B( m35 ) protein is present at an equivalent level to that of wild type. The severity of m35 is due to its poor activity to enhance actin filament turnover. Although this mutant protein weakly binds to F-actin, it has no significant actin-depolymerizing activity (the weakest of the mutants) and no severing activity, which makes actin filaments poorly dynamic. This explanation also can be applied to s1309 , whereas a weak actin-depolymerizing activity detected in a pelleting assay and a fourfold increase in the protein level probably improves its phenotype in the adult muscle as compared with m35 . The phenotype of the r398 mutant probably is caused by a lack of actin-severing activity in the mutant UNC-60B protein that failed to increase the number of barbed ends of actin where polymerization preferentially occurs. Nonetheless, its phenotype in adult muscle is not as severe as e677 and m35 , because the r398 mutant protein has a stronger actin-depolymerizing activity than wild type probably by increasing the off rate of actin subunits from the pointed ends as previously demonstrated for other ADF/cofilins . However, this actin-depolymerizing activity is likely to be important in late development (larvae to adults), because the embryonic myofibrils of r398 were highly disorganized and nearly indistinguishable from those of e677 , suggesting that the severing activity is important during the assembly of myofibrils. In contrast, the s1307 mutation caused higher actin-depolymerizing and severing activities than wild type. Therefore, we can interpret that the phenotype of the s1307 animals are caused by overenhancement of actin turnover, suggesting that the dynamics of actin filaments need to be maintained at a finely tuned level in order to assemble and maintain an organized actin cytoskeleton. This idea is supported by a report that microinjection of a high concentration of cofilin into nascent myotubes can disrupt myofibrils and induce formation of rodlike structures containing actin . However, it remains unclear why all the unc-60 mutations cause similar phenotypes in embryonic muscle despite showing a variety of effects on the properties of UNC-60B protein. One possibility is that actin polymerization during myofibril assembly must be controlled tightly by UNC-60B, so that either hypoactive or hyperactive UNC-60B can easily disrupt the process of proper integration of actin into myofibrils. Even though the phenotypes of unc-60 mutant embryos appeared similar by immunofluorescence microscopy, the other possibility is that the nature of the aggregates of actin in the mutants might be different. Studies on yeast cofilin mutants that are defective in the actin-depolymerizing activity have shown that these mutations cause slower actin filament turnover in actin patches . Therefore, aggregated actin filaments in e677 , m35 , and s1309 may be static, but those in r398 and s1307 may still be dynamic. To understand the physiological relevance of our biochemical data, it would be informative to know the molar ratio of actin and UNC-60B in developing muscle cells. However, this is technically difficult to determine because methods to dissect embryonic worm muscle cells and/or culture systems for worm muscle are not available. The molar ratio of ADF/cofilin to actin varies from 0.17 to 0.64 in embryonic chicken tissues . In 10–d-old chicken embryonic muscles, the ratio of ADF/ cofilin (the sum of ADF and cofilin) to actin is 0.14 . Assuming that embryonic muscles of the nematode maintain the similar ratio to chicken muscles, our data at an UNC-60B to actin ratio of 0.2:1 show that wild-type UNC-60B is able to accelerate actin polymerization and partially depolymerize F-actin , but unable to significantly sever F-actin . Nevertheless, we speculate that severing activity is an important function of UNC-60B because we observed that the r398 mutation that abolished severing activity but not G-actin binding causes disorganization of myofibrils. This discrepancy is probably explained by the cooperative nature of binding between ADF/cofilin and F-actin . We preliminarily have observed that UNC-60B binds F-actin cooperatively by electron microscopy (McGough, A., and S. Ono, unpublished data). In addition, the sigmoidal curves of F-actin severing activity by wild-type and s1307 mutant UNC-60B in Fig. 5 suggest that UNC-60B severs F-actin cooperatively. Therefore, it is possible that severing of a subset of actin filaments in muscle cells is important for actin filament dynamics in vivo. Our studies on mutant UNC-60B provide important insights into the structural basis of the interaction of ADF/ cofilins with actin. Four missense mutations that we identified occur at amino acid residues that are highly conserved among ADF/cofilin species , but none of these residues have been characterized by previous site-directed mutagenesis on ADF/cofilins . Although a mutant maize ADF3, in which both Ala-104 and Tyr-103 are altered, it has been shown to reduce its affinity to both G-actin and F-actin , a role for this alanine on its own has not been characterized previously. However, we should consider that the mutations we identified here were selected for their effects on muscle function from a random mutagenesis and were not designed for a systematic mutagenesis, so that these mutations may alter or disrupt the structure of UNC-60B. It is quite informative that three mutations occur within a putative actin binding helix that has been proposed by molecular modeling and a biochemical study using synthetic peptides . Because these mutations changed small side chains (alanine or serine) to larger side chains (valine, phenylalanine, or leucine), they are likely to interfere with the interaction of the helix with actin, alter the integrity of the helix, or disrupt the intramolecular interaction of the helix with some other regions in UNC-60B. We are interested in the s1307 mutation (S112F) that causes hyperactivity. Recent structural analysis by electron microscopy suggests that this mutant alters rabbit muscle actin filament by a greater extent than wild type , which is a likely reason why this mutant causes stronger F-actin severing activity than wild-type. Currently, we are trying to understand this from molecular modeling of the structure of the mutant protein and reconstruction of the actin filaments that are decorated by the s1307 mutant. Our result that a small COOH-terminal truncation by the r398 mutation impaired F-actin binding and severing activities provides evidence that the COOH terminus of ADF/cofilin is involved in F-actin binding. This is in agreement with the mutational analysis of yeast cofilin that has shown that mutations of charged amino acids in the COOH-terminal α–helix-4 abolished only F-actin binding but not G-actin binding . However, the portion that is truncated in the r398 mutant appeared to be closer to the COOH-terminal end than these mutations in yeast cofilin based on sequence alignments . Currently, it is difficult to predict how the COOH-terminal region is involved in F-actin binding, because this region is quite divergent among ADF/cofilin species and a high resolution structure of a complex of actin and ADF/cofilin has not been determined. Also, it is important that the r398 mutation affects both F-actin binding and severing activities. This result strongly supports the idea that two actin-binding sites are required to change the twist of F-actin resulting in the destabilization of actin filaments . UNC-60B was found to be expressed in embryonic body wall muscle cells before obvious myofibrils were formed, suggesting that UNC-60B is involved in the process of myofibril assembly. Localization of UNC-60B diffusely in the cytoplasm rather than in a structural component suggests its function as a regulator of actin filament dynamics in the cytoplasm. However, we noticed that intensity and location of the immunostaining of UNC-60B was variable depending on the methods of fixation. Among the methods that we tested, freeze-crack–methanol fixation that was used in Fig. 7 gave the brightest and sharpest staining of UNC-60B and appeared to reflect the real localization of UNC-60B. Permeabilization of worms by a detergent (0.1–0.5% Triton X-100) tended to lose UNC-60B and result in unclear diffuse staining of UNC-60B. For example, the staining patterns of UNC-60B in Fig. 8 showed slightly vague images, in which embryos were fixed with formaldehyde and permeabilized with methanol and 0.5% Triton X-100 to optimize the immunostaining of actin. Therefore, although we found localization of UNC-60B to adult myofibrils in Fig. 9 , diffusely localized UNC-60B probably was lost in the course of extensive washing of fixed worms by 0.5% Triton X-100. Actually, when adult worms were fixed and permeabilized by a freeze-crack–methanol method, we observed a diffuse bright staining of UNC-60B in the cytoplasm, whereas the structures of myofibrils were not preserved well (data not shown). Our observation that UNC-60B is mainly localized in diffuse cytoplasm agrees with reports that ADF/cofilin is diffusely distributed in the cytoplasm of cultured chicken skeletal muscle cells . Our finding that mutations in UNC-60B specifically cause defects in myofibril assembly provides the first evidence of an isoform-specific requirement of ADF/cofilin for assembly of a particular differentiated cytoskeletal structure. This is, in part, explained by the muscle-specific expression of UNC-60B. In contrast, UNC-60A was detected in almost all cells in embryos and a wide variety of cells in adult worms including body wall muscle (data not shown). However, more importantly, a functional difference between UNC-60A and UNC-60B probably contributes to the specific function of UNC-60B in muscle cells. We have reported previously that these two isoforms regulate actin filament dynamics in different manners , suggesting that UNC-60B can enhance actin filament dynamics to an optimal range for proper assembly of myofibrils. This speculation is supported by our results that either hypoactive or hyperactive mutant UNC-60B can cause defects in myofibril assembly, Mammals have a muscle-specific isoform of cofilin . M-cofilin is expressed in heart and skeletal muscle and upregulated upon differentiation of cultured myogenic cell lines . Although its intracellular localization and function are yet to be characterized, we expect that M-cofilin has functional similarity to UNC-60B. However, an alignment and a phylogenetic analysis of amino acid sequences of the ADF/cofilin family has shown that UNC-60B does not exhibit outstanding similarity to M-cofilin . The sequence of UNC-60B is most related to UNC-60A and equally similar to three vertebrate ADF/cofilins (ADF, M-cofilin, and nonmuscle-type cofilin). Also, we could not detect any regions commonly unique to UNC-60B and M-cofilin. Therefore, M-cofilin probably has been evolved specifically for the structure and function of skeletal muscle in vertebrates. Nevertheless, because of the conservation of muscle components and architecture in all animals, we believe that there should be a protein, possibly M-cofilin, with a function similar to UNC-60B in mammalian cells. | Study | biomedical | en | 0.999998 |
10225952 | Mice with null mutations in the GFAP or vimentin genes were crossed to generate double mutants. These mice reached adulthood and did not display any overt anatomical or behavioral defects. Genotypes were determined as described . All the mice used in these studies were on C57BL/129 mixed genetic background and were maintained in a conventional animal facility. Mice subjected to spinal cord or brain injury were killed shortly after the injury (2 or 3 d) or were allowed to survive for 2 or 3 wk. In one group of animals the spinal cord dorsal funiculus was transected at the upper thoracic level, and the injury was extended 5 mm rostrally by a longitudinal incision . In other animals, a 27-G needle was inserted through the skull and into the frontal cerebral cortex. After both types of injury, wild-type, GFAP−/−, or vimentin−/− animals did not display any recognizable symptoms after any of the lesions. The following number of animals were used for the spinal cord injury studies (shown as number of animals allowed to survive 2 d, 2 wk): wild-type ( n = 10, 5), GFAP−/− ( n = 8, 4), vimentin−/− ( n = 2, 2), and GFAP−/−vim−/− ( n = 3, 3). For the brain injury, these animals were used (shown as number of animals allowed to survive 3 d, 3 wk): wild-type ( n = 3, 3), GFAP−/− ( n = 4, 3), vimentin−/− ( n = 2, 2), and GFAP−/−vim−/− ( n = 5, 3). Tissues were fixed by transcardial perfusion of anesthetized mice with Tyrode's solution followed by 4% formaldehyde and 0.4% picric acid in PBS or immersion fixation in Bouin's fixative (75 ml of saturated picric acid, 5 ml of glacial acetic acid, 25 ml of 40% formaldehyde). Specimens were cryo-sectioned or embedded in paraffin and sectioned using a microtome. Sections from all animals were stained with hematoxylin and erythrosin/eosin (HE) using standard protocols. Immunohistochemistry with rabbit antiserum to nestin was performed as described . Binding of primary antibodies was visualized with Cy2-conjugated donkey anti–rabbit antibodies (Jackson ImmunoResearch) or HRP-conjugated goat antiserum against rabbit immunoglobulins (Dako A/S). Peroxidase was detected with DAB (Dako A/S). Photomicrographs were scanned and mounted using Adobe Photoshop. Nestin mRNA expression in the injured spinal cord was detected by in situ hybridization using digoxigenin labeled cRNA probes as described by Schaeren-Wiemers and Gerfin-Moser . The animals were allowed to survive 4 d after the incision in the dorsal funiculus. The antisense probe was prepared with T3 RNA polymerase from a NotI linearized plasmid containing a mouse nestin cDNA corresponding to nucleotides 1630–2972 and the sense probe was generated with T7 polymerase from the same plasmid linearized with XhoI. No specific hybridization was seen with the sense probe. Two mice of each genotype were used for the bromodeoxyuridine (BrdU) incorporation experiments. 1 h after the brain injury, which was performed as described above, the mice were injected i.p. with BrdU (100 μg/g body weight). The injections were repeated 17 times for 50 h; injections were applied every second hour with a 10-h injection-free window each night. The mice were anesthetized and transcardially perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde. Vibratome or frozen sections were used for either simultaneous or sequential detection of BrdU and S-100. For BrdU detection sections were treated with 2 M HCl for 30 min followed by 0.25% pepsin in 0.1 M HCl for 2 min at 37°C before incubation with the primary antibody. Mouse monoclonal antibody and goat anti–mouse FITC-conjugated antibody were used. For S-100 immunodetection, rabbit anti–cow S-100 antibody (Z311) was used followed by swine anti-rabbit TRITC-conjugated antibody . The following genotypes and number of mice were used for ultrastructural analysis: wild-type ( n = 4), GFAP−/− ( n = 3), vimentin−/− ( n = 2), and GFAP−/−vim−/− ( n = 3). 3 d after a cortical stab wound, the mice were anesthetized and transcardially perfused with Tyrode's solution followed by 500 ml of a solution containing 2% paraformaldehyde and 2% glutaraldehyde dissolved in PBS. After postfixation overnight, the tissues were cut on an Oxford Vibratome and osmicated for 4 h, dehydrated in acetone, and embedded in Vestopal W. Ultra-thin sections (80–100 nm thick) were cut from injured brain cortex of all animals using an LKB Ultrotome. These sections measured 1.5 × 0.4 mm and included at one side the wound, covered the reaction zone, and displayed compact neuropile at the opposite side. The sections were picked up on Formvar-coated one-slot copper grids, contrasted with lead citrate and uranyl acetate, and examined in a Philips EM 400 electron microscope. Blood vessel diameters were measured in cross sections of thoracic spinal cord segments in a microscope fitted with a scale in the eyepiece. The smallest distance across an individual blood vessels was assigned as the diameter of the blood vessel. The distance from the dorsal spinal cord surface to the bottom of the indentation in the dorsal funiculus was considered as the depth of the indentation below the superficial dorsal vessel (dorsal spinal vein). The genotypes of the animals were unknown to the person performing the measurements. Blood vessel diameters and dorsal funiculus indentations were measured in at least six animals of each genotype and at least 4 sections were analyzed from each animal. The average number of blood vessels wider than 15 μm per section and the average depth of the indentation in the dorsal spinal cord was calculated for each animal, and were used to calculate the mean and standard error of the mean for animals of each genotype. Statistical significance was tested by Student's two-tailed t test. The differences were considered significant if the P values were lower than 0.01. We first analyzed the uninjured nervous system of the mutant mice. Many blood vessels appeared dilated in the brain and spinal cord of GFAP−/−vim−/− mice . The lumina of these structures were lined with von Willebrand factor–immunoreactive endothelial cells indicating that they were true blood vessels rather than, for example, cavities formed by degeneration (data not shown). Measurements of blood vessel diameters in these mice revealed an almost threefold increase in the number of blood vessels with a diameter >15 μm compared to wild-type mice . We frequently noticed that there was an unusually deep indentation in the tissue of the dorsal spinal cord in many of the mutant mice. In wild-type mice, there is often an invagination below the dorsal spinal vein, but measurements of the depth of the invagination revealed a statistically significant increase in the depth compared to wild-type mice in GFAP−/− ( P < 0.005), vimentin−/− , and GFAP−/−vim−/− mice . Endothelial and ependymal cells in the adult CNS express both nestin and vimentin , but not GFAP in wild-type mice. Nestin-immunoreactivity (IR) in these cells appeared normal in GFAP−/− mice (data not shown). However, in vimentin−/− mice, nestin-IR was reduced to very low levels in both endothelial and ependymal cells . Moreover, nestin-IR did not display a filamentous pattern but was spread diffusely throughout the cytoplasm in these cells . The same nestin-IR pattern was seen in GFAP−/−vim−/− mice (data not shown). We next analyzed scar formation in wild-type and mutant mice in response to an incision in the spinal cord dorsal funiculus. The mice were allowed to survive 2 d or 2 wk after the injury, and tissue sections from the site of the injury and from a spinal cord segment 10 mm rostral to the lesion were analyzed. There were no consistent differences in the histology of the scar tissue between wild-type, GFAP−/−, or vimentin−/− mice revealed by HE staining . However, the scar tissue was much less dense in the GFAP−/−vim−/− mice, and the scar tissue was interrupted by numerous fissures most often running in a dorso-ventral orientation . These fissures were filled with blood, tissue fluid or debris . Moreover, in the GFAP−/−vim−/− animals there was a large number of red blood cells within the scar tissue, both 2 d and 2 wk after the injury, indicating more pronounced bleeding and/or defective clearance of blood from the injury site . Nestin expression is rapidly induced in astrocytes after CNS injury and serves as a sensitive marker for reactive astrocytes . Nestin-IR was induced in response to spinal cord injury in mice of all genotypes . However, nestin-IR was more restricted in vimentin−/− or GFAP−/−vim−/− mice compared to wild-type or GFAP−/− mice . Whereas 2 d after the injury nestin-IR cells were seen at the site of the lesion and in the surrounding gray matter in wild-type or GFAP−/− mice, nestin-IR was less pronounced in the gray matter in vimentin−/− or GFAP−/−vim−/− mice . Furthermore, 2 wk after the lesion the levels of nestin-IR in the scar were lower in vimentin−/− or GFAP−/−vim−/− mice compared to wild-type or GFAP−/− mice . Whereas the scar tissue appeared dense after 2 wk in wild-type, GFAP−/− or vimentin−/− mice, the labeled cells were more sparse in GFAP−/−vim−/− mice and nestin-IR was diffusely spread throughout the cytoplasm of the cells . Nestin-IR was induced within 2 d after the injury in segments rostral to the lesion, in the degenerating axonal tract, to a similar extent in mice of all genotypes . However, 2 wk after the injury the level of nestin-IR in the degenerating tract had decreased substantially in the vimentin−/− or GFAP−/−vim−/− mice, whereas it remained strong in wild-type or GFAP−/− mice . To determine whether the reduced nestin-IR in the injured spinal cord was caused by reduced transcription of the nestin gene in vimentin−/− and GFAP−/−vim−/− mice, we performed in situ hybridization with nestin antisense riboprobes in spinal cord sections from animals that underwent a dorsal funiculus incision 4 d before. Nestin mRNA was detected in mice of all genotypes, and no reduction in nestin mRNA levels could be found in vimentin−/− and GFAP−/−vim−/− mice . Scattered cells in the dorsal funiculus expressed nestin mRNA and their distribution was highly reminiscent of the nestin-IR pattern. No specific hybridization was seen with the sense probe . Glial scar formation was also analyzed in the brain. A cortical stab wound was done with a fine needle, resulting in a much more restricted injury than the spinal cord lesion. Interestingly, 3 out of 11 GFAP−/−vim−/− mice died shortly after the injury and the necropsy showed extensive intracranial bleeding that was interpreted as the cause of death (data not shown). In the group of GFAP−/−vim−/− mice that were killed 3 d after the injury, 3 out of 5 mice showed extensive bleeding at the site of injury . Bleeding of comparable magnitude was not seen in mice of other genotypes. In all wild-type, GFAP−/− and vimentin−/− mice, as well as in the GFAP−/−vim−/− mice that survived the operation, the discrete injury caused by the needle did not cause any apparent clinical symptoms and was sealed within 3 wk . Nestin-IR was detected around the cortical lesion 3 d after the injury in mice of all genotypes . However, whereas nestin-IR was comparably strong and revealed distinct reactive astrocytes in both wild-type and GFAP−/− mice, it was weaker and diffuse in vimentin−/− and GFAP−/−vim−/− mice . Nestin-IR was reduced to undetectable levels after 3 wk in mice of all genotypes (data not shown). To further characterize scar formation in the mutant mice, we analyzed the zone immediately adjacent to the central necrotic area of cortical injury by electron microscopy. This area was similar in wild-type, GFAP−/−, or vimentin−/− mice, but differed in GFAP−/−vim−/− mice. The difference was most apparent in the border zone between the severely disarranged tissue close to the wound and the more distant, compact and normal looking brain tissue. Here, the GFAP−/−vim−/− mice exhibited fragmented tissue with a high accumulation of extracellular debris. This debris was diffuse, finely granular or filamentous of moderate electron density . This was in contrast to mice of the other genotypes in which the brain tissue in the corresponding area showed easily identifiable components (e.g., myelinated and unmyelinated axons, synaptosome-like profiles), narrow extracellular spaces and virtually no extracellular debris. To evaluate cell proliferation in the area affected by the cortical injury we have compared BrdU incorporation within a 50-h time window after the injury in wild-type, GFAP−/−, vimentin−/−, and GFAP−/−vim−/− mice. Using confocal microscopy we have counted BrdU-labeled cells within the volume of 5 × 10 6 μm 3 that included the injury area in the middle and was reconstructed from superimposed confocal images. The number of BrdU-labeled cells within this volume ranged from 64 to 87 and no statistically significant differences were found between wild-type and mutant mice. To determine the proportion of astrocytes among the BrdU-labeled cells, S-100 positive cells were counted within the same volume. The number of S-100 positive cells ranged from 65 to 83 and no statistically significant differences were found between wild-type and mutant mice. Fig. 10 provides a comparison between individual and combined BrdU and S-100 immunostaining of the cortical injury area in wild-type and GFAP−/−vim−/− mice. Both contain comparable numbers of BrdU positive, S-100 positive and double positive cells (some of these are indicated by arrows). Thus, the cortical injury in wild-type and mice deficient for GFAP and/or vimentin triggers a comparable induction of cell division. The role of astrocytic IF proteins has been the subject of numerous studies, but has been difficult to establish. In this study we have utilized mice carrying null mutations in the GFAP and/or vimentin genes. We found that GFAP and vimentin have partly overlapping functions in the astrocytic response to injury, and contribute to scar formation. Furthermore, blood vessel dilation was seen in the brain and spinal cord of mice lacking both vimentin and GFAP. In the nervous system of adult wild-type mice, nestin expression is largely restricted to endothelial and ependymal cells . Both of these cell types also express vimentin . The nestin expression pattern was distinctly altered in vimentin−/− or GFAP−/−vim−/− mice. In wild-type or GFAP−/− mice, nestin-IR in endothelial or ependymal cells exhibited a distinct pattern in contrast to the weak and diffuse nestin-IR seen in vimentin−/− or GFAP−/−vim−/− mice . Analysis of reactive astrocytes in vivo and astrocytes in culture from GFAP−/−vim−/− mice revealed the lack of IFs in these cells in spite of nestin expression, demonstrating that nestin fails to form IFs on its own in astrocytes (Eliasson et al., unpublished observations). The in situ hybridization data did not reveal any reduction in nestin mRNA expression in the spinal cord of vimentin−/− or GFAP−/−vim−/− mice compared to wild-type. This implies that the differences detected at the level of nestin-IR are posttranslational, probably caused by the changed equilibrium between unpolymerized and polymerized nestin in a situation when nestin polymerization cannot occur. This is further supported by the results from the Northern blot analysis of the RNA from astrocytes in vitro that showed rather an increase in nestin transcription in vimentin−/− and GFAP−/−vim−/− astrocytes (Eliasson et al., unpublished observations). Two-hybrid analysis has further indicated that nestin cannot bind to itself and nestin also fails to form IFs in SW13 cells devoid of other IF proteins, but incorporates into IFs once vimentin or desmin is transfected into these cells . These data suggest that the altered nestin-IR pattern observed in endothelial and ependymal cells in vimentin−/− or GFAP−/−vim−/− mice reflects the failure of nestin to form IFs in the absence of other IF proteins also in these cells. Many blood vessels were dilated in the brain and spinal cord in GFAP−/−vim−/− mice. Vimentin, but not GFAP, is normally expressed both in endothelial cells and in perivascular cells such as pericytes and smooth muscle cells . A reduction or lack of IFs in endothelial and perivascular cells may decrease the mechanical strength of blood vessels and consequently their capacity to withstand dilation. The observed increase in blood vessel diameter in the CNS of GFAP−/−vim−/− mice may be caused by altered properties of endothelial or perivascular cells (as a consequence of vimentin absence in these cells) and a reduced capacity of the surrounding tissue to withstand blood vessel dilation (the consequence of absence of GFAP and vimentin in astroglial cells). Such a concept is supported by the finding of a progressively deeper indentation beneath the dorsal spinal vein in GFAP−/−, vimentin−/−, and in GFAP−/−vim−/− mice, respectively. Scar formation after a spinal cord or brain injury appeared comparable in wild-type, GFAP−/−, or vimentin−/− mice, but was defective in mice lacking both GFAP and vimentin, indicating that these proteins may have partly overlapping functions in this process. In such mice, the glial scar was less compact, contained more debris compared to the mice of the other genotypes, and the scar tissue was disrupted by fissures. Using BrdU incorporation, we did not detect any differences in cell proliferation in the injury area. Also, the amount of astrocytes, detected as S-100 positive cells, in the injury area was comparable between the mice of different genotypes. These results imply that even in GFAP−/−vim−/− mice, the injury results in a normal response at the level of cell proliferation. Major similarities exist between the epithelial response to injury and reactive gliosis in the CNS. In both cases, production of IF proteins accompanies cell activation and the process results in the closure of the wound. In case of stratified epithelia, such as the epidermis of the skin, the wound closure happens in an orchestrated action of keratinocyte migration from the surrounding healthy tissue and a contraction of the connective wound bed mediated by migrating fibroblasts . Keratinocyte activation and migration parallel induction of the wound repair-associated IF proteins in these cells, namely keratins 6, 16, and 17, which happens within 6–12 h after injury . In the injured CNS, proliferation of stem cells is induced by injury, and the progeny of the stem cells migrate towards the injury where they differentiate to astrocytes . Thus, cell migration and wound contraction may be phenomena playing a major role in wound closure in general. Our preliminary experiments suggest that in the scrape wounding assay in vitro, cultures of GFAP−/−vim−/− astrocytes require longer time to close the defect (unpublished data). Therefore, it is possible that reactive astrocytes without IFs exhibit a cell migration deficit. To address the contractile properties of astrocytes from wild-type and GFAP and/or vimentin deficient mice, we investigated whether primary cultured astrocytes from these mice can contract collagen gels. All type of astrocytes were able to contract the gels and no quantitative differences between wild-type, GFAP−/−, vimentin−/−, and GFAP−/−vim−/− astrocytes were detected (data not shown). The defective scar formation in GFAP−/−vim−/− mice may not only be a result of astrocytic dysfunction but may also relate to the abnormal blood vessels seen in these mice. It is unlikely that the blood vessel abnormality is the major reason for defective scar formation in these mice, since scar formation appeared normal in vimentin−/− mice and only vimentin, and not GFAP, is expressed in cells of the blood vessel. The bleeding seen in GFAP−/− vim−/− mice therefore probably results from the combination of abnormal blood vessels and defective closure of the wound. The amount of debris was clearly elevated in glial scar tissue of GFAP−/−vim−/− mice compared to mice of the other genotypes. This can either be a result of defective clearance of tissue debris or its increased production. Astrocytes are capable of phagocytosis and the abundance of debris in GFAP−/−vim−/− mice may result from impairment of this function. Alternatively, the bleeding observed in GFAP−/−vim−/− mice may result in the formation of more debris in the forming glial scar and its immediate vicinity. Multiple fissures traversed through the scar in the injured spinal cord of GFAP−/−vim−/− mice. This may be a consequence of reduced astrocytic process formation with deficient bridging and sealing of the wound. Absence of IFs in astrocytes is likely to reduce the tensile strength of individual astrocytes, and as a consequence, of the tissue. A low resistance to mechanical forces is likely to inhibit scar formation, especially in the spinal cord that has to adapt to movements of the vertebral column, a situation distinct from that in the skull-enclosed brain. Irrespective of its cause, defective scar formation may lead to repeated tearing of the tissue and recurring bleeding, potentially explaining the abundance of red blood cells in the scar tissue of GFAP−/−vim−/− mice. The defective glial scar formation in GFAP−/−vim−/− mice described here reveals the importance of GFAP and vimentin for this process as well as a certain degree of functional overlap between these two proteins. | Study | biomedical | en | 0.999998 |
10225953 | S . antibioticus ATCC 11891 was used in this work. The microorganism was grown as lawns on glucose/asparagine/yeast extract (GAE) medium (containing 1% glucose, 0.1% asparagine, 0.05% yeast extract, 0.05 K 2 HPO 4 , 0.05% MgSO 4 · 7H 2 O, 0.001% FeSO 4 · 7H 2 O, 100 mM MOPS buffer [pH 7.0], and 2% agar). Plates were inoculated by spreading confluently 0.2 ml of a spore suspension , followed by incubation at 28°C. The developmental stage of the lawns was monitored by visually observing the changes in coloration of the surface of the cultures . For biochemical studies the microorganism was cultured on sterile cellophane membranes which had been overlaid previously onto the solidified culture medium. This cultivation procedure facilitates the harvesting and handling of large mycelial masses while allowing the organism to express all stages of its growth cycle . At different times of incubation, samples of the cultures (exhibiting uniformity of development) were obtained and processed for microscopy as follows. Blocks of agar containing mycelium were cut out from the culture medium and dissected into small pieces (∼3–4 mm in width and 8–10 mm in length). The pieces were fixed overnight at room temperature in 1% wt/ vol osmium tetroxide in 0.1 M veronal acetate buffer (pH 6.0), and postfixed with 0.5% wt/vol uranyl acetate in 0.1 M veronal acetate buffer (pH 6.0) for 2 h. After this, pieces were dehydrated through graded acetone solutions over a 2-h period at room temperature, embedded in Epon 812 resin and polymerized at 60°C for 36 h. Before polymerization, pieces were properly positioned to facilitate vertical sectioning of the whole mycelium. For electron microscopic observations, ultrathin sections of silver-gray interference color (thickness, 60–90 nm) were obtained with an LKB Ultramicrotome III equipped with a diamond knife and mounted on Formvar-coated copper grids. To improve contrast, ultrathin sections were poststained in the dark for 10 min on uranyl acetate drops (2% wt/ vol aqueous uranyl acetate), followed by counterstaining with lead citrate (pH 12, 1.5 min). Ultrathin sections were examined in a Philips EM300 electron microscope at an operating voltage of 60 kV, and photographed with Scientia electron microscopy film (AGFA; developed for 4 min in Kodak D19). For light microscopic observations, thin sections (∼1 μm thick) were mounted on slides, stained in Toluidine blue (0.1% wt/vol Toluidine blue in 0.1% aqueous sodium borate) for 1 min, and examined in a Nikon light microscope. For high-resolution scanning electron microscopy, agar blocks containing mycelium were fixed with osmium tetroxide (1% wt/vol in 0.1 M veronal acetate buffer, pH 6.0) for 2 h, passed through increasing concentrations of acetone, and dried to critical point with a Balzers CPD-030 apparatus. The dried samples were mounted on aluminum stubs, coated with gold by vacuum evaporation (SCD-004 sputter coat; Balzers) and examined with a Jeol JSM-6100 scanning electron microscope. High-contrast photographic negatives were digitized using a Hewlett Packard 4C slide scanner. The digitized images were imported into Scion Image (Beta 2 version for Windows 95; Scion Corp.) for digital analysis. Plots of pixel intensity from zones of interest were obtained by positioning rectangular selections (452 pixels width, 600 pixels height) over such zones, or by tracing linear selections (5 pixels width) perpendicularly to the wall, passing from resin to the middle of the cells. Plots were generated by measuring pixel intensities (intensity range of each pixel 256 gray levels [8 bits]) along the linear selections (Profile Plot function) or in the rectangular selections (Surface Plot function). By using the LUT tool, pseudocolor images were generated in which the different components of the cell displayed an arbitrarily different color. All plots were obtained from hyphae considered to be cut diametrically (i.e., showing a clear-cut cell wall profile consisting of two electron-dense zones separated by a less electron-dense zone). Final images were composed and prepared for printing by using Adobe Photoshop software (4.0 version; Adobe Systems). All the images were printed using a Epson SC-800 printer. At various times of incubation, the mycelium from three plates (8.5 cm diam) was gently scraped from the cellophane with a plastic spatula, pooled, and suspended in 10 mM potassium phosphate buffer (pH 7.0). The suspensions were then sonicated at full power for 2 min on a MSE Soniprep 150 sonicator. Samples of disrupted mycelial suspensions (1.5 ml, by triplicate) were taken, 1.5 ml of 0.5 N perchloric acid was added, and the samples were maintained for 30 min at 0°C in an ice bath. After centrifugation, the pellets were extracted three times with 0.5 N perchloric acid at 70°C. Supernatants were pooled and assayed for RNA by the orcinol method . Pellets were dissolved in 1.0 N NaOH and assayed for protein . For dry cell weight determinations, samples (1.5 ml by triplicate) of disrupted mycelial suspensions were collected in preweighted glass vials and dried at 100°C to constant weight. Standard methods were used for DNA extraction . In brief, samples of mycelium collected from cellophane membranes were suspended in a lysing solution consisting of 5 mM Tris-HCl (pH 8.0), 25% sucrose, and lysozyme (1 mg/ml). After 1 h at 37°C, the solution was successively treated with Pronase (1 mg/ml) and 1% SDS. After phenol/chloroform extraction, the DNA solution was incubated for 1 h at 37°C in the presence of RNase (40 μg/ml) and then precipitated by adding an equal volume of 100% ethanol. The precipitated DNA was washed with 70% ethanol and suspended in TE buffer. The final DNA solution was checked spectrophotometrically for purity and concentration. Equal amounts of DNA from each sample were electrophoresed in 1% agarose gels in TBE buffer at 60–80 mA for 1–2 h. The gels were stained with ethidium bromide and photographed under UV light. The morphological characteristics of hyphal death in S . antibioticus were analyzed by transmission and scanning electron microscopy. With this purpose, S . antibioticus was cultured at 28°C on GAE medium. Between 36 and 96 h of incubation, samples of the cultures were harvested and processed for electron microscopy as described in Materials and Methods. As observed previously (see below), during this period of incubation the cultures were particularly rich in hyphae undergoing cellular degeneration. These hyphae could be distinguished by the presence of large electron-transparent areas in the cytoplasm and by the aberrant shapes they displayed . From inspection of numerous hyphae at different stages of cellular degeneration, a logical progression of cytological changes for hyphal death could be deduced . The first recognizable cytological change characteristic of hyphal death was a disorganization of the nucleoid which loses its condensed, axially disposed form and expands into a large, open network of DNA. During this process, the fibrillar substructure of the nucleoid becomes progressively less electron-dense and more disorganized. As the degree of disorganization of the nucleoid increases, the cytoplasm becomes less electron-dense. Such a clearing of the cytoplasm probably results from degradation of ribosomes and other macromolecular constituents, as is suggested by the marked reduction in the RNA and protein contents which the colony undergoes during this period of development . Finally, what remains of the nucleoid is a large electron-translucent area that shows no signs of fibrillar substructure and fills most of the intracellular space of the hypha. Only small electron-dense aggregates of unknown chemical nature were present in that region of the cytoplasm. While all this is occurring, the plasma membrane shows no signs of ultrastructural disorganization and remains as a continuous, well-stained structure close to the inner face of the wall . Only after the cytoplasm has completely cleared and the fibrillar components of the nucleoid have almost disappeared does the plasma membrane retract from the wall and dissociate into a number of vesicles . Moreover, an undamaged, triple-layered cell wall was persistently seen at all stages of cellular degeneration. All of these ultrastructural changes were particularly evident in the plots of pixel intensity obtained by digital analysis of the images. As Fig. 2 shows, all the plots generated from hyphae undergoing cellular degeneration revealed progressive disorganization of the nucleoid and loss of cytoplasmic electron density with maintenance of plasma membrane and cell wall integrity. In the upper zone of the substrate mycelium and throughout the aerial mycelium, dying hyphae undergo a series of drastic changes in their morphology. As Fig. 3 shows, accompanying degeneration of the nucleoid, the hyphae collapse and undergo distortion of the hyphal shape . The end result is an aberrant hyphal structure, empty of cellular contents but still retaining a continuous, apparently intact cell wall . These morphological changes were also examined by using scanning electron microscopy. As Fig. 4 a shows, normal hyphae typically appear as long, cylindrical cells the surfaces of which are totally smooth. In degenerating hyphae, however, the wall intrudes and forms small depressions at many sites along the hypha . Then, the wall collapses and the hyphae progressively shrink, acquiring an irregular, tubular-deflated appearance . These aberrant, but otherwise characteristic, hyphal shapes probably arise as a consequence of the loss of water and cellular contents in degenerating hyphae or, more likely, of their displacement towards the growing parts of the colony (see below). A series of light and electron microscopic studies was performed to examine the time course of hyphal death during colony development in S . antibioticus . Accordingly, the microorganism was grown in GAE medium. At different times during growth, samples of the cultures were harvested, fixed, and processed for microscopy (Materials and Methods). Large semithin sections were used to examine the general organization of the colony in the light microscope. Ultrathin sections obtained from areas of interest were used for the ultrastructural characterization of the process in the electron microscope. The results obtained are shown in Figs. 5 – 8 . During the first 24 h of incubation , the substrate mycelium grew on the surface of the culture medium by forming a thin compact layer of hyphae. Within it, however, the hyphae penetrated to depths of >60 μm. Throughout these colonies, both above and within the culture medium, the hyphae were indistinguishable ultrastructurally: they appeared very electron-dense and showed no apparent symptoms of cellular degeneration . The general organization of the colonies was basically similar after 36 h of incubation . However, the presence of hyphae surrounded by a thin sheath growing upwards from the substrate mycelium revealed that the aerial mycelium begins form at this stage of development. Ultrathin sections corresponding to these colonies revealed few hyphae with symptoms of degeneration, and these appeared at or near the boundary with the culture medium . After 48 h of incubation, the colonies contained a well-developed aerial mycelium . In semithin sections, the aerial mycelium appeared as a loose network of hyphae that develop upwards into the air . Ultrathin sections through representative zones of these colonies, illustrating the vertical distribution of hyphae undergoing cell degeneration, are shown in Fig. 6 , b–d. No hyphae with symptoms of cellular degeneration were encountered in the aerial mycelium at this stage of development, where a majority of the hyphae showed a dense, heavily stained cytoplasm . Below this zone and near the boundary with the culture medium (not only above, but also within it), the substrate mycelium appeared as an intricate network of hyphae in different stages of cellular degeneration, ranging from hyphae with electron-transparent areas in their cytoplasm to hyphae in which the cytoplasmic components had completely disappeared . At the bottom of the colony (∼130 μm below the surface of the culture medium), a minority of the hyphae displayed a quite different morphological form of cell death, which was identifiable in 5–10% of the hyphae present in that region . These hyphae did not collapse nor did they undergo progressive disorganization of the nucleoid and cytoplasm with maintenance of cell wall integrity. Instead, there was an early rupture of the wall and plasma membrane followed by rapid release of cellular contents into the surrounding medium, as suggested by the almost total absence of hyphae exhibiting stages of cellular degeneration later than those shown in Fig. 6 , b and c. Only very few hyphae showing the initial stages of cell wall degradation could be examined and all these hyphae showed lightly stained cytoplasm with nucleoids of various sizes and shapes irregularly distributed through it. After 72 h of incubation, the colonies displayed abundant sporulation in the aerial mycelium . Ultrathin sections of these colonies did not reveal changes in the substrate mycelium, which appeared composed almost entirely of empty dead hyphae. In the aerial mycelium, however, a fraction of the hyphal population metamorphoses into chains of spores, while the remainder (nonsporulating hyphae) degenerates and dies. Dead hyphae appeared throughout the aerial mycelium, but they were maximally abundant towards the boundary with the substrate mycelium (data not shown). The oldest colonies examined, 5 d old, consisted entirely of dead hyphae and mature spores . All that remained in the older parts of these colonies was an intricate, pseudo-skeletal structure formed by the walls of the dead hyphae. Many such hyphae displayed aberrant morphologies, but near to the boundary with the culture medium a majority of dead hyphae retained their original tubular shape. This is probably because in such a zone of the colony the mycelium was less affected by the emergence of the aerial hyphae. Since the aerial mycelium completely depends upon translocation of water and nutrients from the substrate mycelium , its development will draw fluids from the nearest subjacent hyphae which, as a consequence of this, will undergo collapse and distortion of shape. S . antibioticus was cultured on sterile cellophane membranes overlaid on solid GAE medium. At different times during growth, samples of mycelium were carefully removed from the cellophane membranes and used either to examine the state of DNA by agarose gel electrophoresis or to estimate changes in the macromolecular content of the hyphae. As Fig. 9 a shows, DNA extracted from samples collected during the first 24 h of incubation (lanes 2 and 3) did not shows signs of degradation. However, after 36 h of incubation and continuing through later time points , DNA degradation was apparent as a smear beneath a band of high molecular weight DNA. As can be seen, DNA degradation coincided in time with the presence in the colony of hyphae undergoing nuclear degeneration . On the other hand, there were two time points at which DNA degradation reached a maximum . These corresponded to the same time points at which the colonies were found to form aerial hyphae and to differentiate into spores, respectively . As Fig. 9 c shows, over the period 12–32 h after inoculation the cultures entered a phase of rapid growth during which all the growth parameters increased steadily. This was followed by a long period of slow biomass accumulation (which began with the emergence of the aerial mycelium and extended throughout its development), during which the total contents of RNA and protein in the mycelium varied significantly: there was a marked decrease during emergence of the aerial hyphae (between 32 and 44 h of incubation), followed by a slight increase between 44 and 64 h of incubation and by a moderate but persistent decrease during the period of spore formation. As in most, if not all, bacterial systems exhibiting multicellularity , colony development in streptomycetes is maintained by a tight balance between cell proliferation and cell death processes. Much of what we currently know about hyphal death in streptomycetes comes from an early study on colony development in Streptomyces coelicolor , carried out by Wildermuth almost 30 years ago . In that work, it was reported that the mycelium undergoes extensive breakdown during the life cycle of the colony and that this process is accompanied by the presence of vacuole-like spaces in the cytoplasm , condensation of the nucleoid, and the rupture of the plasma membrane. However, this study was limited in two relevant aspects: these cytological alterations were not precisely placed within the time framework of the overall death process; and there was no information on early cytological changes and, therefore, no clear picture of the sequence of morphological events leading to hyphal death could be deduced from these observations. Since that time, hyphal death has been reported repeatedly to occur as a normal part of colony development in streptomycetes , but no attempts have been made to elucidate the mechanisms underlying this process. Moreover, despite decades of extensive morphological studies on colony development in streptomycetes, it is remarkable that we still know so little concerning the ultrastructural aspects of hyphal death. Therefore, the main objectives of this study were to characterize the sequence of ultrastructural changes leading to hyphal death in S . antibioticus , to identify the zones of the colony where they occur, and to determine the developmental time at which they appear. Our electron microscopy study has revealed some previously unrecognized aspects of hyphal death in streptomycetes. We have discerned that nucleoid degradation is a relatively early event in the hyphal death process and that this degradation clearly precedes the rupture of the plasma membrane. Moreover, we also observed that nucleoid degradation is accompanied by progressive digestion of cytoplasmic contents and distortion of the hyphal shape, and that all this occurs with maintenance of cell wall integrity and without disturbing the general organization of the colony. In addition, we have observed that the hyphae die in specific zones and at specific times during the colony life cycle. Studies on the presence and distribution of degenerating hyphae within the mycelium carried out by electron microscopy and analysis of DNA degradation carried out by gel electrophoresis revealed two rounds of hyphal death during colony development in S . antibioticus . The first round coincided with development of the aerial mycelium. It caused massive death in the substrate mycelium but had no apparent effect on the emergent aerial hyphae. The second round was not triggered until sporulation had been initiated and was more selective, since it only affected the basal, nonsporulating parts of the aerial hyphae. These observations are also interesting because they suggest that hyphal death is somehow included into the developmental program of the colony. All together, the results obtained in this study demonstrate that the hyphae of S . antibioticus do not die via autolysis (see Introduction). Instead, they undergo an orderly process of internal cell dismantling (including extensive genome digestion), followed by shrinkage and distortion of the hyphal shape that resembles PCD in animal development. However, there are some aspects of the cytology and functions of hyphal death in S . antibioticus that distinguish it from PCD in higher organisms. First, dying hyphae do not display features such as reduction in nuclear size, condensation of chromatin, and internucleosomal cleavage of DNA (which gives a characteristic ladder when analyzed by electrophoresis), as is characteristically seen in eukaryotic cells undergoing PCD via apoptosis. This is probably a consequence of the quite different ultrastructural organization of the prokaryotic nucleoid, which lacks a nuclear membrane and contains an extensively folded DNA molecule not arranged into nucleosomes . Second, dead hyphae do not completely disappear, but form part of the colony structure where they still could potentially perform two, nonmutually exclusive roles: they could provide a mechanical support for aerial hyphae to develop far from the surface of the culture medium, and they could serve as a conducting system for passage of water and solutes within the colony (a process of considerable importance for the aerial hyphae which develop in the absence of a surrounding liquid medium). Moreover, as dying hyphae provide nutrient support for development of the aerial mycelium in S . antibioticus , maintenance of cell wall integrity would allow the cytoplasmic contents of these hyphae to be degraded and reused for growth without disturbing the general architecture of the colony. In conclusion, our study has provided morphological evidence for the existence of a process of PCD in a prokaryotic organism. This adds new support to the hypothesis that the basic structure of the cell death processes has been preserved and extended throughout evolution . Establishing the mechanisms and signals that regulate such a process will be a major challenge for the future. In this respect, it is important to note that several bacterial plasmids carrying genes capable of killing their host have been reported recently to be responsible for the death of specific subpopulations of bacterial cells . Interestingly, two such plasmid-encoded killer systems seem to be present in Streptomyces spp. . Finally, the colony growth cycle of the streptomycetes provides a useful prokaryotic system for the study of the mechanism and role of cell death in development. Such studies may provide insights into the role of cell death in more complex eukaryotic systems and may also provide insights into the evolution of this important phenomenon. | Study | biomedical | en | 0.999996 |
10225954 | Quantitation of bone resorption by mature OCs was performed essentially as described by Arnett and Dempster . In brief, OCs isolated from the long bones of post-natal day 1–3 rat pups were put into suspension by curretting the entire bone in culture media (Hepes-buffered M199 pH 7.0 [ GIBCO BRL ] containing 10% FBS [Hyclone], penicillin, streptomycin, fungizone and glutamine [ GIBCO BRL ]). Cells from at least two pups were pooled together for each experiment reported. Large debris were removed by sedimentation at 1 g for 1 min and the supernatant was plated onto 4 mm × 4 mm × 400 μm bovine cortical bone slices preequilibrated with HCO 3 -buffered (1.25 g/liter), M199 in 96-well plates, or directly onto air dried FBS coated glass coverslips. The mature OCs were allowed to attach for 30 min, then most of the more abundant, but less-adherent bone marrow and bone cells were removed by vigorous washing. This procedure generates a sparse culture of cells on the bone slices or coverslips that is enriched for multinucleate, TRAP positive OCs . Varying numbers of mononuclear cells are also present; , and although their identity was not established, some of them do express α v β 3 and form actin rings (see below) and are presumably immature, mononuclear OCs. The bone slices ( n = 4 for each condition) containing mature rat OCs were placed into 24-well dishes with HCO 3 -buffered M199 control media, or the same media containing test compounds (as indicated) and were incubated for 24 h at 37°C in a humidified, 5% CO 2 /95% air atmosphere. Coverslips containing OCs were incubated in Hepes-buffered M199 (pH 6.8) in an air incubator at 37°C as indicated below. Following fixation with 0.25% glutaraldehyde, bone slices were stained for TRAP ( Sigma kit 387-A). Mature OCs were defined as highly TRAP positive cells containing three or more nuclei . The total number of rat OCs on each bone slice was counted using bright field optics on a Nikon Eclipse 800 upright microscope and a 20× objective. After counting, the OCs were removed using 50 mM NH 4 OH and brief sonication. The resorption lacunae on the same bone slices were then visualized by toluidine blue staining . Individual resorption events were distinguished by a dark border of toluidine blue stain surrounding an excavation. The data presented here record each resorption event separately; often several events are apparent in what is classically called a resorption pit . The number of resorption events were counted and area measurements were done via a calibrated MetaMorph image analyzer (Leica) coupled to a Nikon Optiphot microscope. Starting at the bottom left hand corner and working up, the entire surface area of each 4 mm × 4 mm bone slice was scanned for resorption lacunae until the entire surface area of each bone slice was examined. When a resorption lacuna came into the viewing screen, the scanning was stopped and the perimeter of each resorption event was traced . The derived area of each individual measurement was transferred to a spreadsheet. The total number of resorption events per bone slice, total area resorbed, and mean area per event were calculated. The area measurements were sorted by treatment, and the mean area and distribution of event sizes were examined through an appropriate mixed model Analysis Of Variance (ANOVA) complemented with Bonferroni's test when the difference between means was significant at alpha level equals 0.05. The corresponding P values are reported in the text. In addition to means comparison, a variance heterogeneity test was performed on the data following the Brown-Forsythe's test . This test was shown to be robust to the underlying distribution. The corresponding P values are reported in the text. All the analyses were performed using the Statistical Analysis System software (SAS Institute Inc.) on the UNIX platform. Two additional, independent experiments were performed and similarly analyzed, the results of these are presented in the text. Mature rat OCs were isolated and plated on cortical bone slices as described above. Following quantitative analysis of the toluidine blue stained bone slices, they were sonicated in water to remove the stain and any residue. They were dehydrated through a graded ethanol series and left in 100% ethanol overnight. After air drying, the slices were placed in a vacuum desiccator for several hours before being mounted on scanning EM stubs. The mounted slices were sputter coated with 30 nm of gold/palladium. The specimens were examined on a JEOL 5022 scanning EM at 25 KV with a working distance of 20 mm. Resorption lacunae from each slice were identified with scanning EM; representative examples were selected for photography at 750× magnification. For quantitative scoring of the lacunae, the entire surface of two slices per condition, from two separate experiments were analyzed ( n = 4 slices/condition). A semi-quantitative scale was used to indicate whether a resorbed area contained no exposed collagen fibrils (Ø), a few collagen fibrils (+) or many collagen fibrils (++). In addition, the resorbed areas were scored for whether they contained, 1, 2, 3, or more resorption events within the resorbed area. Mouse anti–rat β3 was purchased from PharMingen , HRP-linked rabbit anti-FITC was from DAKO, biotinylated goat anti–rabbit and biotinylated horse anti–mouse were from Vector Labs. The rabbit polyclonal anti-RANK antibodies used were generated against a recombinant RANK extracellular domain (residues 31–211) fused to human IgGγ1 Fc essentially as previously described for OPG-Fc . The rabbit antiserum was affinity purified on a RANK (31-211)-conjugated Sepharose column to generate the mono-specific anti-RANK–specific antibodies used here. Mature rat OCs were isolated and plated on FBS coated glass coverslips as described above. After a brief incubation in control media (∼2 h) the cells were fixed in cold acetone and air dried. The OCs were incubated for 2 h at room temperature with either FITC-labeled OPGL at 20 μg/ml, rabbit anti-RANK at 2.8 μg/ml, or mouse anti–rat β3 at a 1:5 dilution. HRP-linked rabbit anti-FITC, biotinylated goat anti–rabbit or biotinylated horse anti–mouse, respectively, were used to detect the primary reagent. Avidin-Biotinylated enzyme Complex (Vector Labs) was incubated with the biotinylated antibodies, and finally HRP activity was detected using DAB. The coverslips were counterstained with hematoxylin. For competition experiments, a 10-fold excess of unlabeled OPGL (200 μg/ml) was preincubated with FITC-OPGL, or anti-RANK was preincubated with 28 μg/ml of the extracellular domain of RANK both at 4°C overnight. All subsequent incubations were carried out exactly as described above. In addition, an irrelevant mouse IgG incubation served as a general negative control for the staining reagents. Mature rat OCs isolated as above were plated onto FBS-coated coverslips in Hepes-buffered M199 media (pH 6.8) containing 10% FBS. After removal of the nonadherent cells, the coverslips were allowed to incubate for ∼1 h at 37°C before treatments were initiated. Media were replaced with fresh control or test compounds in Hepes-buffered M199 media (pH 6.8) for the times indicated, then the coverslips were fixed, permeabilized, and stained with a modified version of method 1 in Lakkakorpi and Väänänen . In brief, cells were fixed for 10 min in 3% paraformaldehyde, followed by 0.2% Triton X-100 permeabilization for 10 min. A short blocking step in 5% normal goat serum was included before staining for 1 h at room temperature with 5 units/ml Texas red–labeled phalloidin (Molecular Probes). Coverslips were washed in PBS and then counter- stained with DAPI before mounting onto slides using Prolong anti-fade mounting medium (Molecular Probes). The OCs were identified on a Nikon Eclipse 800 upright microscope equipped with epifluorescence optics. OCs were defined as cells containing at least 3 nuclei, and every OC on every coverslip was counted and scored for its type of actin cytoskeletal structure by a blinded investigator. Male BDF1 mice ( n = 5) aged 6–8 wk were maintained on normal chow, or were fed low calcium chow (0.02% vs. 0.6% in standard chow) for 48 h before receiving varying doses of OPGL by intravenous injection in a PBS carrier, or PBS alone as control. Orbital blood samples for ionized calcium determination were obtained 1 h after injection from animals anesthetized with inhaled isoflurane. Blood ionized calcium levels were then determined using a Chiron Diagnostics no. 634 blood ionized calcium/pH analyzer. Data (reported as mean ± SEM) were evaluated by ANOVA with Dunnett's post hoc test to allow for multiple comparisons with control, using JMP statistical software . The sources and preparation of the protein reagents used in this study have been described in detail in Simonet et al., 1997 and Lacey et al., 1998 . In brief, recombinant murine OPGL was expressed and purified from E. coli . FITC-coupled OPGL was prepared using 6-fluorescein-5-(and 6) carboxyamido hexanoic acid succinimidyl ester (Molecular Probes) and murine OPGL as described . A human OPG -hu Fc fusion protein, expressed and purified from Cho cells , was used to inhibit the action of OPGL in various experiments. We previously reported that treatment of mature OCs on bone slices led to the generation of a dose-dependent increase in the number of resorption lacunae . This increase in resorption events could arise by a number of different mechanisms, for example, OPGL might act directly on the OCs, or indirectly through action on other cells in the culture. Whether direct or indirect, OPGL may act as an OC survival factor, and thus increase the total number of OCs in each experiment. Alternatively, OPGL might activate quiescent OCs such that more of the OCs in the culture are active, or OPGL may activate individual OCs to undergo multiple cycles of resorption during the assay period, or both. To investigate the mode of OPGL action, mature OCs isolated from neonatal rat long bones were plated onto cortical bone slices to quantitatively and qualitatively assess the functional consequences of OPGL on bone resorption. The total area resorbed on each bone slice was quantified by image analysis (see Materials and Methods). We found that OCs treated with OPGL resorb >115,000 μm 2 (mean of n = 4 bone slices) compared with untreated, control OCs which resorbed only ∼16,000 μm 2 of the bone surface area , representing about a sevenfold increase in the total area resorbed per bone slice. This marked increase in the total area resorbed was statistically significant compared with each of the controls and was completely inhibited by the addition of OPG. However, by itself, OPG treatment of OCs did not significantly alter the total area of bone resorbed compared with the untreated control . It is noteworthy that both OPG-Fc alone and OPGL plus OPG-Fc treated OCs retain a low level of basal resorption activity. Furthermore, in contrast to the induction of OC differentiation by OPGL , the activation of mature OCs by OPGL occurs in the absence of added CSF-1. In two independent replications of this experiment OPGL caused a similarly significant stimulation of bone resorption. . To address the possibility that OPGL was acting as a survival factor or inducing mononuclear OCs precursors to fuse to form multinucleate OCs during the course of the experiment, the total number of TRAP positive multinucleate OCs were counted on each bone slice at the end of the 24-h incubation period. We found that there was no significant difference in the mean total number of OCs on bone slices treated with control media, media containing OPGL, OPG or the combination of OPGL and OPG, . These results show OPGL does not act by increasing the number of multinucleate OCs in these experiments. Two independent replications of this experiment confirmed that OC numbers do not vary under these different experimental conditions, however, the number of OC per bone slice does vary between different experiments. (Experiment 2, the mean OC number per slice among the four different conditions were not significantly different, P = 0.17; experiment 3, P = 0.20). To show that the overall effect of OPGL treatment on OCs is to activate either more individual OCs, and/or to activate some individual OC more, we have also expressed the area data as the average of the resorbed area per TRAP positive OC . The data show the striking and significant stimulation of OC resorption by OPGL compared with each of the controls . We find that there is about an eightfold increase in the area resorbed per OPGL-treated OCs compared with the control OCs. In two independent replications of this experiment, there was also an approximately eightfold increase in the area resorbed per OPGL-treated OC compared with the controls. If OPGL induces mononuclear (pre)OCs in the cultures to resorb bone, we would expect to observe an increase in the number of very small resorption events compared with control cultures. On the other hand, if OPGL induces fusion of mononuclear cells, many larger resorption events may be generated. To determine whether OPGL treatment altered the area of individual resorption events, we measured the area of each excavation generated by a single round of resorption . When the resorbed areas were closely related, we were able to identify individual boundaries as darkly stained borders separating individual events or as a prominent rim in scanning EM . As seen in Fig. 3 B, the mean area per resorption event generated under the different experimental conditions is not significantly different ( P = 0.14). Furthermore, histograms of individual area measurements suggested that the overall distribution of resorption event sizes under the different conditions were quite similar, with most being between 100 and 3,000 μm 2 . Statistical analyses of the heterogeneity of variances (distribution of sizes) were performed using the Brown-Forsythe's test . The results show that the variance of the event sizes among the four groups was not different ( P = 0.135) demonstrating that there is no significant increase in the proportion of small or large resorption events. Similar analysis on the mean and variance of resorption event sizes from two additional experiments yielded similar results. (Experiment 2, test of means P = 0.24, test of variance P = 0.37; experiment 3, test of means P = 0.58, test of variance P = 0.92.) Scanning electron microscopy (scanning EM) was used to assess if there were any qualitative changes in resorption induced by OPGL. Bone slices were prepared for scanning EM and representative examples of the resorption lacunae observed are shown in Fig. 4 . Resorbed areas generated under control conditions, with OPGL and OPG together, or OPG alone were frequently single or double events. In contrast, resorption areas generated by OCs treated with OPGL were frequently connected with 3 to 7 individual resorption events visible within one contiguous resorbed area . In addition, resorbed areas generated in the presence of OPGL appeared to expose more collagen fibrils regardless of the number of connected events. The generation of physically connected resorption events indicates that single OCs are being induced by OPGL to undergo multiple resorption cycles during the 24-h assay period. To get a better idea about the magnitude of these qualitative observations, we have analyzed the appearance of all of the resorbed areas on four bone slices per condition by scanning EM. To score for exposure of collagen fibrils, we used a semi-quantitative scale where (Ø) equals no fibrils exposed , (+) equals a few fibrils exposed , and (++) equals numerous exposed fibrils . We were able to demonstrate a statistically significance difference (Ø vs. +, ++) between exposure of collagen fibrils by OPGL treated OCs ( n = 115 resorbed areas analyzed on four bone slices from two separate experiments) and the untreated control group and the OPGL vs. OPGL + OPG-Fc group, ( n = 30; P = 0.005). These same bone slices were also scored for the number of resorption events within each resorbed areas by scanning EM. Compared with each of the controls, OPGL treated OCs generated a statistically significant skew toward multiple-resorption events/resorbed area (the number of resorbed areas with 1 and 2 events were compared with those with 3–7 events). To address whether OPGL acts directly on the mature OCs we asked whether OPGL binds specifically to mature OCs, and if the recently described OPGL receptor, RANK, , is expressed by OCs. A modified, but fully active form of OPGL labeled with FITC was bound to fixed OCs on glass coverslips or on bone (data not shown). To amplify the signal from the bound OPGL-FITC, the OCs were incubated with HRP-linked anti-FITC antibody. Essentially all of the multinucleate cells were stained by OPGL-FITC as were some, but not all, of the mononuclear cells . This staining was specifically competed by the addition of excess unlabeled OPGL . To determine if the OPGL receptor, RANK is expressed on the mature OCs we used a polyclonal antibody directed against the extracellular domain of the receptor . Most of the multinucleated OCs, as well as some of the mononuclear cells are positive for RANK . To demonstrate the specificity of this antibody, excess antigen was preincubated with the antibody before incubation with the OCs . As a positive control, we stained OCs with antibodies to the well characterized OC cell surface protein, α v β 3 integrin using a β 3 integrin-specific antibody. Essentially all of the large, multinucleate OCs stained with the β 3 integrin antibody, as did some of the mononuclear cells . In contrast, cells stained with an isotype control antibody were all negative . The staining with anti-RANK is weaker than staining with either OPGL-FITC or anti-β 3 , probably because the OPGL-FITC signal was amplified (see Materials and Methods), and β 3 is expressed at very high levels on OCs. From these combined data, it appears that OPGL binds directly to mature OCs by binding the OPGL receptor, RANK which is clearly present on the mature multinucleate OCs. To resorb bone, the OC must become polarized and form a very tight seal with the substratum (bone) before protons and proteases are released into the specialized extracellular resorption compartment. Numerous laboratories have described the specialized actin ring structure that overlies this zone of tight sealing . (It is also called the clear zone as the actin network in the actin ring appears to exclude organelles from this region.) There is an excellent correlation between actin ring formation and bone resorption , and it appears that actin ring formation is required for bone resorption . Given our evidence suggesting that OPGL directly activates mature OCs we were curious to know if OPGL itself, or perhaps our polyclonal antibody to its receptor RANK would stimulate the formation of actin rings as a prerequisite to induction of the first round of bone resorption during the assay. To quantify actin ring formation in isolated OCs treated under a variety of conditions, we stained fixed OCs for F-actin with Texas red–labeled phalloidin and a blinded investigator tallied the results. Several different types of F-actin containing structures are seen in OCs under all of the experimental conditions, and a few examples are shown in Fig. 6 A. The dot-like structures have been called podosomes, these appear to be short vertical bundles of F-actin which coalesce into larger structures in a time-dependent manner . The structures we identify as partial-, full-, and multiple-actin rings are illustrated in the bottom panels of Fig. 6 A. The tallied number of these latter structures present under the different conditions were summed and the data are presented as a percentage of the total number of multinucleate OCs . About 25% of OCs in control (untreated) cultures at time zero, 30 min and 2 h contained actin rings. OPGL or anti-RANK antibody treatment for as little as 30 min increased the number of actin ring–containing OCs to ∼40–50% of the total. At 2 h, actin ring formation induced by exposure to OPGL or anti-RANK antibodies increased further, reaching ∼50–60%. To demonstrate the specificity of these effects, we used OPG to competitively inhibit OPGL or used OPG alone. Compared with untreated controls, no increase in the number of actin rings was observed under either condition. Finally, when the extracellular domain of RANK was preincubated with the anti-RANK before treatment of the OCs, no stimulation of actin ring formation was observed. As an aside, the mono- and binuclear cells were not included in the analysis shown, as they are not classically defined as (multinucleate) OCs. We did find that the binuclear cells had a similar response to OPGL (2 h control: 29% of the binuclear cells had actin rings; 2 h OPGL-treated: 59% of the binuclear cells had actin rings). It was not possible to unambiguously distinguish mononuclear OCs from other mononuclear cells in the culture, therefore, we could not determine with certainty if OPGL stimulated actin cytoskeletal rearrangements in these cells. Some mononuclear cells clearly contained podosomes and actin ring structures while others contained prominent stress fibers typical of fibroblasts, but these were present in both the control and treated cultures. Taken together, these results show that OPGL, signaling through its receptor, RANK on multinucleate OCs, induces rearrangement of the actin cytoskeleton into actin rings, a structure that is required for OC polarization and the formation of a specialized extracellular bone resorption compartment, and thus OC activity. Because OPGL stimulation of isolated OCs induced rearrangement of the cytoskeleton within 30 min, and increased bone resorption in vitro, we examined whether OPGL could rapidly stimulate bone resorption by activating preexisting OCs in vivo. Randomized groups of mice ( n = 5), were injected intravenously with OPGL at the concentrations indicated . After 1 h, the level of ionized calcium in the blood was determined as a measurement of OC activation, and the results are shown in Fig. 7 . OPGL dose-dependently increased whole blood ionized calcium levels with significant increases seen at doses of 0.05, 0.1, and 0.5 mg/kg. Since we have previously shown that 3 d treatment of mice with OPGL does not increase the number of OCs at the bone surface these results suggest that within the 1-h treatment time, enough preexisting OCs have become activated by OPGL to produce a measurable increase in ionized calcium in the blood. Maintenance of mice on a low calcium diet for 48 h before dosing with OPGL did not abrogate the significant increase in blood levels of ionized calcium (data not shown), indicating that increases in gut calcium absorption are an unlikely alternate explanation for the increased blood calcium levels. In this report we investigated the role of the newly described TNF-related protein, OPGL, in OC activation. Previously, Lacey et al. and Yasuda et al. identified OPGL/ODF as the long sought OC differentiation factor. Direct expression cloning was used independently by the two groups to identify OPGL/ODF as the ligand for OPG/OCIF. OPG is expressed only as a soluble form and is now believed to act as a soluble decoy receptor to regulate the action of OPGL on differentiation of OCs. The data presented in the two reports provide strong evidence that OPGL acts directly on a population of OC progenitors, and together with CSF-1 induces terminal differentiation into mature, active OCs. Our data also showed that OPGL activated mature OCs to resorb bone in vitro , and recent work supports our previous results . In this report we have shown that OPGL or agonist antibodies to its receptor, RANK, act directly on fully differentiated, mature OCs inducing individual OCs to undergo a rapid rearrangement of their actin cytoskeleton into actin rings and to perform multiple cycles of bone resorption as seen by scanning EM. The data demonstrate that many individual OCs are induced to perform multiple cycles of resorption during the assay period, but we also find that more of the OCs in the cultures appear to be activated as suggested by an increase in the number and density of single, isolated resorption events generated by OPGL treated OCs. Because PTH has been shown to induce OPGL expression in primary osteoblasts and osteoblastic cell lines, , it would be interesting to more carefully quantify the relationship between the size and spatial distribution of the resorption events as reported by Murrills et al. . They showed that PTH treatment of rat OC cultures primarily increased the number of resorption foci (defined as resorption lacunae lying within an area of bone covering 1/116th of a bone slice), suggesting that more of the OCs were activated in PTH treated cultures (presumably due to stimulation of OPGL expression by osteoblastic cells present in the cultures). Some of the multiple excavations we observe, especially groups of smaller resorbed areas, may be generated by single large OCs containing multiple actin rings , or by small OCs performing multiple resorption cycles in a focal region of the bone slice as the result of OPGL activation. In addition to the quantitative effects of OPGL on OCs, we also found that the resorbed bone surfaces were quite different: while resorbed areas generated by untreated OCs are fairly smooth and usually single, areas resorbed by OPGL activated OCs are frequently multiple connected excavations, which expose numerous collagen fibrils. These continuous excavations appear to be very similar to those described by Chambers et al. resulting from OC resorption on anorganic bone (hydrazine treated) compared with the intermittent resorption that occurred on whole bone. How these two observations might be related is unknown, but may reflect different OC residence times at the resorption site and/or the probability that the OC will migrate to a new location under the different conditions. It is possible that OC movement and cycle reactivation is mediated by calcium released during each resorption cycle . We found that OPG inhibited the activation of isolated OCs by OPGL in vitro, however basal OC activity was not significantly decreased by OPG alone. However, OPG caused an inhibitory trend in several independent experiments. This might be due to the presence of endogenous OPGL in the system, (either from the cell preparation, or the serum) that excess OPG would inhibit. Dempster reported similar observations at a recent meeting . Nonetheless, even in the presence of excess OPG, OCs retain a low level of basal resorbing activity suggesting that something other than OPGL is responsible for regulating the basal level of OC resorption. We cannot, however, rule out the possibility that some residual ex vivo OC activity is due to prior OPGL exposure of the OCs in vivo. The marked stimulation of bone resorption in these cultures by OPGL does not appear to be mediated by increases in the number of multinucleate OCs as their numbers did not significantly change after the various treatments. In contrast, Fuller et al. have recently reported that OPGL (TRANCE) acts as an OC survival factor in vitro, as has been well documented for CSF-1 . This apparent discrepancy may be explained by the fact that our OC survival measurements were performed on OCs cultured on bone slices, while those of Fuller et al., were on OCs cultured on untreated glass coverslips . It seems likely that under certain conditions, OPGL can act as a survival factor. Finally, in contrast to the action of OPGL on OC differentiation, activation of mature OCs on bone, or stimulation of actin ring formation on glass by OPGL occurs in the absence of added CSF-1. Initial observations that OPG treatment of growing mice induced a very rapid (3 d) increase in bone density, led us to consider that OPG might act to inhibit OC activity in addition to being an antagonist of OC differentiation . Furthermore, OPGL caused hypercalcemia within 2 d in vivo, possibly due to the activation of preformed OCs . By investigating mature OCs in culture and performing very short-term in vivo experiments, we have tried to distinguish between the role of OPGL on OC differentiation and the action of OPGL on stimulating mature OCs in culture, and preexisting OCs in vivo to resorb bone: OPGL clearly plays a role in both OC differentiation and OC activation in vitro . The interpretation of the 1 h in vivo treatment of mice with OPGL is complex, as we cannot rule out the kidney as the source of the increase in blood ionized calcium. However, we found that mice maintained on a low calcium diet for 48 h still show a significant and dose-dependent elevation in blood ionized calcium in response to OPGL (see Materials and Methods; data not shown), thus ruling out the gut absorption as the source of calcium. Given the robust and rapid activation of OCs in vitro by OPGL as evinced by both bone resorption and actin ring formation shown here, it seems most likely that OC activation is involved in vivo as well. OPGL is identical to RANKL/TRANCE, , and it has been previously suggested that RANK is its receptor on OC progenitors . Recently Hsu et al. and Nakagawa et al. provided direct evidence that OPGL exerts its activity on OC progenitors via its receptor RANK. We show here that a monospecific antibody to RANK bound to isolated multinucleate OCs demonstrating that RANK is expressed at the surface by mature OCs. In support of our result, Hsu et al. recently demonstrated that RANK mRNA is expressed by mature OCs in situ. The anti-RANK polyclonal antibody was found to activate OCs as evinced by OC polarization and formation of actin rings, in an apparently not independent manner to OPGL. The most likely explanation is that the anti-RANK antibody was acting as an agonist by binding RANK, causing receptor aggregation and signal transduction . Together these pieces of evidence implicate RANK as the relevant receptor for OPGL mediated cytoskeletal rearrangements and osteoclast activation. At this time, it is unknown how liganding RANK leads to cytoskeletal rearrangement and ultimately to activation of bone resorption in the OC, however, several signaling molecules have been specifically implicated in the cytoskeletal rearrangements associated with OC activation that may also play a role in OPGL activation of OCs. pp60 c-src is clearly a central and key component involved in activation of mature OCs. pp60 c-src is highly expressed in OCs and c-src−/− knockout mice exhibit profound osteopetrosis due to an inability of c-src−/− OCs to become polarized, form actin rings or ruffled borders; all of which are necessary for bone resorption . More recent evidence links the engagement of α v β 3 integrin via pp60 c-src (translocation and activation) to PI3 kinase activation , and association with the F-actin capping/severing protein, gelsolin . Thus for the first time, a specific cytoskeletal protein (gelsolin) and mechanism (reversal of actin capping to support further F-actin polymerization) have been implicated in OC activation by receptor engagement and cell attachment. The stimulation of RANK by OPGL appears to enhance cytoskeletal rearrangements beyond those induced by OC attachment and integrin engagement, and leads to marked stimulation of bone resorption. It will be very interesting to determine if further enhancement of this signaling pathway involving pp60 c-src and PI3 kinase or a completely separate path is responsible for OPGL-RANK–induced actin ring formation and OC activation. Recent data from several groups, suggest that signaling through RANK is mediated by binding to TRAF (TNFR-associated factor) family members. Data from Hsu et al. further suggests that JNK activation downstream of RANK/TRAF interactions may be important for OC-like cell differentiation. Events downstream of OPGL-RANK–mediated OC cytoskeletal changes remain to be investigated. In summary, OPGL binds to individual mature OC- inducing cytoskeletal changes indicative of OC activation and stimulates multiple spatially associated cycles of robust bone resorption in vitro. These effects of OPGL are very selective as they can be inhibited by the natural soluble decoy receptor, OPG, or mimicked by agonistic antibodies to the OPGL receptor, RANK. In addition, OPGL given intravenously induces a rapid increase in blood ionized calcium in mice suggesting that preexisting OCs are activated by OPGL in vivo. Based on these many pieces of evidence, we conclude that in addition to its role in OC differentiation, OPGL is a potent and direct regulator of OC activity in vitro and in vivo. | Study | biomedical | en | 0.999997 |
10225955 | The bait vectors, pBTM116-l-afadin-1 and -2 , were constructed by subcloning the inserts encoding the respective aa residues of l-afadin into pBTM116 . The yeast two-hybrid library constructed from an adult rat brain cDNA was screened using a mixture of pBTM116-l-afadin-1 and -2 as baits as described . β-Galactosidase assay was performed as described . The cDNAs of human nectin-1 and mouse nectin-2α and -2δ were obtained by PCR using human and mouse brain cDNAs as templates, respectively. Nucleotide sequence analysis was performed by the dideoxynucleotide termination method using a DNA sequencer (model 373; Applied Biosystems Inc.). The nucleotide sequence of our isolated human nectin-1 cDNA was different from the originally published sequence , but identical to the recently published sequence . Prokaryote and eukaryote expression vectors, bait vectors, and prey vectors were constructed in pGEX-KG , pCMV-Myc , pFLAG-CMV1 ( Eastman Kodak Co. ), pCAGGS , pCAGGS-FLAG, pBTM116, and pVP16-3 using standard molecular biology methods . pCAGGS-FLAG was constructed by subcloning the insert encoding the preprotrypsin-signal peptide and FLAG epitope of pFLAG-CMV1 into pCAGGS. The glutathione S -transferase (GST)-fusion vectors, containing α-, β-catenins, and the cytoplasmic region of E-cadherin (aa 734–884) , were kindly supplied by Drs. M. Itoh and S. Tsukita (Kyoto University, Kyoto, Japan). Various constructs of l-afadin and nectin shown in Fig. 1 contained the following aa: pBTM116-l-afadin-PDZ, aa 1007–1125; pVP16-nectin-2α-CP, aa 387–467; pVP16-nectin-2δ-CP, aa 403–530; GST-nectin-1-CPN, aa 379–438; GST-nectin-1-CPC, aa 449–518; GST-nectin-1-CPC-ΔC, aa 449–514; GST-nectin-2α-CP, aa 387–467; GST-nectin-2α-CP-ΔC, aa 387–463; GST-nectin-2δ-CP, aa 403–530; GST-nectin-2δ-CP-ΔC, aa 403–526; GST-l-afadin-PDZ, aa 1007–1125; pFLAG-CMV1-nectin-1, aa 27–518; pFLAG-CMV1-nectin-2α, aa 28–467; pFLAG-CMV1-nectin-2δ, aa 28–530; pCMV-Myc-l-afadin, aa 1–1829 (full-length); pCMV-Myc-nectin-1-CP, aa 379–518; pCMV-Myc-nectin-2α-CP, 387–467; pCMV-Myc-nectin-2δ-CP, aa 403–530; pCAGGS-FLAG-nectin-1, aa 27–518; pCAGGS-FLAG-nectin-1-ΔC, aa 27–514; pCAGGS-nectin-1, aa 1–518; and pCAGGS-nectin-2α, aa 1–467. Other constructs contained the following aa: pBTM116-neurabin-II, aa 145–600; and pVP16-neurexin-2α, aa 1658–1715. The GST-fusion proteins were purified by use of glutathione-Sepharose beads ( Amersham - Pharmacia Biotech Ltd.). Affinity chromatographies were done as follows. Anti-Myc epitope Ab-coupled beads were prepared by cross-linking of a mouse anti-Myc mAb with protein A–Sepharose beads ( Amersham - Pharmacia Biotech Ltd.) via dimethylpimelimidate . COS7 cells transfected with pCMV-Myc-nectin-1-CP, pCMV-Myc-nectin-2α-CP, or pCMV-Myc-nectin-2δ-CP were sonicated in a lysis buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EDTA, 10 μg/ml of leupeptin, 1 mM PMSF, and 1 μg/ml of pepstatin A). The lysate was subjected to centrifugation, and the supernatant was incubated with the anti-Myc Ab-coupled beads. After the beads were washed extensively, the beads were used as an affinity column. The purified GST-fusion protein to be tested was applied to the affinity column. After the column was washed with PBS containing 0.1% Triton X-100, the bound proteins were eluted by boiling the beads in an SDS sample buffer [60 mM Tris-Cl, pH 6.7, 3% SDS, 2% (vol/vol) 2-mercaptoethanol, and 5% glycerol]. The sample was then subjected to SDS-PAGE, followed by staining with Coomassie brilliant blue. 35 S-Labeled l-afadin blot overlay was done as described . In brief, 35 S-labeled l-afadin was generated using the TNT T7 quick coupled transcription/translation system ( Promega Corp. ) and used as a probe. The sample to be tested was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in PBS containing 5% defatted powder milk and 1% Triton X-100. The membrane was then incubated at 4°C for 16 h with 40 μl of the 35 S-labeled l-afadin probe in 1 ml of PBS containing 5% defatted powder milk and 1% Triton X-100. After the incubation, the membrane was washed with PBS containing 5% defatted powder milk and 1% Triton X-100, followed by autoradiography using an image analyzer . Rabbit antisera against nectin-1, -2α, and -2δ were raised against GST-nectin-1-CPN, GST-nectin-2α-CP, and GST-nectin-2δ-CP, respectively. These antisera were separately affinity-purified by use of the respective GST-fusion proteins covalently coupled to NHS-activated Sepharose beads ( Amersham - Pharmacia Biotech Ltd.), and used as pAbs. A rat anti–nectin-2 mAb was prepared as described . The specificity of the anti–nectin-2 mAb used here was confirmed as follows. Immunofluorescence microscopic analysis showed that an HeLa cell line stably expressing mouse nectin-2α reacted with this mAb but wild-type HeLa cells did not . In addition, when a cell lysate from mouse mammary tumor MTD-1A cells, which were metabolically radiolabeled with [ 35 S]methionine, was subjected to immunoprecipitation with this mAb, followed by autoradiography as described , a radioactive band with a molecular mass of ∼70–80 kD was specifically immunoprecipitated (data not shown). This band was recognized by another anti–nectin-2 pAb described above (data not shown). A rabbit anti–l-afadin pAb and a mouse anti–l-afadin mAb were prepared as described . Mouse and rat (ECCD2) anti–E-cadherin mAbs were purchased from Transduction Laboratories and TAKARA Shuzo, respectively. Mouse anti-Myc and anti-FLAG mAbs were from American Type Culture Collection and Eastman Kodak Co. , respectively. The bile canaliculi-rich fraction was prepared from mouse liver . This fraction was sonicated in the lysis buffer described above and subjected to ultracentrifugation. After the supernatant was diluted with buffer A (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 μg/ml of leupeptin, 1 mM PMSF, and 1 μg/ml of pepstatin A) to make a final concentration of 0.2% deoxycholate, this sample was incubated with the anti–nectin-2 mAb at 4°C for 3 h. Protein G–Sepharose beads ( Amersham - Pharmacia Biotech Ltd.) were added to this diluted sample, and incubation was further performed at 4°C for 1 h. After the beads were extensively washed with buffer A, the bound proteins were eluted by boiling the beads in the SDS sample buffer, and subjected to SDS-PAGE, followed by Western blot analysis. Immunoprecipitation experiments using cultured cells were done as follows. Mammary tumor MTD-1A cells were sonicated in buffer A. The lysate was then subjected to ultracentrifugation. The supernatant was subjected to immunoprecipitation with the anti–nectin-2 mAb as described above. For COS7 cells expressing the FLAG-tagged protein and/or the Myc-tagged protein, the cells were similarly subjected to immunoprecipitation with the anti-FLAG or anti-Myc mAb. For EL cells expressing the FLAG-tagged protein, the cells were also similarly subjected to immunoprecipitation with the anti-FLAG mAb. MDCK cells were kindly supplied by Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany). EL and L cells were kindly supplied by Drs. A. Nagafuchi and S. Tsukita (Kyoto University, Kyoto, Japan). EL cells were cloned by introduction of the exogenous E-cadherin cDNA to cadherin-deficient L cells . These cells were maintained in DME containing 10% FCS. To prepare COS7 cells transiently expressing the FLAG-tagged protein and/or the Myc-tagged protein, COS7 cells were transfected with the pFLAG-CMV1 construct and/or the pCMV-Myc construct, respectively, using the DEAE-dextran method and cultured for 2 d . To prepare EL cells transiently expressing the FLAG-tagged protein, EL cells were transfected with the pCAGGS-FLAG construct using Lipofectamine reagent ( GIBCO BRL ) according to the manufacturer's protocol. The cells were then cultured for 1 d, replated, and cultured for 4 d. An MDCK cell line stably expressing FLAG-nectin-1 was prepared as described . In brief, MDCK cells were transfected with pCAGGS-FLAG-nectin-1 using Lipofectamine reagent ( GIBCO BRL ). The cells were then cultured for 1 d, replated, and selected by culturing in the presence of 300 μg/ml of Geneticin ( GIBCO BRL ). An L cell line stably expressing full-length nectin-1 (nectin-1-L cells) or nectin-2α (nectin-2α-L cells) was similarly prepared with pCAGGS-nectin-1 or pCAGGS-nectin-2α, respectively, except that the concentration of Geneticin was increased to 500 μg/ml. Immunofluorescence microscopy of cultured cells and frozen sections of various mouse tissues was done as described . Immunoelectron microscopy of mouse intestine absorptive epithelial cells was done using the silver-enhancement technique as described . Cell aggregation assay was done according to the method described by Takeichi with slight modifications. To obtain a single-cell suspension, cells were washed with PBS, incubated with 0.2% trypsin and 1 mM EDTA at 37°C for 5 min, and dispersed by gentle pipetting. Cells were then suspended in HBSS in the presence of 1 mM CaCl 2 or 1 mM EDTA (10 6 cells/ml), placed in 12-well plates precoated with BSA, and rotated on a gyratory shaker at 37°C for indicated periods of time. Aggregation was stopped with the addition of 2% glutaraldehyde. The extent of aggregation of cells was represented by the ratio of the total particle number at time t of incubation ( Nt ) to the initial particle number ( No ). Vinculin was purified from chicken gizzard as described . Protein concentrations were determined with BSA as a reference protein . SDS-PAGE was done as described . The protein markers used were myosin (197 kD), BSA (78 kD), ovalbumin (50 kD), carbonic anhydrase (33 kD), and soybean trypsin inhibitor (28 kD). We first attempted to identify an l-afadin–binding protein(s) by use of the yeast two-hybrid method. We screened 2 × 10 7 clones of a prey cDNA library from rat brain with a mixture of two bait constructs, pBTM116-l-afadin-1 and -2 . Three independent clones, pPrey 0135, pPrey 0139, and pPrey 0140, were obtained. We focused on pPrey 0135 because this clone encoded the cytoplasmic COOH-terminal region of PRR1. pPrey 0135 encoded a 97-aa sequence which was identical to the originally identified human PRR1 (aa 421– 518) except for the absence of a single aa, glutamate, at position 438 . The clone is likely to encode a rat counterpart of human PRR1. PRR has been identified recently as the alphaherpes virus receptor, and PRR1 and -2 have been designed as HveC and HveB, respectively (Table I ). PRR was here renamed nectin. Retransformation of fresh yeast cells confirmed that pPrey 0135 bound to pBTM116-l-afadin-2 containing the PDZ domain. Analysis of other clones will be described elsewhere. l-Afadin has a PDZ domain and nectin has cytoplasmic regions with a COOH-terminal motif of 4 aa residues, E/A-X-Y-V (X indicates W, V, and M for nectin-1, -2α, and -2δ, respectively) (Table I ). It has been shown that PDZ domains bind to unique COOH-terminal motifs of 4-aa residues of integral membrane proteins . We next examined whether l-afadin specifically binds to nectin through the PDZ domain and the COOH-terminal motif. For this purpose, we constructed pBTM116-l-afadin-PDZ containing only the PDZ domain, and pVP16-nectin-2α-CP and pVP16-nectin-2δ-CP, both of which contained the cytoplasmic regions with the COOH-terminal motif . We also prepared pVP16-neurexin-2α (containing the cytoplasmic COOH-terminal region of neurexin-2α) and pBTM116-neurabin-II (containing the PDZ domain of neurabin-II) as controls. Neurexin-2α has a COOH-terminal motif similar to that of nectin, E-Y-Y-V, which has been shown to bind to the PDZ domain of CASK . Neurabin-II is an F-actin–binding protein with one PDZ domain localized at cadherin-based cell– cell AJs , although its PDZ domain–binding protein(s) has not yet been identified. We quantified the yeast two-hybrid interactions by measuring β-galactosidase transactivation. The PDZ domain of l-afadin bound to the cytoplasmic regions of nectin, but not to that of neurexin-2α (Table II ). The PDZ domain of neurabin-II did not bind to any of these proteins. We first examined the in vitro direct binding of l-afadin to nectin by affinity chromatography. A GST-fusion protein of the PDZ domain of l-afadin (GST-l-afadin-PDZ) bound to a Myc-tagged protein of the cytoplasmic region of nectin-1 (Myc-nectin-1-CP) immobilized on protein A–Sepharose beads through the anti-Myc mAb . The stoichiometry of the binding of l-afadin to nectin-1 was ∼1:1. Similar results were obtained with nectin-2α and -2δ (data not shown). To further examine the direct binding of l-afadin to nectin in vitro, GST-fusion proteins of the cytoplasmic regions of nectin were subjected to SDS-PAGE, followed by a blot overlay with 35 S-labeled l-afadin. 35 S-Labeled l-afadin bound to the GST-fusion proteins . However, when the COOH-terminal motif of 4-aa residues of each GST-fusion protein, which were shown in bold characters in Table I , were deleted, 35 S-labeled l-afadin did not bind to the GST-fusion proteins. Consistent with the earlier observation that PDZ domains bind to unique COOH-terminal motifs of 4-aa residues of integral membrane proteins , these results indicate that the PDZ domain of l-afadin directly binds to nectin and that the COOH-terminal motif of 4-aa residues of nectin is essential for this binding. We next examined the binding of l-afadin to nectin in vivo. Western blot analysis indicated that nectin-2α, but not nectin-2δ, was detected in mouse liver (data not shown). An extract from the bile canaliculi-rich fraction of mouse liver was subjected to immunoprecipitation with the anti–nectin-2 mAb, which recognizes the extracellular domains of both nectin-2α and -2δ , followed by Western blot analysis with the anti–l-afadin mAb and the anti–nectin-2α pAb. l-Afadin was coimmunoprecipitated with nectin-2α . When a cell extract from mouse mammary tumor MTD-1A cells was similarly subjected to immunoprecipitation with the anti–nectin-2 mAb, l-afadin was coimmunoprecipitated with nectin-2α and -2δ . Myc-l-afadin and FLAG-nectin-1, -2α, or -2δ were overexpressed in various combinations in COS7 cells and the cell extracts were subjected to immunoprecipitation with the anti-Myc or anti-FLAG mAb, followed by Western blot analysis with these mAbs. Myc-l-Afadin and FLAG-nectin-1 were coimmunoprecipitated . Myc-l-Afadin and FLAG-nectin-2α or -2δ were also coimmunoprecipitated (data not shown). These results indicate that l-afadin binds to nectin in vivo. We examined by immunofluorescence microscopy of frozen sections of small intestine and MDCK cells whether nectin and l-afadin are colocalized. When the frozen sections of small intestine were triply stained with the rat anti–nectin-2 mAb, the rabbit anti–l-afadin pAb, and the mouse anti–E-cadherin mAb, nectin-2 and l-afadin were concentrated with E-cadherin at the junctional complex region of intestine absorptive epithelia, but they were more highly concentrated at the junctional complex region than E-cadherin . In MDCK cells, nectin was detected with the anti–nectin-1, anti–nectin-2α, and anti–nectin-2δ pAbs by Western blot analysis, but not with these pAbs by immunofluorescence microscopy (data not shown). Canine nectin-2 in MDCK cells was not detected with the anti–nectin-2 mAb by immunofluorescence microscopy either (data not shown). Therefore, we prepared a MDCK cell line stably expressing FLAG-nectin-1. In this MDCK cell line, FLAG-nectin-1 was colocalized with l-afadin at the junctional complex region . They were more highly concentrated at the junctional complex region than E-cadherin. These results indicate that nectin is colocalized with l-afadin at the junctional complex region in epithelial cells. To examine the precise localization sites of nectin-2 at the junctional complex region of intestine absorptive epithelia, immunoelectron microscopy was performed. Nectin-2 was localized at ZA, but not at ZO or desmosome . This result is consistent with our previous observation that l-afadin is localized at ZA , and indicates that nectin is colocalized with l-afadin at ZA in epithelial cells. We next examined whether nectin and l-afadin are colocalized in nonepithelial cells. When the frozen sections of heart were doubly stained with the anti–nectin-2 mAb and the anti–l-afadin pAb, both of the proteins were colocalized at intercalated discs (cell–cell AJs) and not observed at costameres (cell–matrix AJs) . This result suggests that nectin and l-afadin are colocalized at cell–cell AJs in nonepithelial cells. To confirm this result, we examined their colocalization in EL cells expressing E-cadherin. EL cells were cloned by introduction of the exogenous E-cadherin cDNA to cadherin-deficient L cells . We have shown previously that l-afadin is colocalized with E-cadherin at cell–cell AJs in cultured EL cells . In this cell line, nectin-2 was also colocalized with l-afadin at cell–cell AJs . These results indicate that nectin is colocalized with l-afadin at cadherin-based cell–cell AJs in nonepithelial cells. We then examined the function of the interaction of nectin with l-afadin. We prepared EL cells transiently expressing the FLAG-tagged full length of nectin-1 (FLAG-nectin-1-EL cells) or the FLAG-tagged, COOH-terminal 4 aa– deleted mutant of nectin-1 (FLAG-nectin-1-ΔC-EL cells). Immunoprecipitation analysis revealed that l-afadin was coimmunoprecipitated with FLAG-nectin-1, but not with FLAG-nectin-1-ΔC . In FLAG-nectin-1-EL cells, FLAG-nectin-1 was colocalized with l-afadin and E-cadherin at cell–cell AJs . In FLAG-nectin-1-ΔC-EL cells, however, nectin-1-ΔC was not recruited to cell–cell AJs where E-cadherin was localized . Nectin-1-ΔC was not colocalized with l-afadin. These results indicate that nectin is recruited to cell–cell AJs through interaction with l-afadin in EL cells. It has been shown previously that nectin-2α and -2δ show cell aggregation activity . To first confirm this result and to then examine cell aggregation activity of nectin-1, we prepared L cells stably expressing full-length nectin-1 (nectin-1-L cells) and -2α (nectin-2α-L cells). By use of these cell lines, we examined cell aggregation activity of nectin as described . Nectin-1 as well as nectin-2α showed cell aggregation activity in a time-dependent manner . This activity was not affected by the presence or absence of Ca 2+ in the medium, indicating that cell–cell adhesion activity of nectin-1 and -2α is Ca 2+ independent. In the last set of experiments, to understand how nectin is recruited to cadherin-based cell–cell AJs through interaction with l-afadin, we examined the in vitro binding of nectin to the known components of cell–cell AJs, including α-, β-catenins, vinculin, and E-cadherin, by affinity chromatography. Under the conditions where Myc-nectin-1-CP bound to GST-l-afadin-PDZ, it did not bind to vinculin or any GST-fusion protein of α-, β-catenins, and the cytoplasmic region of E-cadherin (data not shown). Similar results were obtained with nectin-2α and -2δ (data not shown). Recently, we found that l-afadin does not bind directly to α-, β-catenin, or the cytoplasmic region of E-cadherin . Thus, although nectin is recruited to cadherin-based cell–cell AJs through interaction with l-afadin, the mechanism of this recruitment remains unknown. Nectin constitutes a family consisting of three members, nectin-1, -2α, and -2δ, and belongs to the Ig superfamily . The Ig superfamily encompasses diverse molecules that share a common structural homology . This superfamily includes CAMs and receptors for cytokines and growth factors. In contrast to cadherin, IgCAMs are far less well characterized with respect to their linkage to the actin cytoskeleton . NCAM and L1, which are major IgCAMs expressed in neural tissue, regulate neurite outgrowth and guidance by the interaction with the actin cytoskeleton . Other several IgCAMs redistribute or form “caps” on the surface of cells in an energy-dependent manner when cross-linked by divalent Abs . Capping requires the reorganization of the actin cytoskeleton, indicating that IgCAMs are linked to the actin cytoskeleton. However, no F-actin–binding protein which interacts with IgCAMs and is specifically localized at cell– cell adhesion sites has been reported. We have first shown here that l-afadin, an F-actin–binding protein, binds to the three members of the nectin family both in vitro and in vivo. The binding of l-afadin to nectin-1, -2α, and -2δ is stoichiometric and their affinities are apparently similar as estimated by the in vitro binding assay using the recombinant proteins and the immunoprecipitation experiment using COS7 cells, although it is not clear why nectin-1 shows ∼10-fold higher binding activity than nectin-2α and -2δ in the yeast two-hybrid assay. We have not directly shown here that nectin is associated with the actin cytoskeleton through l-afadin, but this possibility is likely because l-afadin binds F-actin in vitro and in vivo . We have shown here that nectin is colocalized with l-afadin at cadherin-based cell–cell AJs, but not at cell– matrix AJs, in various tissues and cell lines. Moreover, we have shown that nectin is recruited to cadherin-based cell– cell AJs through interaction with l-afadin. It is not known how these two proteins are recruited and colocalized with the cadherin-catenin system at cell–cell AJs. We have found recently that l-afadin does not directly bind to α-, β-catenin, or the cytoplasmic region of E-cadherin . We have shown here that nectin does not interact directly with any of these proteins either. These results suggest that the nectin-l-afadin system is colocalized with the cadherin-catenin system through a still unidentified factor(s). Recently, we have isolated another l-afadin–binding protein, named ponsin, which is ubiquitously expressed and colocalized with vinculin at cell–cell and cell–matrix AJs . Furthermore, ponsin binds vinculin. However, because ponsin forms a binary complex with either l-afadin or vinculin but hardly forms a ternary complex with l-afadin and vinculin, there should be an additional system which associates the nectin-l-afadin system to the cadherin-catenin system. It may be noted that, in intestinal absorptive epithelial cells where ZO, ZA, and desmosome are well separated, nectin is specifically localized and more highly concentrated at ZA than E-cadherin which is distributed along the entire lateral membrane. This unique localization is also found for vinculin , l-afadin , and ponsin . ZO and ZA in the junctional complex of polarized epithelial cells are closely aligned from the apical side to the basal side, suggesting that there are molecular interactions between these two junctional structures. Evidence is accumulating that the cadherin-catenin system plays essential roles for the assembly of the junctional complex . It has been shown recently by use of an α-catenin–deficient colon carcinoma cell line that the interaction of α-catenin with vinculin is required for the organization of ZO . Furthermore, it has been shown that the junctional organization is impaired in vinculin-null F9 cells . The unique localization properties of nectin, l-afadin, ponsin, and vinculin suggest that the nectin-l-afadin system plays a role in the assembly of the junctional complex in cooperation with the cadherin-catenin system. We have confirmed that nectin-2α has cell–cell adhesion activity as described and have shown that nectin-1 also has this activity. In contrast to cadherin, most IgCAMs regulate cell–cell adhesion in a Ca 2+ -independent manner. Consistently, both nectin-1 and -2 show Ca 2+ -independent cell–cell adhesion. These results indicate that nectin is a Ca 2+ -independent CAM which is associated with l-afadin and specifically localized at ZA in epithelial cells and at cadherin-based cell–cell AJs in nonepithelial cells. Nectin-1 and -2 have been shown to be expressed in most tissues examined thus far . We have found here that the three members of the nectin family are expressed in MDCK cells. It remains to be clarified why the different nectin family members are expressed in the same cells, but the three members of the nectin family may be functionally redundant because of their common properties, including Ca 2+ -independent cell–cell adhesion activity, l-afadin–binding activity, and localization at cadherin-based cell–cell AJs. We have analyzed here the binding regions of l-afadin and nectin and found that the PDZ domain of l-afadin and the cytoplasmic regions of nectin directly interact with each other. PDZ domains are modular domains that bind to specific COOH-terminal peptide sequences . Many PDZ domain–containing proteins and their binding partners have been isolated recently, and peptide sequences for various PDZ domains have been reported. Using the oriented peptide library technique, PDZ domains are assigned into classes according to their peptide-binding specificities . The PDZ domain of AF-6 (s-afadin) is classified as the class II, selecting peptides with hydrophobic or aromatic aa residues at position −2 relative to the COOH terminus. The PDZ domain binds preferentially to a peptide which terminates in the sequence, E-F-Y-V . Nectin terminates in the sequence, E/A-X-Y-V (X indicates W, V, and M for nectin-1, -2α, and -2δ, respectively). Our finding is consistent with these earlier observations , but we have shown here by the yeast two-hybrid assay that the PDZ domain of l-afadin does not bind to neurexin-2α which terminates in the sequence, E-Y-Y-V. A recent study of the third PDZ domain of PSD-95/SAP90 indicates that X residues at position −1 in the consensus sequence (X-S/T-X-V) and the upstream residues of the tetrapeptide determine the specificity and affinity for the binding of the PDZ domain to its binding partner . By analogy, unique aromatic or hydrophobic X residues at position −2 in the sequence (E/A-X-Y-V), such as W, V, and M, may be necessary to bind to the PDZ domain of l-afadin. It is also possible that the upstream residues of the tetrapeptide are crucial for the specificity and affinity for the PDZ domain. It has been shown recently that the PDZ domain of AF-6 (s-afadin) binds to neurexin as well as the Eph receptor tyrosine kinase family members , but this result is not consistent with ours and the reason for this discrepancy is not known at present. | Study | biomedical | en | 0.999998 |
10225956 | Mouse mAbs HECD-1 and SHE78-7 to human E-cadherin (Takara Shuzo Co., Ltd.), rat mAb NCD-2 to chicken N-cadherin , mouse mAb to p120 ctn (Transduction Laboratories), mouse mAb M2 to FLAG , rabbit polyclonal antiserum to FLAG (SC-807; Santa Cruz Biotechnology, Inc. ), mouse mAb to αE-catenin (Transduction Laboratories), rat mAb α18 to αE-catenin , mouse mAb 5H10 to β-catenin , and rabbit polyclonal antiserum to β-catenin were used. Anti-MUC1 antibody MY.1E12 was a gift from T. Irimura (University of Tokyo, Tokyo, Japan). Antibodies used for detection of primary antibodies were as follows: goat Cy-3–labeled species-specific antibody to mouse IgG (AP-124C; Chemicon International, Inc.), donkey biotinylated species-specific antibody to rabbit IgG , FITC-labeled streptavidin , sheep HRP-linked species-specific antibody to mouse IgG and rat IgG (NA932; Nycomed- Amersham ), goat HRP–linked antibody to rabbit IgG (NA934; Nycomed- Amersham ), and Sepharose 4B–linked goat antibody to mouse IgG . The following protein kinase inhibitors were used: staurosporine (#19-123; Upstate Biotechnology), calphostin C (C-159; Research Biochemicals Inc.), tyrphostin (EI-215; Biomol), genestein (G-103; Research Biochemicals International), and herbimycin A (OP-12, Kyowa Medex Co., Ltd.). UCN-01 was a kind gift of T. Tamaoki (Kyowa Medex Co., Ltd.). O -sialoglycoprotein endopeptidase was purchased from Cedarlane Labs., Ltd. Human colon carcinoma cell lines Colo 205 and HT-29 , and MDCK cells were used. These cells were cultured in a 1:1 mixture of DME and Ham's F12 supplemented with 10% FCS (DH10). For transfer of Colo 205 cells, an aliquot of the suspended cell culture was moved to new dishes with fresh DH10 medium. When necessary, they were trypsinized as described below. For HT-29 and MDCK cells, they were rinsed with 1 mM EDTA in Ca 2+ - and Mg 2+ -free saline, then treated with 0.05% crude trypsin and 1 mM EDTA in the same solution for 5 min at 37°C, and finally suspended in DH10. Colo 205 cells were transfected by electroporation or by use of adenoviral expression vectors. For electroporation, trypsinized cells (4 × 10 5 ) were suspended in 200 ml of Hepes-buffered (pH 7.4) saline with 1 mM Ca 2+ and 1 mM Mg 2+ (HBS). 20 μg of an expression vector was added to the suspension, which was electrified at 1,160 μF at 250 V. For adenovirus-mediated transfection, 10 6 cells were suspended in 500 μl of DH10 with 5 × 10 7 plaque-forming units of adenovirus, and incubated for 4 h. Cells were washed twice with DH10, and after 48 h samples were collected. MDCK cells were transfected by electroporation under the same conditions as for Colo 205 cells, except that 1,060 μF at 200 V was used. HT-29 cells were transfected with adenoviral vectors only. Colo 205 cells were washed twice with Ca 2+ - and Mg 2+ -free Hepes-buffered saline supplemented with 1 mM EDTA, and completely dissociated into single cells by pipetting. Then, 2 × 10 5 cells were resuspended in 1 ml of DH10 with or without 1 μg/ml of SHE78-7, a blocking antibody to human E-cadherin, and placed in a Nunc 6-well plate . Cells were incubated for 3 h at 37°C on a gyratory shaker at 80 rpm. For trypsin treatment to induce cell adhesion, cells were rinsed twice with DH (DH10 without FCS), and incubated in DH with crystalline trypsin of various concentrations at 37°C. For biochemical analysis, the incubation was generally stopped at 30 min, and the cells were rinsed twice with HBS containing 0.1% trypsin inhibitor and subjected to further analysis. 0.01% trypsin in the presence (TC) or absence (TE) of 1 mM Ca 2+ treatments were performed as described previously . Using mouse p120 ctn cDNA as a template, we generated FLAG-tagged p120 ctn (FLf) 1 and other constructs by PCR. The following primers were used: primer N, 5′-GAATTCATGGACGACTCAGAGGTG-3′; primer A, 5′-GAATTCATGTTAGCAAGCTTGGATAGTTTG-3′; primer CR, 5′-GAATTCGATATC CTA CTTGTCATCGTCGTCCTTGTAGTC AATCTTCTGCATCAAGG-GTGC-3′; primer ΔAR, 5′-CTGCAGGAAATCCACTGTATCATT-3′ Primer CR and ΔAR were antisense primers. The primer CR contained an EcoRV restriction site, and complementary sequences for stop codon and FLAG epitope DYKDDDDK at the 5′ portion, as underlined. Primers N and CR were used for amplifying FLf, and primers A and CR for ΔN346f, a mutant molecule in which the NH 2 -terminal 346 amino acids (aa) of p120 ctn were deleted. For construction of other NH 2 -terminal deletion mutants ΔN101f, ΔN157f, ΔN244f, and ΔN323f, the fragment FLf was internally digested with BglII/EcoRV, NcoI/EcoRV, SmaI/ EcoRV, and Bsu36I/EcoRV, respectively. We assume that translation of the constructs will start at the next ATG downstream of the deletion. For FLΔRf and ΔN346ΔRf, in which aa 641–819 were deleted, oligonucleotides corresponding to aa 346–640 were synthesized by PCR by use of primer A and ΔAR, digested by PstI, and inserted into the internal PstI sites of FLf or ΔN346f. The obtained fragments were subcloned into the pCA-pA expression vector and pAdV-CA-pA adenoviral construction vector . For construction of cN/JM(−), encoding a chicken N-cadherin mutant in which the juxtamembrane portion of the cytoplasmic region is deleted, the catenin binding region with COOH-terminal 70 aa and the extracellular–transmembrane region were amplified by PCR, respectively, using pCMV-cN/FLAG-pA as a template. For amplifying the catenin binding region, primers 5′-CGCCGTACGACCATG AGATCT AATGAGGGACTTAAAGCAGCC-3′ and 5′-GCGGCCGCTTACTTGTCATCGTCGTCCTTGTAGTC-3′ were used. For the extracellular–transmembrane region, primers 5′-GCGCGTACGACCATGTGCCGGATAGCGGGAACGCCG-3′ and 5′-CGCG AGA TCT CTGACGCTCCTTATCCGGCG-3′ were used. Obtained products were linked through the BglII sites underlined above, and subcloned into pCA-pA or pAdV-CA-pA. All PCR-generated fragments of DNA obtained above were completely sequenced, and no sequence error was detected. Recombinant adenovirus AdV-CA-lacZ expressing β-galactosidase with the CAG promoter was a gift from K. Moriyoshi (Kyoto University, Kyoto, Japan). Construction of recombinant viruses was performed according to methods described previously . In brief, HEK 293 cells cultured with DH10 in 6-well plates (Iwaki Co.) were cotransfected with viral genome fragments (0.2 μg) and linearized adenoviral shuttle vector plasmids (1 μg) by use of Lipofectamine™ . The next day, the cells were divided into collagen-coated 24-well plates (Iwaki Co.). 10 d later, wells became full of dead cells, caused by viral propagation, and the debris was screened for proper protein expression by immunostaining and immunoblotting with the anti-FLAG antibody M2. We obtained AdV-CA-FLf, AdV-CA-ΔN101f, AdV-CA-ΔN157f, AdV-CA-ΔN244f, AdV-CA-ΔN346f, which expressed different forms of p120 ctn proteins under the control of the CAG promoter , and AdV-cN/JM. The full-length N-cadherin expression vector AdV-Ncad was described previously . The recombinant adenoviruses were amplified, and purified by CsCl 2 step gradient centrifugation . FCS was added to the purified adenovirus solutions at a final concentration of 10%. Aliquots of the virus solution were stored at −80°C until used. Colo 205 cells were removed from dishes by pipetting, rinsed twice in HBS, and lysed in TBS-Ca (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM CaCl 2 ) supplemented with 1% Triton X-100, 1% NP-40, 1 mM PMSF, 1 mM NaVO 3 , and 50 mM NaF (TBS-Triton) for 30 min at 4°C on a rocking platform. The soluble fraction was collected by centrifugation and preincubated with 50 μl of secondary antibody–conjugated beads and 5% BSA on ice for 20 min. After preincubation, incubations with primary and secondary antibodies were sequentially performed on ice for 1 h each, followed by washing three times with TBS-Triton. Finally, samples were boiled in 150 μl of SDS sample buffer with 7.5 μl of β-mercaptoethanol for 5 min. Proteins were separated in SDS-PAGE and electrophoretically transferred to membranes. The transferred proteins were detected by the enhanced chemiluminescence system . Immunohistochemical detection of cadherin and catenins was performed as described in Watabe-Uchida et al. . Immunoprecipitated materials were washed twice with phosphatase reaction buffer (50 mM Tris-HCl, pH 8.5, 50 mM MgCl 2 , 1 mM PMSF, 1 mM DTT, 0.1% Triton X-100, 0.1% NP-40). 27 μl of the reaction buffer and 3 μl of alkaline phosphatase (#567-744; Boehringer Mannheim GmbH ) were added to the precipitate, and the mixture was then incubated at 30°C for 1 h. After incubation, 1 μl of 0.5 M EDTA and 30 μl of 2× SDS sample buffer were added, and the mixture was boiled for 5 min. For 32 P-metabolic labeling, cells were cultured in phosphate-free DME with 0.5 mCi of 32 P (NEX-053, New England Nuclear Life Science Products, Inc.) for 24 h. Then, cells were harvested and trypsinized when necessary. From their detergent extracts prepared as above, p120 ctn was immunoprecipitated and separated by SDS-PAGE. From the gels, the labeled p120 ctn -protein band was excised after comparing with their autoradiograms, and the collected gel pieces were homogenized in 500 μl of freshly prepared 50 mM NH 4 HCO 3 . After addition of 25 μl β-mercaptoethanol and 5 μl 10% SDS, the samples were boiled for 5 min and agitated at room temperature for 2 h. Supernatants were collected after centrifugation at 15,000 rpm for 5 min, mixed with 20 μg RNase A and 250 μl ice-cold TCA (100% wt/wt), and incubated on ice for 1 h. After centrifugation, the pellets were air-dried and proteins were digested by incubating with 50 μl 6 N HCl at 110°C for 90 min. Digested products were air-dried again and resuspended in buffer (2.2% formic acid, 7.8% glacial acetic acid), pH 1.9, with unlabeled phosphoserine, phosphothreonine, and phosphotyrosine. Samples were spotted on TLC plate and separated by two-dimensional electrophoresis at 1.5 kV for 40 min with the pH 1.9 buffer, and at 1.0 kV for 30 min with buffer (5% glacial acetic acid, 0.5% pyridine), pH 3.5. Finally, labeled phosphoamino acids were visualized by the BAS-1000 image analyzing system (FUJIX Inc.). Colo 205 cells grow as dispersed cells occasionally forming loose, small clusters as seen in catenin-deficient cells , and they only lightly attach to the culture dish. Despite this behavior, they express an apparently normal set of E-cadherin, αE-catenin, β-catenin, and p120 ctn proteins . Moreover, the expression levels of these proteins are similar to those in HT-29 cells, which can organize epithelial sheets . To determine whether the cadherin–catenin system in Colo 205 cells is entirely inactive, we rotated the cultures to facilitate cell aggregation. Under these culture conditions, Colo 205 cells clumped into larger aggregates and this clumping was inhibited by addition of the E-cadherin blocking antibody SHE78-7 , indicating that E-cadherin is functional. However, their aggregates were still loose and never formed tightly associated compact aggregates as generally produced by the cadherin adhesion system . These observations suggest that Colo 205 cells expose functional E-cadherin molecules on their surface, but their cadherin system has some deficit in exerting its full activity to organize compact cell aggregates. Interestingly, when the cultures of Colo 205 cells were starved by not refreshing the culture medium for a few days, they occasionally formed compact aggregates , implying that their cadherin system is reversibly impaired and can be reactivated under certain physiological conditions. As an initial attempt to investigate how the cadherin system is impaired in Colo 205 cells, we examined the effect of various biochemical reagents, including kinase inhibitors and activators, on their aggregation. Among the reagents tested, staurosporine showed a marked effect. It induced compact cell clustering in Colo 205 cultures within a few hours after administration , during which the initial sign of adhesion induction was observed within 2 h. This effect was saturated by 6 h. The compaction of cell clusters was E-cadherin–dependent, as it was inhibited by SHE78-7 . Staurosporine also slightly promoted spreading of cells, but this was not inhibited by SHE78-7 . Since staurosporine is known to inhibit protein kinase C (PKC), we tested other PKC inhibitors, but none were effective (Table I ). Tyrosine kinase inhibitors were also negative, except herbimycin A showed a weak compaction-inducing activity, but only when cells were cultured overnight with this inhibitor (data not shown). PKC activators and phosphatase inhibitors, such as phorbol esters and okadaic acid, also had no effects (data not shown). Thus, among the reagents tested, staurosporine exhibited an exceptionally strong effect on Colo 205 aggregation. This effect of staurosporine was reversible as its removal caused redispersion of Colo 205 cells (data not shown). Besides metabolic inhibitors, we found that trypsin is a strong inducer for compact Colo 205 aggregation. When low concentrations of trypsin (0.01–0.001%) were added to serum-free cultures, Colo 205 cells became tightly associated with each other, deforming their morphology . This adhesion induction was quick, beginning within 15 min after the addition of trypsin, and the effect was almost saturated at 30 min. The trypsin-mediated induction of aggregation was completely blocked by SHE78-7 , indicating that it was an E-cadherin– dependent process. Under these conditions, which included a physiological concentration of Ca 2+ , cadherins are not digested with trypsin . Trypsin concentration of 0.001% was sufficient for the above adhesion induction, whereas 0.0001% was not effective. Thus, we found two different classes of reagents to induce compact Colo 205 aggregation, trypsin as a quicker inducer and staurosporine as a slower inducer. Previous studies indicated that mucins, such as MUC1 and epiglycanin , reduce cell–cell adhesion presumably by steric hindrance. As Colo 205 cells express MUC1 , trypsin may remove such antiadhesive mucins. To check this possibility, we immunofluorescently stained for MUC1 before and after trypsin treatment, and found that this proteoglycan was equally present on the surface of both trypsinized and untrypsinized cells , even being localized at intercellular contact sites in the adhesion-induced aggregates. Treatment of cells with O -sialoglycoprotein endopeptidase, which inactivates epiglycanin and enhances adhesion of mammary carcinoma cells , showed no effects on Colo 205 aggregation (data not shown). From these observations, we assumed that the effect of trypsin was not to remove steric hindrance molecules, but to digest some signaling proteins on the cell surface, such as receptors, affecting intracellular physiological states. This idea is supported by results of other experiments described below. Immunostaining for E-cadherin in Colo 205 cells show a diffuse distribution of this molecule on their surface . When compaction was induced in their aggregates by staurosporine or trypsin treatment, E-cadherin became highly concentrated into cell–cell contact sites . Catenins displayed a similar distribution . These observations suggest that the E-cadherin–catenin complex can be redistributed to cell–cell contact sites under the above compaction-inducing conditions. To study if any changes were induced in the cadherin– catenin system following the above treatments, we immunoprecipitated E-cadherin from Colo 205 cells untreated or treated with staurosporine or trypsin. The amount of catenins coprecipitating with E-cadherin was not changed after these treatments . However, we noted a significant change in the pattern of the p120 ctn band. In untreated Colo 205 cells, p120 ctn was detected as a broad, diffuse band that appeared to comprise multiple components , although the pattern varied slightly from experiment to experiment. After compaction-inducing treatments, the band pattern was altered. Its most intense portion was shifted to the position corresponding to the bottom (electrophoretic front) of the original band . Similar electrophoretic patterns of these proteins were observed in Western blots of the whole cell lysates . The changes in the p120 ctn band pattern coincided with the onset of adhesion induction by each reagent, starting within 15 min with trypsin and within 2 h with staurosporine . A certain level of p120 ctn -band shift was already detected at 5 min of incubation with trypsin. When trypsin was removed from the culture, the original electrophoretic profile of p120 ctn was eventually restored within overnight incubation , together with a recovery of the dispersed cell morphology (data not shown). Removal of staurosporine gave similar results. Next, we asked whether the p120 ctn change occurred before, or as a result of, adhesion induction. We treated Colo 205 cells with 0.01% trypsin in the presence (TC) or absence (TE) of 1 mM Ca 2+ . TC treatment leaves E-cadherin–mediated adhesion intact, whereas TE destroys E-cadherin–dependent adhesion . We found that both treatments resulted in a similar p120 ctn -band shift . This finding indicates that the p120 ctn band change was not brought about as a result of adhesion induction but directly through trypsin treatment signals. We also found that the trypsin-induced p120 ctn -band shift took place even when E-cadherin was blocked with antibodies . This was also the case in the staurosporine treatment (data not shown). When other kinase inhibitors listed in Table I were tested, only herbimycin A induced a similar p120 ctn -band shift at a 24-h incubation period (data not shown). We sought to understand the molecular nature of the p120 ctn -band shift. When p120 ctn immunoprecipitates collected from untreated Colo 205 were incubated with alkaline phosphatase, the diffuse p120 ctn band was transformed into a sharp band positioned at the front of the original , which comigrated exactly with the p120 ctn from staurosporine or trypsin treated cells. This suggests that the band shift observed may have been brought about by p120 ctn dephosphorylation also. We then analyzed phosphorylated residues in p120 ctn by 32 P-metabolic labeling. The results showed that the major phosphorylated residues were serine . However, their labeling intensity was however, not significantly reduced after trypsin treatment of cells. Antiphosphotyrosine antibodies detected only weak signals from the p120 ctn bands in these cells (data not shown). It is possible that the p120 ctn -band shift is caused by dephosphorylation of a subset of the phosphorylated residues. Alternatively, the quantitative differences in phosphorylation suggested by the phosphatase treatments may be underrepresented by amino acid analysis methodology. The above findings suggest that the process of p120 ctn -band shift could be associated with the induction of compact Colo 205 aggregation. To pursue this possibility, we attempted to modify the activity of p120 ctn by expressing a series of its deletion constructs attached to a FLAG-tag at the COOH terminus in Colo 205 cells . Transient expression of these molecules was achieved by electroporation of cDNAs or infection by adenoviral expression vectors. Generally, the adenoviral infection yielded much higher cDNA transfection efficiencies. Successfully transfected cells were detected by immunofluorescence staining with antibodies to the FLAG-tag. We first tested the full-length p120 ctn as a control and found no particular effect on the aggregation of Colo 205 cells . Then, we transfected them with ΔN346f, leaving the Armadillo repeat domain intact. Notably, cell groups expressing this construct were transformed into tightly associated aggregates in which the introduced molecules were sharply concentrated at cell–cell contact sites . Then we tested other deletion constructs. Concerning shorter NH 2 -terminal deletions, ΔN323f displayed the same adhesion-inducing effect as ΔN346f , and ΔN244f also showed a positive effect, but their aggregation appeared looser than that observed with the former . On the other hand, ΔN157f and ΔN101f had no effect on cell adhesion . These observations indicate that the critical sites are located between 245 and 323. We tested two other constructs, FLΔRf and ΔN346ΔRf, in which the Armadillo repeat domain was partially deleted, and found that they had no effect on cell aggregation . The last two molecules did not bind to E-cadherin, as previously found , whereas all the others tested did. Consistently, FLΔRf and ΔN346ΔRf were distributed only in the cytoplasm whereas the others were located along the cell membrane, as well as the cytoplasm . Cell membrane distributions were categorized into two groups: the adhesion-inducing mutant molecules were concentrated into cell–cell contact sites whereas the noneffective proteins were located randomly along the cell membrane. In the preceding experiments, the p120 ctn -band shift was correlated with adhesion induction. Therefore, we also analyzed the band profile of the NH 2 terminus deletion constructs. In Western blotting of the lysates of cells transfected with these constructs, all NH 2 -terminally deleted molecules showed broad electrophoretic bands , although the proportion of the bottom to the upper components in the band tended to increase in the adhesion-inducing construct ΔN346f. When FLf and ΔN346f were collected by immunoprecipitation and treated with phosphatase, only the bottom component remained undigested in both samples , as found in endogenous p120 ctn , indicating that the bottom bands of different p120 ctn constructs were equivalent to each other in terms of phosphatase resistance. We examined whether or not the binding of p120 ctn to E-cadherin was altered by NH 2 -terminal deletions. We transfected Colo 205 cells with FLf or mutant ΔN346f, and immunoprecipitated E-cadherin from them . Anti-FLAG antibody D8 detected only the ectopic proteins, and the antibodies recognizing the COOH-terminal region of p120 ctn detected both the transfected and endogenous molecules . Comparison of the band profiles in these samples with those in Western blotting of whole cell lysates indicates that the proportion of the E-cadherin–bound p120 ctn to its entire pool was not different between FLf and ΔN346f. The small difference seen in electrophoretic mobility between the endogenous and full-length ectopic molecules is probably due to their difference in species origin, the former from the mouse and the latter from the human. These results suggest that the binding affinity of p120 ctn for E-cadherin was not altered by the NH 2 -terminal deletion. We tested two other epithelial lines, HT-29 and MDCK cells, to ask whether they also respond to the NH 2 terminus–deleted p120 ctn constructs, as well as the compaction-inducing reagents. Human colon carcinoma HT-29 cells, which express normal levels of E-cadherin and catenins , organize into epithelial sheets in confluent cultures . When these cells are dispersed with trypsin in the absence of Ca 2+ and seeded into new plates they require a lag period to reestablish epithelial sheets, e.g., at 18–24 h after cell transfer, the cultures still contain many round, dispersed cells , although they eventually established epithelial sheets at 48 h. We analyzed p120 ctn in these HT-29 cells, and found that its band pattern dynamically changed with the cycle of cell transfer. p120 ctn derived from freshly trypsinized HT-29 cells showed a single band . 24 h after the transfer p120 ctn was shifted to an upper position . At 48 h, when cells formed confluent epithelial sheets, the p120 ctn band returned to the lowest position . We tested if the p120 ctn -band shift observed after overnight culture could be canceled by retreatment with trypsin (0.001%; 30 min) or staurosporine (7 nM; 6 h) by adding them to HT-29 cells precultured for 18 h. The results showed that both treatments abolished the mobility shift of the p120 ctn band . The range of the p120 ctn -band shifting was similar to that found in Colo 205 cells. In correlation with this p120 ctn band change, tight cell–cell association was induced in HT-29 cells preincubated for 18 h and then incubated with staurosporine (7 nM) for 6 h or with trypsin (0.001%) for 30 min , as seen in the case of Colo 205 cells. On the other hand, MDCK, which is widely used as a model of polarized epithelial cells, reorganized epithelial sheets within 18 h after transfer. Addition of staurosporine or trypsin to such MDCK cultures had no effects on cell– cell association , although we observed subtle morphological alterations in the treated cultures. Analysis of p120 ctn in MDCK cells detected two major bands as reported by Mo and Reynolds , the upper band likely corresponds to the p120 ctn band in human colon carcinoma lines . The p120 ctn pattern in MDCK was not changed by trypsin-mediated cell transfer. When MDCK cells were treated with staurosporine, the electrophoretic mobility of p120 ctn was slightly enhanced , as described by Ratcliffe et al. . However, this position shift of the p120 ctn band was subtle compared with that observed in the above carcinoma lines. Trypsin treatment of MDCK cells did not affect the mobility of p120 ctn . Next, we transfected the above cell lines with the NH 2 terminus–truncated ΔN346f, as well as with the control FLf immediately after cell transfer, and examined the effects after 24 h. ΔN346f, but not FLf, enhanced HT-29 cell–cell association . ΔN346f-expressing cells formed compact aggregates, whereas FLf had no effects. Rather, we observed that high-level overexpressions of FLf tended to disperse epithelial sheets (data not shown). In MDCK cells, neither construct affected the cell–cell contact morphology, they simply accumulated at cell–cell contact sites . These findings suggest that in particular cells such as Colo 205, p120 ctn inhibits cadherin-mediated adhesion under certain physiological conditions. If this is the case, cadherin activity might be restored under circumstances where it cannot interact with p120 ctn . To test this possibility, we transfected Colo 205 cells with two different constructs of N-cadherin cDNA, one encoding its entire portion and the other encoding a mutant molecule in which the juxtamembrane region of the cytoplasmic domain, which is known to contain the p120 ctn -binding site , has been deleted . Immunoprecipitation with anti–N-cadherin antibodies from the transfected cells confirmed that the mutant N-cadherin was unable to associate with p120 ctn , but coprecipitated normally with β-catenin. Morphological and immunostaining examinations of these transfectants showed that the expression of full-length N-cadherin had no effect on cell–cell adhesion, suggesting that cadherins were generally blocked in the Colo 205 environment, irrespective of their type. In contrast, the mutant N-cadherin induced a strong aggregation of Colo 205 cells . These findings suggest that cadherins can function normally in these cells unless they interact with p120 ctn , supporting the idea that p120 ctn can act as an inhibitor of cadherin function. This work was initiated to understand why Colo 205 cells cannot undergo typical E-cadherin–dependent association despite their normal expression of E-cadherin and associated proteins. We found that the treatment of these cells with two independent reagents, trypsin and staurosporine, could reactivate the E-cadherin adhesion system. Although the initial mechanisms for their actions remain unknown, we assume that trypsin removes or truncates some cell surface proteins involved in intracellular signaling, leading to an activation or suppression of their downstream cascade and via this pathway, modulates the cadherin system. The response of Colo 205 cells to the trypsin treatment was rapid, suggesting that the proposed signaling system probably does not require transcription of new genes. As another possible mechanism of the trypsin action, we considered that this enzyme might have facilitated cell adhesion by digesting such surface components as mucins, known to physically prevent cell–cell contacts. However, this possibility is less likely, because we could induce compact cell aggregation by intracellular manipulation of Colo 205 cells without enzymatic treatment. Moreover, no correlation was found between the distribution of MUC1, a major Colo 205 mucin, and adhesion induction. Concerning staurosporine, its action could be connected with the proposed signaling cascade directly or indirectly. Of the many kinase inhibitors tested, only staurosporine effectively induced Colo 205 cell adhesion within a short incubation period. This suggests that staurosporine has a specific target in its action on Colo 205 adhesion, although we cannot specify it because this antibiotic can inhibit multiple classes of enzymes. It was reported that retinoic acid treatment results in a similar adhesion induction , suggesting that this reagent is another effector to activate the cadherin system. Since the initial or intermediate steps of the proposed signaling system could be complex, we focused our analysis on its putative terminal step, asking if the cadherin– catenin complex per se was modified during the adhesion induction, and we identified an alteration in the electrophoretic mobility of p120 ctn . This change could be triggered in the absence of E-cadherin–mediated adhesion, implying that it resulted directly from the trypsin or staurosporine-triggered signaling cascade. A trypsin/staurosporine-sensitive mobility shift of p120 ctn was also observed with another carcinoma line HT-29. Although transient in these cells, the shift nonetheless correlated perfectly with adhesion induction. In contrast, MDCK cells neither showed such modulation of p120 ctn , nor responded to the adhesion-inducing reagents. Although staurosporine treatment slightly enhanced the electrophoretic mobility of p120 ctn , even in MDCK cells , the magnitude of the band shifting in this case was much smaller as compared with that for Colo 205 and HT-29 cells. These findings prompted us to further examine the role for p120 ctn by structure–function analysis, and we found that NH 2 -terminally truncated p120 ctn constructs by themselves could reactivate the E-cadherin system. This observation implies that p120 ctn inhibits cadherin-mediated adhesion in Colo 205 cells. Thus, we postulate that the NH 2 -terminally deleted p120 ctn lacks this activity, and competes with endogenous molecules, ultimately resulting in the reactivation of the cadherin system. This hypothesis is strongly supported by the finding that ectopic N-cadherin induced aggregation of Colo 205 cells only when it was unable to bind to p120 ctn . This finding also suggests that different types of cadherin, in general, cannot function in the intracellular environment of Colo 205 if they bind to p120 ctn . The correlation between the band shifting of p120 ctn and adhesion induction suggests that the putative inhibitory activity of this molecule might be elicited by certain biochemical modifications. In Colo 205 cells, the original p120 ctn band was broad. Although multiple splicing products of p120 ctn are expressed in many carcinoma lines , the broad band observed in Colo 205 cells appears to result from phosphorylation at various levels because phosphatase treatment of the Colo 205 p120 ctn resulted in the generation of a sharp single band. This phosphatase-treated p120 ctn was similar in electrophoretic mobility to that found in the adhesion-induced Colo 205 cells. These findings suggest the possibility that p120 ctn acquires the inhibitory activity when hyperphosphorylated, and the staurosporine/trypsin-induced signals reduce the level of phosphorylation. We found that the major phosphorylated residue in p120 ctn of Colo 205 cells was serine, consistent with a previous observation by Ratcliffe et al. . Our results do not exclude the possibility that other residues are also phosphorylated at low levels, as it has been reported that tyrosine is phosphorylated in p120 ctn of other cell lines . The net amount of serine phosphorylation in p120 ctn did not appear reduced after its electrophoretic mobility shift, although phosphoamino acid analysis may not adequately detect small changes in phosphorylation status. If dephosphorylation is indeed involved in this process, one possibility is that only a specific subset of phosphorylation sites are required for the p120 ctn change in motility. The NH 2 -terminally deleted p120 ctn , which induced cell adhesion, still contained slower-migrating, phosphatase-sensitive components, although their proportion tended to be reduced. Possibly, the key phosphorylation sites directly affecting the electrophoretic mobility of p120 ctn are located outside the NH 2 -terminal region. These observations also suggest that hyperphosphorylation may be insufficient to confer the adhesion inhibitory activity on p120 ctn if its NH 2 terminus is absent. A model for the signaling cascade to regulate p120 ctn phosphorylation has been proposed by Ratcliffe et al. . How does p120 ctn inhibit cadherin function? It is interesting to note that the juxtamembrane domain of the cadherin cytoplasmic region where p120 ctn binds can inhibit cell adhesion when overexpressed , and also is required for clustering of cadherin molecules which is thought to be essential for cadherin-mediated cell adhesion. Another report shows that deletion of a similar region in E-cadherin resulted in activation of this adhesion molecule, enhancing its lateral interactions in the cell membrane of a leukemia line , suggesting that this domain can inhibit lateral clustering of cadherins. p120 ctn is the only molecule known to bind to the juxtamembrane domain of cadherins and could be the major effector of these activities. For example, p120 ctn may directly regulate lateral clustering of cadherins and this activity may be blocked by hyperphosphorylation or other mechanisms controlled by the NH 2 terminus. In support of this hypothesis, our immunostaining observations suggest that clustering of E-cadherin into cell–cell contact sites is enhanced by the adhesion-induction treatments. Alternatively, p120 ctn may have no function in the default state and might serve as an inhibitor of cadherin function only when posttranscriptionally modified. The results of our N-cadherin experiments, as well as the above leukemia study , indicate that cadherins can be active without p120 ctn . We have demonstrated a p120 ctn -dependent inhibition of the cadherin system in carcinoma cells. It is known that cadherin-mediated adhesion is perturbed in tumors in a variety of ways, including mutation of cadherin or catenin genes, and these events have been implicated in cancer invasion and metastasis . Here, we have identified a novel physiological mechanism of inhibiting cadherin function. Many metastasizing tumors maintain strong cadherin and catenin expression. Such tumor cells must have a mechanism to destabilize cell–cell adhesion, at least transiently, for detaching from the original tumors. Biochemical modification of p120 ctn could be used for such processes. Colo 205 cells may be an extreme case in which the p120 ctn -dependent inhibitory mechanism is constitutively turned on, whereas in HT-29 it operates only transiently, as these cells can eventually form normal looking epithelial sheets. The mechanism for the transient modification of p120 ctn in HT-29 remains unknown. Finally, the question arises as to how such an activity of p120 ctn is involved in normal cellular events. Cell–cell adhesion is dynamic and requires regulation during many types of morphogenetic processes. p120 ctn could be implicated in such regulation as suggested by previous observations. The juxtamembrane domain of the cadherin cytoplasmic domain to which p120 ctn binds was implicated in cell motility and in axon outgrowth . Overexpression of p120 ctn in Xenopus embryos perturbs gastrulation . p120 ctn , whose action can be modulated posttranscriptionally, could play a role in the control of such adhesion-related cellular behavior. As we suggested in light of the effect of trypsin, there might be a signaling cascade originating at the cell surface to regulate the p120 ctn activity. Identification of components of such cascades is an important future issue for unraveling the morphogenetic regulatory mechanisms of cell–cell adhesion. | Other | biomedical | en | 0.999997 |
10225957 | A random and oligo-dT–primed mouse adult brain cDNA library was screened with a 32 P-labeled chicken ten-m cDNA (cten22, 1.8 kb; Chiquet-Ehrismann, R., manuscript in preparation) which encodes EGF-like repeats and part of the COOH-terminal cysteine-rich sequence. Filters were hybridized and washed as previously described . The cDNA insert of clone DT1 was subcloned into pBluescriptSK (Stratagene) and both DNA strands were sequenced using the ABI Prism Dye Terminator kit (Applied Biosystems). The sequences were analyzed using a 373A automatic sequencer (Applied Biosystems). Using the insert of DT1 the brain library was rescreened to obtain cDNAs extending in the 3′ and 5′ direction. This rescreening was repeated until the entire coding region was cloned. All clones were subcloned and both DNA strands were sequenced. The ten-m2 , ten-m3 , and ten-m4 cDNAs were isolated by screening the brain cDNA library with the PstI/EcoRI fragment of DT19 (731–1,833 bp) at low stringency as previously described . Full-length cDNA fragments were isolated and characterized from the same mouse brain cDNA library. Sequence alignments were performed using the BLAST program and PileUp from the GCG (Genetics Computer Group) package. Poly(A) + RNA was isolated from brain, heart, liver, kidney, skeletal muscle, testes, and spleen of 7-wk-old and thymus of 3-wk-old 129/Sv mice. 20 ml of proteinase K buffer (20 mM Tris-HCl, pH 7.4, 0.5% SDS, 0.1 M NaCl, 1 mM EDTA, 200 mg/ml proteinase K) was added to cell pellets and DNA was sheared by treating cells with a polytron for 1 min. 1 ml of oligo-dT cellulose ( Pharmacia ) suspended (vol/vol) and equilibrated for 30 min in high salt buffer (10 mM Tris-HCl, pH 7.4, 0.4 M NaCl, 1 mM EDTA, 0.1% SDS) and 1.25 ml 5 M NaCl (0.4 M final concentration) were added to the cell lysate and incubated on a rocking platform for 1 h at room temperature. Polyadenylated RNA bound to the oligo-dT cellulose was washed three times in high salt buffer and subsequently put on an Econo column ( Pharmacia ). After three additional washings of the oligo-dT with high salt buffer, polyadenylated RNA was eluted with 1 ml of RNase-free water. For Northern analysis, 4 μg of polyadenylated RNA was electrophoretically separated, blotted onto Hybond membrane ( Amersham ), UV cross-linked, and probed in Church buffer at 65°C. Filters were washed twice in 0.2× SSC/1% SDS at 65°C and exposed to x-ray film for 1 or 4 d at −80°C. The following oligolabeled probes were used: for ten-m1 a fragment ranging from nucleotide 75 to 1833, for ten-m2 a fragment ranging from nucleotide 1 to 2006, for ten-m3 an EcoRI fragment ranging from nucleotide 762 to 1408; for ten-m4 a fragment ranging from nucleotide 1 to 1108. The RNA loading was controlled by probing blots with a GAPDH cDNA probe. To obtain secretion of the recombinant proteins, all cDNA fragments were linked to the BM-40 signal peptide via an NheI site in the BM-40 sequence . To express the entire extracellular domain of Ten-m1, the tetrapeptide APLA derived from the BM-40 signal peptide region was followed by amino acid E526 of the Ten-m1 sequence. The constructs for the expression of the alkaline phosphatase (AP) fused to either three or eight EGF domains or the entire extracellular domain (AP-ten-m1), respectively, started with an APLVGSSG sequence, followed by I23 of the human placental AP sequence, which terminated before the hydrophobic glypiation signal through a stop codon encoded by an HpaI site . To express both fusion proteins the NH 2 -terminal AP domain with the BM-40 signal peptide was cleaved at the HpaI site (which destroyed the stop codon) and ligated via a linker segment to the ten-m1 cDNA fragment encoding the EGF domains or extracellular domain, respectively. In these fusion proteins the AP domain was linked to the Ten-m1 fragment via an SSGG sequence to the G359 of the Ten-m1 sequence. The fusion proteins were terminated by the introduction of stop codons after E624 for the AP-3EGF recombinant protein and after I796 for the AP-8EGF recombinant protein, respectively. To express the intracellular and transmembrane domain fused to the AP the first 370 amino acids of Ten-m1 were connected via the linker sequence IKLAYVRSSG to I23 of the AP sequence. Human embryonic kidney cells (HEK 293 cells; American Type Culture Collection) were transfected with the constructs in an eukaryotic expression vector containing a CMV promoter (pRC/CMV; Invitrogen) and a puromycin resistance gene. Puromycin-resistant clones were isolated by ring cloning. Positive clones were identified by SDS-PAGE and Coomassie blue staining according to standard protocols and maintained as described . The AP expression was determined as described by Flanagan and Leder . For determining whether the intracellular and transmembrane domain localized the AP module on the outer cell surface, cells were treated with trypsin for 30 min at 37°C and then allowed to adhere on gelatinized glass coverslips for 30 min, washed, fixed, and stained for AP activity. Conditioned medium containing the entire extracellular domain of Ten-m1 (Ten-m1sec) was dialyzed against TBS and applied to 5% of the volume of DEAE-Sephacel equilibrated with the same buffer. The proteins were eluted by a gradient from 0 to 500 mM NaCl in TBS. Ten-m1sec– containing fractions were identified by SDS-PAGE and Coomassie blue staining, pooled, and concentrated to <1 ml by centrifugation in 2- or 3.5-ml concentrators with membranes with a cutoff of 10 kD (Amicon or Pall-Filtron). The concentrated protein was applied to a Superose 6 column equilibrated with 50 mM Tris-HCl, pH 7.6, 500 mM NaCl, 2 mM EDTA, 0.5 mM NEM, and 0.5 mM PMSF. Ten-m1sec–containing fractions were dialyzed against 50 mM NaHCO 3 , 150 mM NaCl and stored at −80°C. 2 mg purified Ten-m1sec was coupled to 2 ml CNBr-activated Sepharose ( Pharmacia ) according to the manufacturer's protocol. The remaining activated groups were blocked with 1 M ethanolamine, pH 8. The affinity matrix was incubated with 10 ml of anti–Ten-m1sec antiserum and washed with PBS (50 mM Na-phosphate, pH 7.4, 150 mM NaCl), with PBS containing 500 mM NaCl, and was prepared for elution with PBS containing 10 mM Na-phosphate, pH 8, 150 mM NaCl. Antibodies were eluted with 50 mM diethylamine, pH 11.5, and immediately neutralized with 1/10 volume of 1 M Na-phosphate, pH 6.8. The eluted and neutralized antibodies were supplemented with 1/10 volume of 5% BSA and dialyzed against PBS. The purified antibodies represented ∼1% of the immunoglobulin fraction of the unpurified antiserum and, according to enzyme-linked immunosorbent assays, exhibited ∼10% of their antigen-binding capacity. Brain of 8-wk-old mice was homogenized in 5 vol of 20 mM Tris-HCl, pH 8, containing 11% sucrose, protease inhibitors (5 mM EDTA, 5 mM N -ethylmaleimide, 5 mM benzamidine), and freshly added 1 mM PMSF on ice using a Dounce homogenizer. After centrifugation at 16,000 g the insoluble material was washed once with the same buffer, then washed with a buffer containing 150 mM NaCl instead of sucrose, and extracted with 20 mM Tris-HCl, pH 8, 150 mM NaCl, protease inhibitors, and 3% Triton X-100. Sucrose extracts were supplemented with 3× SDS-PAGE buffer, whereas Triton X-100 extracts were precipitated with acetone and the pellet was dissolved in 1× SDS-PAGE buffer (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol). A critical variable for the proper solubilization and migration of Ten-m1 was the reducing agent in the sample buffer. In initial experiments SDS-PAGE buffer with 5% mercaptoethanol was used. Under these conditions recombinant Ten-m1sec barely entered the stacking gel. In further studies with tissue extracts and recombinant Ten-m1sec, mercaptoethanol was replaced by 10 mM dithioerythrol which improved the migration behavior of Ten-m1sec and discrete bands could be observed. However, also under these conditions the recombinant Ten-m1sec molecules were not always separated into monomeric structures. Western blots of tissue extracts were performed by transfer of proteins separated by SDS-PAGE to PVDF membranes (Hybond-P; Amersham ) in Tris/glycine buffer containing 10% methanol for 1 h with 100 V with the Bio-Rad mini gel system. The membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20), incubated with affinity-purified polyclonal antibody against Ten-m1sec in TBST, and developed with horseradish peroxidase–conjugated secondary antibody in TBST containing 5% nonfat dry milk and the ECL+ detection system ( Amersham ) according to the manufacturer's protocol. Western blots for detecting recombinant proteins were performed by transferring proteins separated by SDS-PAGE to supported nitrocellulose (Bio-Rad) in Tris/glycine buffer containing 10% methanol for 1 h with 100 V with the Bio-Rad mini gel system. The blots were blocked with 1% BSA, incubated with an anti–calf intestine AP antiserum ( Sigma Chemical Co. ), and developed with AP-conjugated secondary antibodies. HEK 293 cells transfected with Ten-m1ap were washed with PBS, lysed at 4°C in 2 ml TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) supplemented with 0.7% Triton X-114, and centrifuged at 4°C at 5,000 rpm (2,000 RCF). 550 μl supernatant was layered on top of 800 μl 6% sucrose in TBS, incubated for 3 min at 30°C, and centrifuged in a swing out rotor with 30°C warm buckets for 3 min at 3,000 rpm (1,000 RCF). The Triton X-114–rich bottom phase (∼30 μl) was precipitated with acetone and dissolved in 100 μl SDS-PAGE sample buffer. From the Triton X-114–depleted upper phase 500 μl was recovered, cooled to 4°C, mixed with 30 μl 14% Triton X-114 in TBS, and layered on top of another 800 μl 6% sucrose in TBS, incubated 3 min at 30°C, and centrifuged. 400 μl of this second upper phase was precipitated with 110 μl 55% TCA solution. The precipitate was washed with acetone and dissolved in 100 μl SDS-PAGE sample buffer. 15-μl aliquots were used for protein stainings with Coomassie blue and for Western blots. Far Western blotting was performed as described with minor modifications. Recombinant Ten-m1sec, neurocan, and BSA were separated by electrophoresis on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 1% BSA in 10 mM Tris-HCl (pH 8) buffer (containing 150 mM NaCl, 0.05% Tween 20, 2 mM CaCl 2 , and 2 mM MgCl 2 ; TBST-CM buffer) at 4°C overnight, washed five times with TBST-CM buffer and then incubated with purified 30 nM AP-ten-m1 or AP, respectively, in the TBST-CM buffer containing 1% BSA for 3 h at room temperature, respectively. Afterwards membranes were washed with TBST-CM buffer five times and stained for 15 min in 0.1 M Tris-HCl, pH 9.5, buffer containing 0.1 M NaCl, 0.05 M MgCl 2 , 1.75 μg/ml BCIP, and 4.5 μg/ml NBT ( Boehringer Mannheim ) for AP activity. Immunofluorescence analyses were carried out using affinity-purified rabbit antibodies against Ten-m1. All tissue specimens (testes, lung, brain, eyes, and kidneys) were derived from 6-wk-old mice, frozen on dry ice, and sectioned (6 μm) on a MICROM/ Zeiss ( Zeiss ) cryostat. Sections were fixed in 4% paraformaldehyde for 5 min at room temperature and washed in PBS. Affinity-purified antibodies against Ten-m1 were diluted 1:25 in PBS containing 2.5% ovalbumin. Normal rabbit serum (1:150) served as the negative control. Fluorescence labeling was performed with Cy3-conjugated goat anti–rabbit immunoglobulins ( Sigma Chemical Co. ). Fluorescent specimens were mounted in 90% glycerol containing 1 mg/ml β-phenylenediamine. Microscopy was carried out with an Axiophot fluorescence microscope ( Zeiss ). Frozen 8-μm-thick tissue sections were prefixed in 4% paraformaldehyde for 5 min. Staining with AP-ten-m1 or AP was performed as described with some minor modifications. In brief, sections were blocked with 1% BSA in HBSS for 1 h at room temperature and then incubated with either 30, 60, 120, or 240 nM AP-ten-m1 or AP, respectively, in HBSS buffer including 1% BSA at room temperature overnight. Sections were washed five times with HBSS buffer, fixed with 60% acetone, 3% formaldehyde, 20 mM Hepes (pH 7.5) for 30 s, washed with 150 mM NaCl, 20 mM Hepes (pH 7.5; HBS) three times, heated at 65°C to inactivate endogenous AP for 15 min in HBS buffer; rinsed with 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl 2 and stained at room temperature for 5 h in the same buffer containing 10 mM l -homoarginine, 0.17 mg/ml BCIP, and 0.33 mg/ml NBT ( Boehringer Mannheim ). Afterwards sections were dehydrated, cover-slipped, and photographed. Samples were stored in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.4) at concentrations of ∼1 mg/ml and diluted to 0.2 M ammonium hydrogen carbonate, pH 7.9, shortly before use (final concentration 5–10 μg/ml). The diluted Ten-m1sec solution was subsequently mixed with an equal volume of 80% glycerol immediately before spraying on to freshly cleaved mica pieces. They were dried in a high vacuum and rotary shadowed with 2 nm platinum/carbon at a 9° angle by means of electron bombardment heating, followed by coating with 10 nm carbon from above . For negative staining 5 μl of purified Ten-m1sec in TBS (typical concentration 3 μg/ml) was adsorbed onto 400 mesh carbon-coated grids for 1 min, washed with two drops of water, and stained with two drops of 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were observed in a Jeol 1200 EX transmission electron microscope operated at 60 kV accelerating voltage. Images were recorded on Kodak SO-163 plates without preirradiation at a dose of typically 2,000 electrons/nm. Evaluation of the data from electron micrographs was done as described previously . Figure 1 : Proposed cytosolic, transmembrane (framed), and linker domain of Ten-m 1–4. Conserved amino acid residues are in shadowed boxes. The dibasic motif conserved in all four mouse Ten-m proteins, in Drosophila Ten-m and Drosophila Ten-a is indicated by asterisks in a box below the sequence. Other amino acids shown in this box are conserved in these six molecules. Figure 2 : COOH-terminal domain of mouse Ten-m 1–4. Conserved amino acid residues are in shadowed boxes. To obtain the primary structure of ten-m/odz in mouse, a cDNA library derived from an adult mouse brain mRNA was screened using an radiolabeled chicken ten-m cDNA fragment (cten22, 1.8 kb; Chiquet-Ehrismann, R., unpublished observations) as a probe. The initial screening led to the isolation of one cDNA clone (DT1) which was purified and sequenced. The deduced amino acid sequence of DT1 showed a 35% homology with Drosophila Ten-m/Odz and contained three EGF-like repeats and part of the cysteine-rich domain also present in Drosophila Ten-m/Odz. Therefore, we designated this cDNA mouse homologue of Drosophila ten-m/odz and named the gene ten-m1 . The full-length cDNA was obtained by isolating 10 overlapping cDNA clones. The deduced Ten-m1 polypeptide contained 2,731 amino acid residues. When the ten-m1 cDNA fragment DT19 was hybridized to the brain cDNA library at low stringent condition, several clones showing a weak hybridization signal were isolated and sequenced. The deduced amino acid sequence of three clones contained the eight EGF-like repeats and part of the cysteine-rich domain of ten-m molecules. Despite the high homology of 63, 51, and 52% to Ten-m1, respectively, the three cDNA clones were clearly different from ten-m1 and from each other. To obtain full-length cDNAs, overlapping cDNA clones were isolated from the same library. The cDNA sequences were designated ten-m2 , ten-m3 , and ten-m4 , which had open reading frames of 2,764, 2,715, and 2,771 amino acid residues, respectively, and the translated amino acid sequences showed an overall similarity between 56 and 70%. None of the four ten-m sequences contained a signal peptide. Approximately 300–400 residues after the start codon, all four sequences showed a continuous stretch of 34 amino acids lacking charged residues, which could serve as transmembrane domain . About 200 amino acids COOH-terminal of the hydrophobic amino acids were eight consecutive EGF modules followed by a large COOH-terminal sequence which was rich in cysteines and devoid of any known modular motifs . The absence of a signal peptide and the presence of a stretch of hydrophobic amino acids suggested that the family of ten-m proteins may be expressed as type II transmembrane molecules. According to this model, the NH 2 -terminal 300–400 amino acids serve as cytosolic domain. They showed on average the lowest level of identity between the four mouse ten-m sequences, ranging between 34 and 46%. The extracellular part of ten-m molecules would consist of a linker domain of ∼200 amino acids, a region with eight EGF-like domains of ∼250 amino acids, and a COOH-terminal domain of ∼2,000 amino acids . The linker domain of all mouse ten-m proteins contained several dibasic amino acid residues which could serve as potential sites for proteolytic processing of the molecules. One of these sites is conserved in all four mouse ten-m sequences and the Drosophila Ten-m/Odz and Drosophila Ten-a . The Drosophila Ten-m/Odz shows higher identity to the mouse ten-m sequences in the linker domain (26–29%) than in the cytosolic domain (19–21%). Between the mouse ten-m sequences, the similarity of the cysteine-free linker domain ranged from 43 to 48%, the EGF domains ranged from 65 to 72%, and the large cysteine-rich COOH-terminal domain ranged from 58 to 68%. The similarity to Drosophila Ten-m/Odz over the entire COOH-terminal domain ranged from 30 to 33%. When the ten-m protein sequences were compared with the cDNA and protein data bank, DOC4 was identical to Ten-m4. In addition, the NH 2 -terminal part of the human γ-heregulin, identified in the breast cancer cell line MDA-MB-175 , had 95% identity to the NH 2 -terminal fragment of mouse Ten-m4/DOC4. The COOH terminus of the ten-m proteins contained several amino acid repeats which showed similarities with the rearrangement hot spot elements (rhs) of Escherichia coli and with a wall-associated protein (WAP) of Bacillus subtilis . To obtain evidence for the proposed model in which ten-m proteins are expressed as type II transmembrane molecules, a fusion cDNA was constructed in which the entire putative extracellular part of Ten-m1 was replaced by an AP module . The AP module was derived from the GPI-linked placental AP by introducing a stop codon in front of the hydrophobic COOH-terminal GPI sequence . The fusion cDNA was cloned into an expression vector containing a puromycin resistance gene and transfected into HEK 293 cells. 24 puromycin-resistant cell clones were isolated, grown on glass coverslips, fixed, and stained for AP activity. Whereas untransfected HEK 293 cells showed no AP expression, all transfected clones tested stained strongly for AP at the plasma membrane . Recombinant cells treated with trypsin for 30 min and then cultured for 30 min to allow adhesion showed significantly reduced AP activity . Due to the extensive trypsin treatment and short incubation time for adhesion, only a few cells were attached on the gelatinized glass coverslips which showed no or very poor cell spreading . The supernatant of the transfected clones showed no AP activity. To obtain biochemical evidence for the membrane localization of the fusion protein, the expressing cells were analyzed by Triton X-114 phase partition experiments. Aqueous solutions of 0.7% Triton X-114 separate into two phases when warmed from 4 to 30°C. The dense Triton X-114 containing detergent-rich phase has been shown to be enriched in integral membrane and membrane-anchored proteins, whereas the Triton X-114–depleted phase is enriched in soluble proteins . In such experiments the fusion protein of 116 kD, which is the expected size for a protein consisting of the NH 2 -terminal 370 amino acids of Ten-m1 and the 67-kD AP module, was distributed preferentially to the detergent-rich phase . The majority of the proteins released from the cell layer at 4°C with 0.7% Triton X-114 remained at 30°C in the detergent-depleted phase . These data show that the NH 2 -terminal and the hydrophobic sequence of Ten-m1 can direct the AP to the plasma membrane. This, together with the finding that the AP activity can be found on the cell surface, suggests that Ten-m1 is a type II transmembrane protein. To analyze the structure of the extracellular domain of Ten-m1, the cDNA sequence starting with the first EGF module was linked to the BM-40 signal peptide cDNA , cloned into an expression vector, and transfected into HEK 293 cells. 96 samples of serum-free conditioned culture medium derived from individual cell clones were collected and separated by SDS-PAGE under reducing conditions. Three recombinant cell clones expressed a protein with an apparent molecular mass of 225 kD after reduction which was not present in untransfected or mock-transfected controls. The protein was purified by ion exchange and gel permeation chromatography . Under nonreducing electrophoresis conditions, the recombinant protein barely entered the separating gel. To confirm the identity of the recombinant Ten-m1, the purified protein was subjected to NH 2 -terminal sequencing. The amino acid sequence obtained was APLAEIMD which represents the last four amino acids of the fused BM-40 signal peptide and the first four amino acids (526–529) of the fused Ten-m1 protein. Purified recombinant molecules were visualized by electron microscopy after spraying from glycerol spraying/ buffer mixtures and after adsorption to carbon films and negative staining . By both techniques mainly particles with a structure compatible with dimeric molecules were visible. They consisted of two elongated globular domains connected by a thin extended rod. 24% of these dimeric particles were, in addition, visible in close proximity to each other. The size distribution of the spherical moiety exhibited Gaussian profiles with a long diameter d1 of 13.2 ± 1.3 nm and a short diameter d2 of 7.8 ± 1.3 nm. Values are corrected for overestimation of ∼3 nm due to decoration with platinum. Negative staining revealed similar values (d1 12.7 ± 0.8 nm, d2 6.9 ± 0.7 nm), but in addition, each globular domain was resolved into three globular subdomains with similar sizes of 5.4 ± 0.9 nm . The extended rod connecting the spherical domains had a total length of 5.9 ± 2.9 nm after negative staining. This rod appeared more variable in structure after rotary shadowing with a total length of 17.9 ± 4.9 nm where the middle part occasionally formed a short branch of 8.8 ± 2.8 nm extending sideways. Tandem arrays of multiple EGF-like modules often appear as rod-like structures . Therefore, the short extended rods in the Ten-m1 extracellular domain most likely represent the EGF-like modules, which are located at the NH 2 termini of the recombinantly expressed Ten-m1 monomers. This hypothesis was further supported by the fact that the second and fifth EGF-like domains had an odd number of cysteines which might enable the formation of intermolecular disulfide bonds. To test this biochemically, the AP cDNA was fused to the BM-40 signal peptide and to either the first three or all eight EGF-like domains of ten-m1 . The EGF-like modules ended COOH-terminally at glutamic acid 624 and isoleucine 796, respectively. The fusion constructs were cloned into an expression vector and stably transfected into HEK 293 cells. Control cells were transfected with an expression construct containing the AP cDNA only fused to the signal peptide. Individual clones which showed AP activity in the supernatant were expanded. Serum-free conditioned media from several clones were analyzed by Western blot assay using AP specific antibodies. Fig. 4 B shows that cells transfected with AP only express a protein of ∼67 kD under nonreducing as well as reducing conditions. Cells transfected with the AP-3EGF repeat construct secreted a recombinant protein of apparent molecular mass of 110 kD when the proteins were separated under reducing conditions . When the AP-8EGF repeat construct was transfected the secreted protein had an apparent molecular mass of 150 kD under reducing electrophoresis conditions . Under nonreducing conditions both molecules showed approximately twice these sizes . Untransfected or mock-transfected cells did not secrete immunoreactive AP. These data suggest that the EGF-like modules of Ten-m1 with an odd number of cysteines can form intermolecular disulfide bonds, leading to the homodimerization of Ten-m1. Northern blot analyses using the cytoplasmic and transmembrane part of the cDNAs as probes revealed widespread expression of ten-m1 , 2 , 3 , and 4 genes in various mouse tissues . All four ten-m genes showed tissue-specific expression of variously spliced mRNAs. Alternative splicing was most prominent in the testes. The highest levels of all four mouse ten-m mRNAs were observed in brain. Fig. 5 B shows a short exposure of Northern blots of brain mRNA. All four ten-m genes are expressed in alternatively spliced forms. To assess protein expression of Ten-m1, a specific antiserum was produced by immunizing rabbits with the recombinant extracellular domain of Ten-m1. The antiserum was further purified by affinity chromatography with covalently immobilized immunogen. The results obtained by the specific antibodies in Western blots depended strongly on the reducing agent in the sample buffer and the treatment of the samples (see Materials and Methods). After exhaustive reduction of detergent extracts from mouse brain with dithiothreitol, two major protein bands with apparent molecular masses of 270 and 225 kD were observed . Often also higher molecular mass proteins, probably representing incompletely reduced protein dimers, were recognized by the purified antibody , occasionally even after prolonged incubations in the reducing sample buffer. Whereas the 270-kD form might correspond to the complete Ten-m1 molecule, the 225-kD form might represent an alternatively spliced or proteolytically processed molecule. It is also possible that the 225-kD form is a soluble form of the molecule, since it migrated at a similar position as the recombinant extracellular domain of Ten-m1 . A clear pattern of protein bands recognized by the purified antiserum could only be observed in brain extracts, but not in extracts of other tissue such as thymus, lung, and kidney (not shown). A lower abundance of Ten-m1 protein in tissues other than brain would be in agreement with the results of the Northern blot assay. To localize Ten-m1 protein expression in mice, immunofluorescence assays were performed on a variety of tissue sections. Strong signals were observed in many regions of the mouse brain . The cerebellum showed intensive staining in the molecular layer and weaker staining around cells in the granular layer. The staining around the cells appeared membrane associated and sometimes speckled. This localization strengthens the notion that Ten-m1 is a type II transmembrane protein with the COOH terminus on the extracellular side (antibodies were raised against the extracellular domain), as already suggested by sequence analysis and results obtained with recombinant fusion proteins. Purkinje cells and white matter were negative for Ten-m1 expression . The hippocampus had strong staining in the molecular layer and the neuronal layer of the CA3 region. Weak staining was observed in the CA1 region and dentate gyrus . Several other regions of the brain including the deep cerebellar nuclei, thalamic nuclei, cortex, and brain stem showed strong staining for Ten-m1 (not shown). Double immunostaining using anti–β3 tubulin antibodies which specifically label neurons revealed colocalization in many areas (not shown). Expression of Ten-m1 was also seen in the outer and inner segments of the retina where rods and cones showed very strong staining and around the basal epithelial cells of the cornea . The corneal surface showed a filamentous, strong staining which either could identify soluble Ten-m1 present in the tear film moistening the corneal surface or could be an artifact. Lung showed high expression in smooth muscle cell layers of bronchi, veins, and arteries and low expression on alveolar epithelial cells . In kidney, the staining for Ten-m1 was restricted to the glomeruli and vessels in the medullar region (not shown). Immunostaining of adult testes revealed Ten-m1 expression around spermatides but no expression was detected in spermatogonia and mature sperms . Staining of control sections with a nonimmune serum was negative (data not shown). Transmembrane proteins often have the capability to bind to molecules outside the cell and thereby act as receptors. To search for ligands that bind to Ten-m1, the cDNA encoding the entire extracellular domain was linked to an AP module equipped with a signal peptide resulting in the fusion protein AP-ten-m1 . Fig. 8 B shows that AP-ten-m1 and AP alone, which was used as a control, are secreted proteins with molecular masses of ∼300 and 67 kD, respectively. As ten-m1 is expressed in many tissues , AP-ten-m1 fusion protein should be able to detect its ligand(s) directly in a variety of tissue sections. Cryosections were treated with AP-ten-m1, washed, and then tested for bound AP activity of the fusion protein using an AP substrate precipitating on the cells. Whereas each concentration of AP-ten-m1 produced a specific staining pattern in tissues , equimolar concentrations of unfused AP did not show any AP reaction products . The binding of AP-ten-m1 in brain sections was high in the molecular layer of the cerebellum and around Purkinje cells. Little but significant staining was observed in the granular layer . The hippocampus showed a clear signal in the molecular layer and a weak signal was visible in the CA3 region, CA1 region (not shown), and dentate gyrus . Strong reactivity was also observed in many other regions of brain including cortex and several deep cerebellar nuclei (not shown). Kidney sections showed very intense staining in glomeruli and vessels of the medullar region (not shown). The lung sections had AP reactivity in the smooth muscle cell layers of bronchi, arteries, and veins (not shown). Incubation of tissues with the AP module did not reveal any staining reaction . The similar staining pattern of the Ten-m1 antiserum and the AP-ten-m1 probe prompted us to test whether Ten-m1 may be able to bind to itself. To test this hypothesis, purified Ten-m1 extracellular domain was applied in a Far Western blot using AP-ten-m1 as a probe. Under nonreducing conditions the extracellular domain of Ten-m1 had an apparent molecular mass of ∼400 kD as shown by amido black protein staining and Western blot with purified anti–Ten-m1 antibodies . The AP-ten-m1 probe bound specifically the Ten-m1 on the blot as revealed by an AP catalyzed staining reaction . Neither recombinant neurocan nor BSA was recognized by AP-ten-m1 fusion protein . AP alone did not bind to any of these proteins . Given the fact that Ten-m1 appears to interact in a homophilic fashion, it is noteworthy that by rotary shadowing and negative staining of purified Ten-m1 extracellular domain ∼24% of the Ten-m1 particles showed a clear interaction with each other . The Drosophila ten-m and odz genes are identical and define a new class of pair rule genes which do not function as transcription factors. In this paper we report the cloning and characterization of mouse ten-m/odz cDNAs. The mouse has at least four genes, named ten-m1–4 , which are homologous to the Drosophila ten-m/odz gene. The primary sequence of all four ten-m proteins lacked a signal peptide, and contained a stretch of hydrophobic amino acid residues ∼300–400 amino acids COOH-terminal of the start codon, eight EGF-like domains, and a large COOH terminus. Based on the cysteine and the putative N-linked glycosylation patterns, we divided the COOH terminus of the molecule tentatively into a cysteine-rich part, a strongly N-glycosylated part, and a part with four cysteines conserved in ten-m1 and 4, but not in ten-m2 and 3 . Sequence comparison with the data bank revealed homologies with several genes including Drosophila ten-m/odz , Drosophila ten-a , human γ-heregulin, and mouse DOC4 . Drosophila Ten-a shares homologies with the NH 2 -terminal part of Ten-m/Odz and the mouse ten-m proteins but lacks the large COOH-terminal region. Ten-a is expressed in the CNS, muscle, and eye. Its function is not known . The NH 2 -terminal part of γ-heregulin, a protein produced by the human breast cancer cell line MDA-MB-175 , shows 95% identity to the 562 NH 2 -terminal amino acids of mouse Ten-m4. Since the COOH-terminal part of γ-heregulin is identical to heregulin β3, the expression of γ-heregulin is very likely the result of a gene rearrangement between the 5′ region of the ten-m4 gene and the heregulin β gene. The DOC4 gene is identical to ten-m4 and can be activated by cell stress–induced transcription factor CHOP . The COOH terminus of the ten-m proteins contained several amino acid repeats which showed similarities with the rearrangement hot spot elements of E . coli and with a wall-associated protein (WAP) of B . subtilis . Experimental evidence suggests that these proteins may be involved in sugar binding . The mRNAs for all four ten-m genes were present in all tissues analyzed so far. The highest levels of ten-m mRNAs were found in brain similar to the expression of Ten-m/Odz in Drosophila . In many tissues the mRNA bands appeared as different sizes, suggesting that the ten-m genes are alternatively spliced. This was supported by the isolation of further ten-m cDNA clones corresponding to the different mRNA bands seen in Northern analysis (Oohashi, T., and R. Fässler, unpublished observations). The expression of Ten-m1 protein was further demonstrated by Western blotting and immunohistochemistry. Immunostaining for Ten-m1 confirmed the widespread expression. Moreover, Ten-m1 was expressed in a very restricted manner in the tissues analyzed. For example, in kidney Ten-m1 is only found in the glomeruli and in vessels of the medulla, in lung the expression is particularly high in the smooth muscle layers of the bronchi, veins, and arteries and in the testes Ten-m1 is present in spermatides but not in spermatogonia, mature sperms, or Sertoli cells. The brain sections revealed a distinct signal around neuronal cells (hippocampus, cerebellum, cortex) and a very strong signal in fiber tract regions. A similar staining pattern was also observed in the CNS of developing and adult flies. Similarly to Drosophila ten-m/odz , ten-m1 mRNA is expressed in nerve cell bodies and the protein in axons, suggesting that Ten-m1 may also have an axonal localization signal (Zhou, X.-H., T. Oohashi, and R. Fässler, unpublished observations). The lack of a signal peptide and the presence of a single stretch of hydrophobic amino acids COOH-terminal of the start codon suggested that the ten-m proteins in mouse are expressed as type II transmembrane proteins . The recombinant expression of various parts of Ten-m1 alone or fused to the AP reporter provided strong evidence that Ten-m1 is a dimeric type II transmembrane molecule, in which the subunits are covalently linked to each other by cysteines in EGF-like domains 2 and 5 . This finding is unexpected since Ten-m can be isolated as a soluble protein from the supernatant of Drosophila Schneider cells. It is also in contrast to a report describing the isolation of Odz as a type I phosphotyrosine-containing transmembrane receptor from the same cell line. Although we do not have direct evidence for secreted versions of the ten-m proteins, the presence of dibasic amino acids in the region which links the transmembrane domain to the first EGF-like domain suggests that ten-m proteins may be cleaved from the membrane. This could explain the presence of Ten-m/Odz in the supernatant of Drosophila Schneider cells . Interestingly, we isolated splice variants of ten-m cDNAs which lacked the linker region (Oohashi, T., and R. Fässler, unpublished observations), suggesting that ten-m proteins may be expressed in a form which does not allow cleavage of the extracellular domain. Our finding that the NH 2 -terminal part of Ten-m1 including the short hydrophobic domain has the ability to locate an AP reporter protein at the outside of the cell membrane suggests that this region of the protein contains all the information necessary to insert the protein into membranes as a type II transmembrane molecule. These results are different from the sequence interpretation of Odz which described a transmembrane domain COOH-terminal of the EGF repeat domain. However, this sequence is interrupted by charged amino acids and, therefore, is not entirely hydrophobic, which is unusual for transmembrane domains. Furthermore, the transmembrane domain of Odz is poorly conserved in the mouse ten-m proteins. In addition to charged amino acids, all four mouse ten-m proteins contain a conserved potential N-glycosylation site in this region. Finally, the secreted form of the recombinant mouse Ten-m1 contained this stretch of amino acids but we never observed integration of the recombinant protein into the cell membrane of transfected HEK 293 cells. Therefore, we believe that even in Drosophila Odz this region cannot be used as a transmembrane domain for a type I insertion. The ultrastructural analysis of the extracellular domain of Ten-m1 and the expression of recombinant fusion proteins composed of an AP reporter and either the first three or all eight EGF-like domains revealed that ten-m proteins are expressed as dimers covalently linked by a pair of free cysteine residues in EGF repeats 2 and 5. Negative staining of the extracellular domain in the EM revealed that the large COOH terminus of Ten-m1 is divided into three globular domains preceded by a fine rod which represent the EGF repeat domains. The EGF repeat domain of all ten-m proteins is composed of eight EGF-like domains which lack Ca 2+ binding consensus motifs and are most similar to the EGF-like repeats of the ECM molecule tenascin-C . The predicted length of the complete EGF repeat domain is 16 nm and of the first five EGF-like repeats 10 nm . This is in agreement with the EM observation that the extended rods which connect the two monomers are ∼9 nm long. In EGF-like domains 2 and 5 the third cysteine is replaced by either a tyrosine or phenylalanine residue. This cysteine substitution is conserved in all four mouse ten-m proteins and in the Drosophila Ten-m/Odz. In a normal EGF repeat, the first cysteine would make a disulfide link to this third cysteine residue. Since it cannot be engaged in such a link in EGF-like domains 2 and 5 of the Ten-m proteins, it conceivably can form an intermolecular disulfide bond with another ten-m molecule. To our knowledge it has never been reported that an EGF repeat domain can covalently interact with another EGF domain. However, it cannot be excluded that an EGF-like domain with only five cysteine residues will loose the typical EGF-like folding pattern when it is engaged in intermolecular covalent interactions. Clearly, more structural studies are needed to resolve the folding pattern of the EGF-like repeats in its dimerized form. The only other molecule which shares this incomplete cysteine pattern in modules within an EGF repeat array is Drosophila Ten-a , which appears to have a topography very similar to ten-m molecules, but terminates shortly after the EGF repeat domain. The EGF repeat domain of Ten-m/Odz and Ten-a are more similar to each other than to any of the four mouse ten-m sequences (Oohashi, T., and R. Fässler, unpublished observations). Therefore, in addition to the ten-m family it is possible that a ten-a family of mammalian orthologues exists. The finding that ten-m proteins are expressed as type II transmembrane molecules and the immunolocalization of Ten-m1 and DOC4/Ten-m4 on cells suggest that they function as cellular signal transducers. Additional support for a signaling function of the ten-m proteins comes from the fact that Odz was isolated in Drosophila as a phosphotyrosine-containing transmembrane protein . The high structural similarity of Drosophila Ten-m/Odz together with our experimental findings with the recombinant expression of the NH 2 terminus of mouse Ten-m1 would suggest that Ten-m/Odz is also expressed as a type II rather than a type I transmembrane protein. The presence of several tyrosines in the putative intracellular domain of the mouse ten-m molecules as well as of the Drosophila Ten-m/Odz would allow phosphorylation to occur. Tyrosine phosphorylation of intracellular domains of transmembrane proteins is often associated with intracellular signaling events . The potential intracellular domains of the mouse ten-m molecules contain between 8 and 10 tyrosine residues, respectively. To be able to visualize potential Ten-m1 ligand(s) in tissue sections and show binding on Far Western blots, we expressed and purified a recombinant fusion protein in which the AP module was fused to the extracellular domain of Ten-m1. This fusion protein bound strongly to specific regions in several tissues which, especially in the nervous tissue, overlapped with the immunostaining. Far Western blots and electron micrographs indicated that like other membrane molecules in the nervous system, for example NCAM and NgCAM/L1 , Ten-m1 is able to interact homophilically. Such interactions could influence a wide variety of processes including cell migration, neurite extension, and fasciculation. However, since the staining patterns of immunohistochemistry and affinity histochemistry were not completely overlapping, heterophilic interactions of Ten-m1 with other, so far unidentified ligands are likely to occur also. Additional heterophilic interactions have been observed as well for NCAM and especially for NgCAM/L1 which is able to interact with several other molecules including laminin, proteoglycans, other neural cell adhesion molecules, and integrins . The presence of an RGD containing motif in Drosophila Ten-m/Odz together with results from cell adhesion and cell spreading assays using Ten-m/Odz–derived peptides suggested an interaction with specific splice variants of the Drosophila PS2 integrins . We could not observe an altered adhesion or spreading behavior when we did adhesion assays with a β1 integrin– deficient cell line and recombinant mouse Ten-m1 (Fässler, R., unpublished observations). Furthermore, none of the mouse ten-m proteins contains an RGD motif which is frequently used for integrin engagement . The mechanism by which ten-m molecules may transduce signals from the extracellular environment into the cell is not obvious. The activation of many signaling receptors is mediated by ligand-induced dimerization. Since ten-m molecules are already dimerized before ligand binding, an alternative activation could be associated with a conformational change induced by ligand binding which allows intracellular protein interactions and phosphorylation. A similar signaling cascade has been suggested for integrins . Furthermore, the cytoplasmic domains of the ten-m proteins contain several proline-rich regions which could serve as docking domains for intracellular SH3-containing proteins . Another way to transmit signals into the interior of the cell could occur via proteolytic cleavage of the ten-m molecules in the linker domain close to the cell surface. All mouse ten-m proteins contain several dibasic sites in the linker domain and one of them is conserved in all sequences as well as in the Drosophila Ten-m/Odz and Ten-a. Dibasic sites have been observed frequently as recognition sites for a family of mammalian proteases with similarity to bacterial subtilisin . A proteolytic cleavage in this domain would release the cytosolic part of the molecule from a dimeric into a monomeric state, which might alter its ability to interact with other molecules or its accessibility for kinases and phosphatases. Evidence for a proteolytic cleavage was reported for Drosophila Ten-m by Baumgartner et al. . Another observation which indicates proteolytic processing events in the linker domain is the occurrence of a soluble form of γ-heregulin in the supernatant of the breast cancer cell line MDA-MB-175 . Since γ-heregulin and mouse Ten-m4 share marked homologies in their NH 2 termini, including their linker regions, the potential for shedding of the extracellular domain may be a characteristic of all the ten-m family members. Do signaling cascades also modulate pair rule gene activity in Drosophila ? During the syncytial blastoderm stage gap genes and pair rule genes are directly regulated by the spatial and temporal expression of upstream transcription factors. In the cellular blastoderm stage, when cell membranes form and cellular boundaries are established, the regulation of gene expression may become dependent on signal transduction. In elegant genetic and biochemical experiments it was shown that several pair rule genes including even-skipped , fushi tarazu , and runt are regulated by the soluble ligand unpaired which activates a Janus kinase (JAK; hopscotch)/signal transducer and activator of transcription (STAT; Stat92E) signaling pathway . Although current models of the JAK/STAT signaling pathway propose that the activation of JAK, phosphorylation of STAT, and finally regulation of gene transcription are preceded by ligand-induced dimerization or multimerization of transmembrane receptors, experimental evidence for this speculation is lacking, and other signal transduction mechanism, like conformational changes in transmembrane molecules, cannot be ruled out. In Drosophila , pair rule genes including ten-m/odz initiate a genetic program which is essential for the normal segmentation. The expression pattern of Ten-m/Odz in adult flies suggests further functions in many organs which have not yet been elucidated. Our data using immunostaining of various tissues derived from adult mice also indicate that the ten-m family has a widespread expression. At present, however, we are unable to exclude that the antiserum raised against the extracellular domain of Ten-m1 does not cross-react with other family members. A possible function for Ten-m4 has been reported by Wang et al. who searched for stress response gene which are dependent on the activity of the transcription factor CHOP. Using representational difference analysis, they isolated the DOC4 cDNA which is identical to the ten-m4 cDNA. Although DOC4/Ten-m4 expression is induced by cellular stress, it is not clear which function is propagated by this protein. To obtain direct evidence for possible roles of ten-m proteins during development and disease we have isolated the mouse ten-m genes and have generated mutant mice. The analysis of these mice will provide further insights into the function(s) of these interesting molecules. | Study | biomedical | en | 0.999996 |
10225958 | Rat anti–mouse occludin mAb (MOC37) was raised and characterized as described previously . Mouse anti-neurofilament 200-kD mAb and rat anti-FLAG mAb were purchased from Boehringer Mannheim Biochemicals and Eastman Kodak Co. , respectively. Mouse L cells and their transfectants were grown in DME supplemented with 10% fetal calf serum. Kidney total RNA was isolated according the method described by Chomczynski and Sacchi . Poly(A) + was prepared from the total RNA using oligo-dT cellulose ( New England Biolabs, Inc. ). First strand cDNA was prepared from this poly(A) + RNA with Superscript™ II reverse transcriptase ( GIBCO BRL ) and used for PCR. Claudin-11/OSP cDNA was amplified from mouse kidney first strand DNA by PCR using the previously reported sequence of mouse OSP . The amplified cDNA was subcloned into pGEM-T Easy Vector ( Promega ). The lack of sequence errors was confirmed by DNA sequence analysis using a Dye Terminator Cycle Sequence Kit (Applied Biosystems, Inc.). Mouse claudin-11/OSP was tagged with FLAG-peptide at its COOH terminus. To construct FLAG-tagged claudin-11/OSP-expression vectors, EcoRI site was introduced at the 3′-end of claudin-11/OSP cDNA by PCR, and amplified fragments were subcloned into pBluescript SK(−)- Flag-tag. The insert was excised by SalI-XbaI digestion followed by blunting with T4-polymerase, and then introduced into pCAGGSneodelEcoRI , which was provided by Dr. J. Miyazaki (Osaka University). Mouse L cells were used for transfection as described previously , and the clones stably expressing tagged protein were screened by fluorescence microscopy with anti-FLAG mAb. The expression of claudin-11/OSP in various mouse tissues was examined by Northern blotting using a Mouse Multiple Tissue Northern Blot ( Clontech ). The DNA fragment of mouse claudin-11/OSP (entire ORF) was radiolabeled with [ 32 P]dCTP and used as a probe for Northern blotting. Hybridization was performed in ExpressHyb™ Hybridization Solution ( Clontech ) at 68°C for 12 h. The membranes were washed with 2× SSC containing 0.1% SDS at room temperature for 30 min and then with 0.1× SSC containing 0.1% SDS at 50°C for 30 min. The membranes were exposed to imaging plates for 12 h, and the signals were visualized using a BAS2000 Bio-Imaging Analyzer (Fuji Photo Film Co. Ltd.). A polypeptide, CNRFYYSSGSSSPTHAKSAHV, corresponding to the COOH-terminal cytoplasmic domain of mouse claudin-11/OSP was synthesized, and coupled via cysteine to keyhole limpet hemocyanin. This conjugated peptide was used as an antigen to generate polyclonal antibodies (pAb) in rabbits. The antiserum was affinity-purified with glutathione- S -transferase (GST) fusion protein with the COOH-terminal cytoplasmic domain of claudin-11/OSP. Lysates of E. coli expressing GST/claudin fusion proteins were subjected to one-dimensional SDS-PAGE (12.5%), according to the method of Laemmli , 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 second antibodies and streptavidin-conjugated alkaline phosphatase ( Amersham Corp. ). Nitroblue tetrazolium and bromochloroindolyl phosphate were used as substrates for detection of alkaline phosphatase. L transfectants plated on glass coverslips were fixed with 1% formaldehyde in PBS for 10 min at room temperature. Mouse brain and testis were frozen using liquid nitrogen, and frozen sections ∼10–20 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. These samples were processed for immunofluorescence microscopy as described previously . Specimens were observed using a fluorescence microscope, Zeiss Axiophot photomicroscope ( Carl Zeiss, Inc. ), or a Bio-Rad MRC 1024 confocal fluorescence microscope (Bio-Rad Laboratories) equipped with a Zeiss Axiophot photomicroscope. For each stereoscopic image , 30 optical sections (0.3–0.4-μm interval) were accumulated in the computer. For conventional freeze-fracture analysis, tissues or cultured L fibroblasts were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 3 h at room temperature, 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). The samples were then immersed in household bleach, and replicas floating off the samples were washed with distilled water. Replicas were picked up on formvar-filmed grids, and examined with a JEOL 1200EX electron microscope (JEOL) at an acceleration voltage of 100 kV. The immunoelectron microscopic technique for examining freeze-fracture replicas was described in detail previously , except that samples were frozen in a high-pressure freezer (Baltec HPM010; Bal-Tec). Immunoelectron microscopy using ultrathin cryo-sections was performed essentially according to the method developed by Tokuyasu . Samples were examined with a JEOL 1200EX electron microscope (JEOL) at an acceleration voltage of 80 kV. Using the previously reported nucleotide sequence of mouse OSP , we amplified a full-length cDNA encoding mouse OSP by PCR, and confirmed that its open reading frame encoded a protein of 207 amino acids with a calculated molecular mass of 22.1 kD. OSP showed rather weak sequence similarity to claudins: it was almost equidistantly related to previously identified members of the claudin family (claudin-1 to -8; ∼30% identity at the amino acid sequence level to each member). As shown in Fig. 1 , comparison between OSP and claudin-1 revealed that identical amino acids were almost evenly distributed throughout these molecules. Next, we introduced cDNA encoding OSP with a FLAG- sequence at its COOH terminus into cultured L fibroblasts which lacked TJs or the expression of claudins . Immunofluorescence microscopy of the stable transfectants with anti-FLAG mAb showed that expressed FLAG-OSP was concentrated at cell–cell borders as planes or on thin cellular protrusions . This mAb gave no signal from parent L fibroblasts. Then, these stable L transfectants expressing FLAG-OSP were fixed with glutaraldehyde and examined by conventional freeze-fracture electron microscopy . In these cells, TJ strand/groove-like structures were frequently observed to be arranged in a parallel manner, whereas in parent L cells these structures were not detected. These strands were associated with the P-face, and were mostly discontinuous with intervening spaces of various widths . On the E-face, complementary continuous grooves were identified, containing scattered particles . The OSP-induced strands did not bifurcate frequently and showed a tendency to run parallel to each other. These characteristics of OSP, i.e., sequence similarity to claudins and ability to induce TJ strand/groove-like structures in L fibroblasts, led us to regard OSP as a member of the claudin family, and we tentatively designated it as claudin-11 after consulting the human gene nomenclature committee ( http://www.gene.ucl.ac.uk/nomenclature/ ). (In the updated human gene database, at least 15 members of the claudin family were found, all of which were already assigned to claudin-1 to -15.) We next examined the expression of claudin-11/OSP in various tissues by Northern blotting. As shown in Fig. 3 A, claudin-11/OSP mRNA was detected as a 2.3-kb band in large amounts in the brain and testis and in only a trace amount in the kidney, suggesting that this type of claudin is involved in the TJ formation specifically in the brain and testis. Then, to examine the subcellular distribution of claudin-11/OSP, we generated polyclonal antibodies (pAbs) in rabbits using a synthesized peptide corresponding to COOH-terminal 20 amino acids of claudin-11/ OSP as an antigen. By immunoblotting, one affinity-purified pAb (pAb CL11-2) specifically recognized the GST fusion protein with COOH-terminal cytoplasmic domain of claudin-11/OSP, but not that of claudin-1 to -8, which were produced in E. coli . Using this pAb, we first examined the subcellular distribution of claudin-11/OSP in the brain. When frozen sections of mouse brain were immunofluorescently stained with anti–claudin-11/OSP pAb, a large number of intensely stained linear structures were seen scattered in random directions in the cortex . In deeper regions, these linear structures were occasionally arranged in a parallel manner to form thick bundles . In the brain, TJs are known to be developed in vascular endothelial cells. Since these TJs were stained positively with anti-occludin antibody, we then stained frozen sections of brain doubly with anti–claudin-11/OSP pAb and anti-occludin mAb. However, as shown in Fig. 4 , c and d, the claudin-11–positive linear structures did not overlap with occludin-positive endothelial TJs. Then, frozen sections of the brain cortex were doubly stained with anti–claudin-11/OSP pAb and anti-neurofilament mAb, examined by confocal microscopy, and stereoscopic images were generated by computer . At higher magnification, each claudin-11/OSP-positive linear structure was seen to run in a gentle spiral around a neurofilament-positive axon. These images suggested that these claudin-11/OSP signals were derived from myelin sheaths surrounding individual axons. As reported previously , conventional freeze-fracture electron microscopy of glutaraldehyde-fixed mouse brain revealed that so-called interlamellar strands of oligodendrocytes were arranged in a gentle spiral around axons . Similarly to TJ strands in L transfectants expressing claudin-11/OSP , these interlamellar strands were associated with the P-face, and were mostly discontinuous with intervening spaces of various widths . When freeze-fracture replicas from unfixed brains or optic nerves were immunolabeled with anti–claudin-11/OSP pAb, these interlamellar strands were specifically labeled . Furthermore, as shown in Fig. 7 , when ultrathin cryo-sections of the brain were incubated with the same pAb, transverse sections of myelin sheaths were labeled radially, which may correspond to the so-called radial component of myelin . Finally, frozen sections of mouse testis were doubly stained with anti–claudin-11/OSP pAb and anti-occludin mAb. As shown in Fig. 8 , a and b, both claudin-11/OSP and occludin were concentrated and precisely colocalized in a linear fashion at the most basal region of lateral membranes of adjacent Sertoli cells where TJs were reported to be well developed. Endothelial cells of microvessels were occludin-positive, but claudin-11/OSP-negative . Immunofreeze-fracture electron microscopy revealed that claudin-11/OSP was exclusively localized on well-developed TJ strands of Sertoli cells . Interlamellar strands (radial components) of myelin sheaths are of great interest in terms of the physiological functions of oligodendrocytes . Although their appearance on freeze-fracture images is similar to those of epithelial and/or endothelial TJs, TJ-specific proteins, especially occludin, have not been shown to be localized at these interlamellar strands. On the other hand, we recently identified novel gene family members, claudin-1 to -8, which are major constituents of TJ strands, and found that single or several claudins are copolymerized to form TJ strands in various tissues . However, Northern blotting revealed that they were not expressed in large amounts in the brain as compared with non-neuronal tissues . In this study, we found that OSP (oligodendrocyte-specific protein) shows a sequence similarity to claudins, although the degree of identity was at most ∼30% . OSP was first identified as a protein specifically expressing in oligodendrocytes, but it has not been characterized in detail . We have demonstrated that OSP has the ability to form TJ strand-like structures in L fibroblasts, and that it is exclusively localized at interlamellar strands. Based on these observations, we concluded that interlamellar strands can be regarded as a variant of TJ strands composed of a new claudin, claudin-11/OSP. As previously reported, when claudin-1 or -2 was singly expressed in L fibroblasts, TJ strands were induced, and through their ramification a huge network of strands was formed . In contrast, the claudin-11/ OSP-induced strands scarcely branched in L fibroblasts, and showed a tendency to run parallel to each other. Interestingly, interlamellar strands run parallel without branching , while typical TJ strands in epithelial cells frequently ramify to form networks . These findings favored the notion that claudin-11/ OSP is a major constituent of interlamellar strands (and also TJ strands in Sertoli cells) and that the frequency of ramification of strands depends on the intrinsic nature of each claudin species. It is intriguing that TJ strands in oligodendrocytes (interlamellar strands) share a specific claudin species, claudin-11/OSP, with those in Sertoli cells. In the testis, well-developed TJ strands in Sertoli cells (so-called Sertoli junctions) closely protect spermatogenic cells from the external environment, which is known as the blood-testis barrier . Similarly to the brain, Northern blotting analysis indicated that the testis does not express claudin-1 to -8 in large amounts . Therefore, it is likely that in the testis claudin-11/OSP is a major component of TJ strands, which function as a tight barrier, and that in the brain interlamellar strands of oligodendrocytes also function as a barrier to closely isolate the extracellular compartment within myelin sheaths. In the central nervous system, neurons are also closely protected from the external environment, but this blood-brain barrier is known to be established by TJ strands in vascular endothelial cells in vertebrates . However, in invertebrates such as insects, TJs of the perineurial sheath in the central nervous system and/or pleated septate junctions (possible invertebrate equivalent of TJs) of glia cells were reported to be directly involved in the blood-nerve barrier . It is still premature to discuss this issue further, but it would be interesting to examine the phylogenetical relationship between claudin-11/ OSP and the other claudins in future studies. Sertoli junctions were initially called “the junctional specializations of Sertoli cells” , and thought to be different from typical TJs in other epithelial cells . These Sertoli junctions were characterized by (a) discrete bundles of filaments running parallel to the junction membranes, (b) cisternae of the endoplasmic reticulum located deeper to the layer of filaments, and (c) the parallelly arranged strands that did not anastomose extensively. However, recent findings on the localization of ZO-1 , symplekin , and occludin in Sertoli junctions indicated that this specialization is a variant of TJs found in many other epithelia. This study, i.e., the occurrence of claudin-11/ OSP in Sertoli junctions, conclusively supported this notion. Another issue that we should discuss here is the sequence differences in the cytoplasmic tail between claudin-11/OSP and the other claudins. All of claudin-1 to -8 end in -Y-V at their COOH termini , whereas only claudin-11/OSP ends in -H-V. In this sense, claudin-11/OSP is rather distantly related to the other claudins. The COOH-terminal -E-S/T-D-V motif in the Shaker K + channel (-ETDV)/NMDA R2 subunit (-ESDV) and -E-Y-Y-V motif in neurexins were shown to be responsible for their binding to PDZ domains of Dlg/PSD-95 family proteins and LIN-2/CASK, respectively . Considering that three PDZ-containing MAGUK family proteins, ZO-1, ZO-2, and ZO-3, are exclusively concentrated at the cytoplasmic surface of TJ strands in epithelial and endothelial cells , it is expected that the COOH-terminal -Y-V in claudin-1 to -8 binds to some of the PDZ domains of the MAGUK family proteins to recruit them to TJ strands. So far, occludin has been regarded as a membrane binding partner for ZO-1 and ZO-2 , but it was shown that even in occludin-deficient TJ strands ZO-1 (and also ZO-2; unpublished data) was still recruited . These findings are consistent with the above expectation, and led to the further speculation that the COOH-terminal -H-V of claudin-11/OSP shows distinct affinity to ZO-1, ZO-2, and/or ZO-3 from the other claudins. Actually, ZO-1 and ZO-2 were not concentrated at the interlamellar strands of oligodendrocytes . In this study, we found that although the amino acid sequence of claudin-11/OSP is fairly diversified from the eight previously identified claudins, it has the ability to induce TJ strands in L fibroblasts and constitutes the interlamellar strands in oligodendrocytes as well as TJ strands of Sertoli cells in the testis. Molecular manipulation of claudin-11/OSP including targeted gene disruption will more directly unravel not only the physiological functions of this unique claudin family member in the brain and testis, but also the etiologies of some myelin-related as well as spermatogenesis-related disorders. | Study | biomedical | en | 0.999998 |
10225959 | Pharmacological agents: 2,3-butanedione 2-monoxime (BDM; 1 Sigma Chemical Co), an inhibitor of actin–myosin interactions, and 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine (H7; Sigma Chemical Co. ), a broad specificity kinase inhibitor, were dissolved in water as stock solutions of 500 and 30 mM, respectively. KT 5926 ( Calbiochem Corp. ), an inhibitor of myosin light chain kinase, was dissolved in DMSO as a stock solution of 6 mM. Stock solutions were used to make working dilutions of each drug in serum free tissue culture medium. Freshwater molly fish, Poecilia sphenops , from a local pet shop, were kept in tanks at ambient room temperature. Marine killifish, Fundulus heteroclitus , were kept in recirculating seawater tanks at the Marine Biological Laboratory (Woods Hole, MA). Primary cell cultures of fish epidermal keratocytes from both species were similarly prepared as previously described . Molly fish cells were used in all experiments except for the high-resolution microscopy studies, which used killifish cells. Nonwrinkling, elastic substrata in Rappaport chambers (22-mm diameter Pyrex tubing cut into 8-mm cylinders) were made as previously described , and used for quantitative traction mapping. Vaseline/lanolin/paraffin wax (VALAP) was substituted for vacuum grease to make a better cover glass seal at the chamber's base. Traction mapping is a computer deduction of the spatial distribution of cell traction stresses, based on reproducing the observed patterns of multiple particle displacements in a two-dimensional material with elastic properties. An individual traction stress generated by the cell against the substratum is represented as an arrow, whose length and direction is proportional to the magnitude and direction of that stress. Confidence in the traction data is measured by a statistical test of the data robustness after the introduction of random Gaussian noise to the bead displacement data. Since addition of random noise to the displacements could be in any direction, it was necessary to repeat this operation for a sufficiently large sample of trials to yield a statistical sample of related traction maps. The results of 10 of these independent simulations are summarized as a cluster of four maximum likelihood error vectors at each node. The box defined by the pointed ends of the four bootstrap vectors gives a measure of the uncertainty of both the direction and magnitude of the computed traction stress. That is, if one does a series of independent trials in which the maximum likelihood displacement arrows are perturbed by adding noise as described above, then the pointed end of the recomputed traction vectors lie inside this box 66% of the time. In particular, the “angle of spread” defined by the bootstrap vectors is a graphical device to indicate on the map how well traction directions may be resolved above background noise. Thus, a spread of 360° is indicative of a poor signal/noise ratio. Conversely, one would place high confidence in a spread of as little as 30°. Wrinkling rubber was used for qualitatively monitoring traction forces in the presence of actomyosin-perturbing drugs and was prepared as previously described , with the following modification. Wrinkling rubber was prepared by glow discharge vulcanization of a small drop of silicone oil (3–4 μl of dimethylpolysiloxane, 12,500 centistokes; Sigma Chemical Co. ), placed at the center of a cover glass that had been precleaned in 8 M HCl, rinsed in distilled water, and dried. Cells in suspension, concentrated in a small drop of tissue culture medium, were allowed to attach to cover glasses with and without an island of wrinkling silicone rubber ∼4 mm in diameter. A cover glass with adherent cells was assembled into a microscope slide flowchamber . The flowchamber represented an improved means of maintaining uninterrupted imaging of cells during changes of the incubating medium. Only the flat, wrinkle-free central area of the silicone rubber island was used for cell observations. Relative wrinkling levels were measured in an unbiased and semiautomated way. First, a Sobell edge-finding algorithm was applied to the raw video images to render pixels occupied by wrinkles as black (zero gray level). The area of the film wrinkled was then expressed as the percentage of black pixels counted in each panel, after manually correcting for the presence of cell edges and other black, nonwrinkle objects. The relative degree of wrinkling before and after drug application was displayed as a histogram normalized to 100% wrinkling for control cells. It was not feasible to make large numbers of traction maps for the drug experiments due to the constraints imposed by full traction mapping. These include sensitivity of elastic films to changes in medium in the Rappaport chamber, sampling error implicit in studying small numbers of cells, and time and effort required to map many cells . Instead, we applied the semiquantitative wrinkling assay , modified here for a flowchamber, which is ideally suited to sampling many cells simultaneously while maintaining uninterrupted observation of cell traction force generation during repeated changes in medium. The choice of spacer in the chamber was a compromise between a thin spacer that minimized the “dead volume” (∼200 μl) of the chamber and discouraged unstirred layers during perfusion, and a thick spacer that permitted adequate perfusion of adherent cells located in the narrow region of the chamber between the silicone rubber island and the chamber roof. Unattached cells and loose cell debris were washed away by flushing several changes of tissue culture medium through the flow chamber before image recording. Drug solutions were added to the slide in increasing concentrations by freely dropping them onto the space enclosed by a “berm” of grease on one side of the chamber. A strip of filter paper was used to wick away solutions from the other side of the slide, drawing a stream of fluid through the chamber by capillarity. Only in this way could a single cell be observed at high magnification or a group of cells studied at low magnification during multiple chamber perfusions without interrupting the continuity of video recording. Microscopy, image processing, and analysis were performed as previously described . Video images from a Dage Newvicon camera were captured as digital TIFF files on a Matrox LC imaging card, driven by the time-lapse dialogue of MetaMorph ( Universal Imaging Corp. ). Stacks of TIFF files were replayed as movies to detect motion. Individual TIFF files were removed from stacks for image analysis on Image-1 software ( Universal Imaging Corp. ). Fig. 2 , a and b, shows the relationship between the phase contrast and traction images of a keratocyte locomoting with nearly constant speed and direction. Note that the major traction stresses are directed perpendicular to the cell's direction of locomotion, in a pincer-like pattern. In Fig. 2 c, the small “angle of spread” (good coherence) of four summary bootstrap vectors at a single traction node in the lateral lamella (rectangular inset) indicates strong confidence in substantial, equatorially directed pinching tractions at this location. Conversely, the large angle of spread at a node in the leading lamella (circled inset) indicates traction stresses of a magnitude below the assay's threshold of detection. For comparison, a single keratocyte is shown on wrinkling rubber in Fig. 2 d. The large wrinkle parallel to the direction of locomotion is qualitatively consistent with the equatorially oriented pincer-like tractions exerted in the gliding mode of locomotion. A stable pattern of tractions during 2 min of steady rectilinear gliding locomotion for one cell is seen in Fig. 3 . Minor deviations from a mean pattern may reflect temporal changes in cell shape, adhesion distribution, and velocity as well as the local properties of the substrata. Chrzanowska-Wodnicka and Burridge have shown that 20 mM BDM, and 15 μM KT 5926 can each inhibit the wrinkling of silicone rubber substrata by BalbC 3T3 cells in response to lysophosphatidic acid (LPA), (while 300 μM H7 inhibited myosin light chain incorporation of 32 P). To test whether traction force development in keratocytes is also dependent on the contractility of actomyosin, we carried out a similar wrinkling study. Fig. 4 , a–c, illustrates a single cell locomoting on glass before, during, and after chamber perfusion with 10 mM BDM. Within 5 min of exposure to BDM, keratocytes adopt a stationary but ruffling “fried egg” phenotype , may exhibit a partial loss of the fan shaped lamellipodium in this less polarized morphology but otherwise remain adherent, and spread. Removal of the drug quickly restores the fan shape . Fig. 4 , d–f, illustrates a population of cells on glass before (d), during, and after perfusion with a concentration of 750 μM H7. Again, nonpolarized, immotile “fried egg” cells predominate within 7 min of adding drug . Gliding fan shapes are restored after washing out the drug . Similar morphological and locomotory changes occur in the presence of 100 μM KT 5926, but not with the solvent (DMSO) control. Table I shows how keratocytes locomoting on glass respond to increasing concentrations of BDM, and this data illustrates the extreme sensitivity of a sample of keratocytes to concentrations of BDM >4 mM. Overall morphology and locomotion for this sample of cells is expressed as an index of motility (IM), where IM = (number of locomoting fan-shaped cells/total number of adherent cells) × 100. In general, as the drug concentration and exposure time increased, the IM approached 0 as a greater proportion of the cells lost polarity and stopped locomoting. The effects of BDM are reversible. Cells readily recovered their locomotory phenotype and reestablished a high IM within a short time after drug washout. Individual cells remained adherent to the substratum in the presence of BDM concentrations up to 100 mM, and withstood the shear forces generated by the flowchamber. This indicates that cell adhesion is maintained even when motility is inhibited by BDM. A similar effect on motility was observed for cells on glass in the presence of increasing concentrations of H7, with a maximum effect seen at ∼750 μM (data not shown). Fig. 5 shows the principle of the wrinkling assay for a single keratocyte. The prominent wrinkle characterizing control cells is parallel to the direction of locomotion . This is consistent with the strong pincer-like equatorial tractions previously mapped . This wrinkle relaxed substantially in the presence of 50 mM BDM , indicating a considerable reduction in equatorial tractions; the effect is reversible by removing the drug . A sample of control cells generated a tight network of multiple traction wrinkles (data not shown). Wrinkles diminished after perfusion with BDM or KT 5926 and were reestablished within minutes of washout. Fig. 6 summarizes the relative degree of wrinkling assayed for these two drugs, normalized to control cells. There was a 75–80% reduction in wrinkling in the presence of 100 mM BDM and 0.1 mM KT 5926, with recovery to ∼56% of control values after drug washout. The number of cells sampled in a single field of view was similar for each of the drugs tested, and this number remained constant despite repeated perfusions of the same chamber (mean cell count = 73.8 ± 11.4 for nine trials in three chambers). Slight variation in cell number over time could be accounted for by the passage of locomoting cells into and out of the field of view, by the presence of clumps of cells, or by the occasional detachment of cells during perfusion. Both BDM and KT 5926 reduced wrinkling, but at markedly different concentrations, consistent with the order of efficacy in a similar assay using BalbC 3T3 cells . A clear alteration of the steady state traction distribution is seen in keratocytes that become spontaneously attached to the substratum at their trailing edges. When this happens, the body of the cell continues to move forward, with the result that tails of varying length develop and the cell assumes a fibroblast-like morphology. Fig. 7 , a and b, shows two time points in the process of tail growth for a single cell. This figure clearly shows that, within 50 s, pinching tractions reorient to provide both pinching and significant rearward-directed “propulsive” tractions in the cell body. At the same time, forward-directed “frictional” tractions become visible in the tail. Note that tractions remain negligible at the narrow “midbody” as well as at the leading edge. Fig. 7 , c and d, shows traction maps for two additional stuck cells, one with a very short tail and one with a very long tail. In all cases, the tail remained in place while the cell body continued to slowly advance and exert pinching tractions. The speed of advance of the cell body was diminished compared with the gliding cell, but the magnitude of the mean squared traction generated by the cell was increased by 10–20% (mean squared traction is the area average of the norm of the traction vector). However, in addition to the pinching tractions, the cell body of the stuck cell exerts a distinct component of propulsive (rearward-directed) traction. These propulsive tractions have the same location as the pinching tractions, i.e., in the wings of the cell. Surprisingly, tractions at the leading edge of a stuck cell are not significantly altered from those of the gliding cell (i.e., the tractions at the leading edge remain negligible when the cell is stuck). In all cases of stuck cells, adhesion at the tail eventually ruptured. Subsequent to such rupture, all stuck cells recovered a normal fan shape and resumed the steady state gliding mode. The length of the tail when rupture occurred was unpredictable . The frictional tractions occurring in the tail of stuck cells had a maximal magnitude of ∼12 × 10 3 dyn/cm 2 . The total force propelling the cell body forward against the retarding load of the tail was on the order of 1 mdyn. This value was obtained by integrating stress over the adherent tail of the cell. Fig. 8 a shows a representative traction map for a cell turning clockwise (the turning radius is greater than four cell diameters). This figure shows that during long radius turning there is minimal redistribution of tractions compared with the traction map for steady state locomotion . This is consistent with little or no change to the cell's shape. A similar pattern (in mirror image) was seen for cells turning in the opposite direction (not shown). Fig. 8 b shows the traction map for another cell turning clockwise about a pivot point (O) (the turning radius is less than one cell diameter). Fig. 8 c shows the cell's circular trajectory, based on centroid tracking of successive nuclear profiles (black polygon) over 24 min. Overlapping nuclear profiles recorded during this maneuver are shown superimposed on one another in Fig. 8 d. Note that in executing this turn the cell rotates as well as translates towards the top of the page (left to right from the cell's viewpoint). Cells were induced into this circling behavior by incorporating beads with adsorbed poly- l -lysine into the substratum. A similar pattern (in mirror image) was seen for cells turning in the opposite direction (not shown). An analysis of the traction image of a sharply turning cell will be presented in the discussion. These new results can be employed to enhance our understanding of the keratocyte traction patterns. Our previous results showing the predominant equatorial traction pattern exhibited by gliding cells (the “pinching” pattern) was paradoxical in that the major traction stresses revealed were exerted perpendicular to the direction of keratocyte locomotion . The current study shows how this paradox can be resolved by hypothesizing that the propulsive tractions driving locomotion are normally canceled by adhesive tractions resisting locomotion. This hypothesis was constructed from new data and analysis of tethered cells, in which the propulsive and adhesive components of the traction pattern are clearly well separated. The resolution of net tractions into components can also be applied to turning cells. The traction patterns associated with cells undergoing sharp turns differs markedly from the normal pinching traction pattern and can be accounted for by postulating an asymmetry in contractile activity of the opposed lateral wings of the cell. To understand the motion of a cell from a mechanical standpoint, the forces acting on the cell must be analyzed. The total force acting on a contact-dependent locomoting cell consists of several components. These are schematically depicted in Fig. 9 (top) and comprise reactions (a) to rearward-directed propulsive tractions (a′) exerted by the cell's molecular motors, viscous drag (b) between the cell and the aqueous medium, and reactions (c) to forward-directed frictional tractions (c′) caused by tension in the adhesive bonds to the substratum. Note that the propulsive and frictional components as shown in Fig. 9 (top) may not always be so clearly separated, but rather may be locally commingled such that they are difficult to resolve. The net force required to move a keratocyte smoothly forward in low viscosity medium is negligible because both the inertia of the cell and the viscosity of the aqueous medium are so small. Thus, we conclude from Newton's second law that there is always a nearly perfect global balance of the propulsive and frictional reactive components exerted on the cell by the substratum. Furthermore, by Newton's third law (action and reaction), this means that there will also be a global balance in the forces exerted on the substratum by the cell. Hence, integrating the vectors of a traction image over the entire region of cell-substratum contact will always yield zero. A similar analysis shows that the total torque exerted by the cell on the substratum is also zero. The global balance of forces pertaining in any traction image can be exploited to help identify the magnitude, direction, and location of the specific active stresses provided by molecular motors, and to separate these from corresponding properties of those frictional stresses that resist motion. The “stuck” keratocyte is a case in point. Occasionally, gliding keratocytes become transiently stuck by strong adherence of their trailing margin and develop a remarkably fibroblast-like shape, having a fan-shaped lamella and a distinct tail. Stuck cells provide a natural laboratory wherein the frictional tractions become concentrated to the tail region. Because of the global force balance, the concentration of “frictional” tractions in the tail of such cells implies a reverse enrichment of “propulsive” traction in the main cell body. In such cases, the traction map of the cell body of a stuck cell provides a glimpse of isometric motor activity with much reduced contamination by friction. To help in subsequent analysis of stuck , gliding , and turning cells, we have schematically separated the various contributions to the observed traction pattern into three traction components, pinching, propulsive, and frictional, such that vector addition of these components produces the measured traction pattern. For stuck cells , we first show the so-called pinching tractions, which are concentrated in the wings of the cell and are oriented perpendicular to the usual direction of locomotion. Next, we sketch the propulsive tractions. These are also concentrated in the wings but are oriented so as to push the substratum backward and provide forward thrust to the cell body. Thirdly, we schematically show the frictional tractions concentrated in the tail. Adding these three components together produces a resultant that can be compared with the observed traction images of stuck cells . The pinching and propulsive tractions are identified as being the direct result of motor activity because they are seen to persist under the moving cell body in stuck cells and because they do not oppose forward locomotion. An interesting property of the traction image of stuck cells is that the locus of maximum active traction stress is in the vicinity of the lateral edges of the cell (similar to the gliding cell). The leading edge of the keratocyte is unable to develop detectable propulsive traction stresses, even when the cell is stuck and trying to pull free. Note that the lack of propulsive traction development at the leading edge is similar to the gliding cell . This reinforces the assertion that the major propulsive engines of the keratocyte are localized in the wings. By integrating the rearward-directed component of the traction vector field in several stuck cells, we find that the net propulsive thrust of a keratocyte is ∼1 mdyn (10,000 pN). This compares favorably with the force (∼5 mdyn) required to stall a locomoting keratocyte with a calibrated microneedle and is somewhat greater than the force required to extend a neurite . The root mean squared magnitude of the tractions for a stalled cell (5,000 dyn/cm 2 ) is actually slightly larger than that reported previously for gliding cells . Thus, there is no deficiency in the ability of stuck cells to generate and apply force to the substratum (if anything, these cells are superior to gliding cells in this regard). The stuck phenotype is a case in which the processes governing adhesive debonding at the tail are primary determinants that subsequently induce extensive changes in patterns of force generation and cellular morphology. Similarly, tail release in fibroblasts stimulates retraction-induced spreading . The traction distribution in the stuck phenotype permits an explanation of the paradoxical traction pattern characteristic of gliding keratocytes. A model of the relationship between the stuck and gliding phenotypes is illustrated by comparing Figs. 9 b and 10 a. Essentially, this model shows that the entire difference between these two phenotypes can be explained by a simple change in the distribution of the frictional tractions exerted by adhesive bonds. There is no need to invoke any difference in the activity of the molecular motors (this is implied by the similar magnitude of the root mean squared traction stresses for both stuck and gliding cells). Fig. 10 a illustrates a schematic decomposition of the traction pattern for gliding cells into its components: pinching, propulsive, and the frictional (adhesive) tractions. The observed superposition of these three components is shown as the resultant tractions. Note that the propulsive and frictional components are hypothesized to be identical, to a first approximation, but in opposite directions. (Since gliding cells are adherent, propulsive tractions must exist to overcome this adhesion, although the equatorial tractions may also be employed to peel away the adhesions at the lateral edges, see below.) Based on the visualization of propulsive tractions in the stuck cell, these tractions for the gliding cell are also placed in the wings. Thus, in this hypothesis, the forward directed frictional tractions and rearward directed propulsive tractions perfectly cancel each another. This local cancellation, in which only the pinching tractions remain, explains why gliding cells seem to move so effortlessly. The rearward propulsive tractions necessary to drive locomotion, as well as the frictional tractions resisting movement, are largely hidden in the composite traction image. The decomposition in Fig. 10 a is an oversimplification because local cancellation of propulsive and frictional tractions is clearly not always complete. Some rearward-directed, propulsive traction components are seen in the wings of the cells in traction images shown in Figs. 2 and 3 . However, the notion of local cancellation of propulsive and frictional tractions explains the paradoxical pinching pattern characteristic of gliding keratocytes. A consistent and unexpected finding in all our images of gliding cells is the absence of propulsive tractions at the front of the cell. This does not exclude the possibility that there might be very small propulsive tractions at this locus (“small” in this context is <10% of the pinching tractions). Our data therefore suggests that the leading edge protrudes forward in a process that requires minimal propulsive traction at the front edge . This is surprising when compared with the implied force generating role of filopodia and lamellipodia in other cells, e.g., neurite growth cones. However, in fibroblasts, the situation is not fully resolved: Dembo and Wang find that fibroblasts can exert strong traction under the lamellipodium, while Galbraith and Sheets and Harris et al. found that strong pulling tractions are absent within 5 μm of the leading edge. Several additional observations indirectly support the basic idea that protrusion does not require pulling molecular motors situated at the leading edge. These suggest that forward progress of the keratocyte cell body (nuclear mound), which does require pulling tractions, is decoupled from leading edge extension. de Beus and Jacobson showed that prevention of new β1-integrin–mediated adhesion inhibited lamellar protrusion while permitting continued retraction of the trailing margin. Anderson et al. showed that in the presence of cytochalasin B, the cell body continued moving forward for twice the distance covered by the lamellipodium before all motility ceased. One intriguing model for cytoskeletal force production in keratocytes is the dynamic network contraction (DNC) model based on elegant ultrastructural and fluorescence microscopy studies. In principle, this model must account for the traction image measured for gliding cells. That is, the contractility proposed to be generated by the model in combination with the adhesion distribution for gliding keratocytes should produce the active tractions required for locomotion . These active tractions are shown as the vector sum of the pinching and propulsive tractions , but not the adhesive tractions. Proposed revisions to the DNC model, as discussed below, are given in Fig. 11 b. The dominant feature of the traction images for gliding keratocytes are the pinching tractions. In addition, it is important to realize that equatorial contractility is so pronounced that the entire rear lateral actin meshwork is pulled in, as deduced from the curvature of photoactivated actin bars . As articulated, the DNC model has marked anisotropy producing predominantly propulsive tractions in the direction of locomotion that are used to pull the cell body forward, but it does not directly address the origin of the strong equatorial contractility. One possible way to account for these strong, pinching tractions is to postulate isotropic lamellar contractility . Another possibility is that contractility underlying these tractions may be based on a “sarcomeric-like” contraction, dependent on actomyosin bundles running parallel to the equator that could be produced by dynamic network contraction. Svitkina et al. rejected this structure as being important because its slight convex curvature was thought to generate a force component that resists locomotion. This curvature probably results from the fact that bundles are strongly, but transiently, anchored at the rear lateral margins, often with focal adhesion-like contacts . Nevertheless, the sarcomeric-like mechanism seems like an attractive candidate to explain the main feature of the keratocyte traction image, a strong pinching component perpendicular to the direction of cell motion. Thus, we have depicted it in the schematic of the contractile mechanism . A second characteristic of traction distributions is that the propulsive and pinching tractions are localized predominantly in the wings of the keratocyte. This is seen from the fact that even when cells are tethered, the propulsive tractions appear in the wings and not under the front leading lamella. Dynamic network contraction, which is postulated to produce forces along the axis of locomotion, should be most active in these regions, and we have depicted it as producing propulsive tractions in these regions. There is a large reduction in propulsive traction in the vicinity of the front leading edge. Since adhesion is indicated in this region , the contractility in this part of the lamella must be downregulated to explain this reduction in traction. This is consistent with the relatively high actin network density and low myosin cluster density in the front portion of the lamella . Such reduced contractility could lower cortical tension along the extending margin and lead to enhanced protrusion as required by the graded radial extension model of keratocyte movement . One clear role for the pinching tractions and the structures that produce them is to maintain the fan shape characteristic of keratocytes, since agents that inhibit the pinching tractions also lead to loss of this shape. Furthermore, the use of BDM and KT 5926 suggests that the strong equatorial tractions present in the normal gliding cell are dependent on actomyosin interactions. Significantly, the concentration dependence of BDM's effects , reflected by the rate of spreading in postmitotic Ptk2 cells and by the inhibition of platelet nonmuscle myosin II, is similar to the concentration dependence we observe when BDM inhibits keratocyte motility on glass (Table I ). Three independent assays thus confirm that half maximal inhibition occurs in the presence of 2.5–4 mM BDM. This argues strongly for the dependence of keratocyte shape and locomotion (both are contributing factors in our index of motility) on the presence of functional nonmuscle myosin II. The strong equatorial tractions correspond to regions where close contacts, as revealed by interference reflection microscopy in Fig. 11 a (center), diminish in area at the rear lateral margins (arrows), consistent with the notion that these tractions may play a role in peeling away rear adhesions. This may be compared with the ease with which a piece of adhesive tape may be peeled from a surface by peeling from an edge or corner, rather than the center. In the method reported here, tractions can be calibrated absolutely and the maximal traction stresses correspond well to the failure stress characteristic of amoebae close contacts , supporting the idea that these pincer-like tractions are sufficiently strong to assist in peeling away the trailing margin. The occurrence of pinching tractions may be the result of the biological context (the epithelium of the scale) in which these cells find themselves. Such tractions result from contractile actomyosin structures that are needed for normal epithelial tissue structure and wound healing responses, but become remodeled when individual keratocytes break away from the cell sheet and undergo rapid locomotion. That is, these forces are normally employed to maintain and remodel the epithelial sheet and only when a single cell is released are they partially used to achieve a high, uniform velocity. In this sense, keratocyte locomotion may not be fully efficient. The situation may be analogous to fibroblasts in that the tractions produced by fibroblasts are much stronger than needed for locomotion, prompting Harris et al. to postulate that their primary role was to remodel the extracellular matrix. The difference in orientation of the major traction forces between keratocytes and fibroblasts is most likely related to the relative difficulty of adhesive tail detachment in these cell types, but it also reveals an important difference in the regulation of force orientation between the two cell types. Even when keratocytes become stuck and develop tails, the pinching tractions persist, suggesting that this traction orientation is characteristic of this epithelial cell type. The properties of traction images can also be exploited to clarify the mechanism by which cells regulate adhesion and contractility to control and maintain the direction of locomotion. A turning motion can be considered as the sum of a gliding motion and a pivoting motion about the center of mass. That is, when cells move in circular paths, they must rotate as well as translate, which means that it is necessary for them to exert propulsive torque. In cells that execute very gradual turns, the contribution of the pivoting motion is minor and the underlying traction distribution does not differ substantially from that of a keratocyte “gliding” in a straight line . However, in the case of cells that turn sharply , we are able to discern a stereotypical morphology and pattern of traction that differs radically from that of the gliding cell. To find the simplest way of explaining the turning phenotype, we will analyze the traction image using the decomposition of tractions for the stuck cell as a starting point. Examination of the stuck cell illustrates that the only way to obtain the necessary propulsive torque to turn a cell is to change the propulsive tractions on one side , so that they are slightly stronger than on the other side (R). Note that the propulsive tractions have to change because they are the only ones that have a nonzero moment arm about the center of mass. For simplicity, we propose to obtain this increase while keeping the total magnitude of the motor activity of the cell constant. Geometrically, this means that we can redirect (rotate) some of the pinching tractions on one side of the cell (L) so that they now have a rearward-directed component. In Fig. 10 b, we show pinching and propulsive traction distributions similar to that of a stuck keratocyte (strong in the wings, diminishing towards the center), except that on the left side of the cell (L), we have converted some of the pinching activity into propulsive activity for the reasons described above. As a result of the asymmetric pinching tractions, the cell experiences a force that tends to move it sideways (towards the cell's right), like a crab. Simultaneously, the propulsive tractions exert a clockwise torque and also a force that moves the cell forward in the usual way. This is similar to the pattern of forces acting on a rowboat that is moving crosswind with one oar slightly longer than the other. In Fig. 10 b, we show the necessary frictional (adhesive) tractions that must go along with this kind of propulsive activity. The basic requirement is that these tractions are needed to balance total force in both the x and y directions, and are also needed to balance torque about the center of mass. The force balance basically fixes the magnitude and direction of the total friction force. The concentration of the friction in the left wing of the cell is then dictated because the friction needs to have the longest possible moment arm to achieve torque balance. Finally, we show the vector addition of the three preceding traction fields given as the resultant in Fig. 10 b. This figure reproduces the main features of the experimentally derived traction map for a clockwise-directed turning cell . We conclude from this analysis of the turning cell that the observed behavior can be explained by postulating a slight asymmetry in the motor activity of one side of the cell versus the other. How might molecular mechanisms be translated into the mechanics required to turn the cell? Changes in cell stiffness accompany turning cells . Perturbation experiments have demonstrated that altering the normal patterns of adhesion and contractility will redirect the cell. Disrupting lamellipod adhesion with low doses of antiintegrin antibodies in Xenopus keratocytes results in a redirection of cell movement . In addition, when Anderson et al. asymmetrically disrupted the cytoskeleton by traumatic microinjection on one side of the cell body, keratocytes changed their direction of locomotion. Releasing calcium locally near the lateral edge of a keratocyte causes it to turn towards the source of calcium . These observations demonstrate that asymmetric alterations in contractility and/or adhesion can be the basis for directional control of locomotion. One of the most fascinating challenges will be to relate the changing traction patterns required for cell motility and morphogenesis to the way in which the adhesions and motor activities that produce traction are regulated. | Study | biomedical | en | 0.999998 |
10225960 | The purification of the novel laminin 12 (α2β1γ3) was carried out as follows. Human placental chorionic villi were frozen in liquid nitrogen, ground in a Waring blender, and then washed in 1 M NaCl. Unless otherwise noted, all subsequent steps were performed at 4°C. The final tissue pellet (200 g, wet weight) was suspended by stirring for 48 h in 1 liter of extraction buffer (0.5 M NaCl, 10 mM EDTA, and 625 mg/liter N -ethylmaleimide, 150 mg/liter phenylmethylsulfonyl fluoride, and 50 mM Tris-HCl, pH 7.8). The soluble fraction was collected after centrifugation (30,000 g , 60 min) and precipitated with 300 g/liter ammonium sulfate. The precipitated proteins were collected by centrifugation (30,000 g , 60 min) and dissolved in chromatography buffer (2 M urea, 25 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.8). The sample was then dialyzed against the same buffer. After dialysis, 0.5 vol of buffer-equilibrated DEAE-cellulose (DE-52; Whatman) was added and the mixture was shaken overnight. Material not bound to DEAE-cellulose was collected by filtration on a Buchner funnel (Whatman; filter 4) and precipitated by addition of 300 g/liter ammonium sulfate. The proteins were collected by centrifugation (30,000 g , 60 min), redissolved in Concanavalin A buffer (0.5 M NaCl, 5 mM CaCl 2 , 5 mM MgCl 2 , and 50 mM Tris-HCl, pH 7.8), and dialyzed against the same buffer overnight. The fraction was applied to a 2.5 × 5 cm Concanavalin A–Sepharose column ( Pharmacia ). Unbound material was removed by extensive washing while bound proteins were eluted by successive washing with 10 mM α- d -mannopyrannoside, 1 M α- d -glucopyrannoside, and finally 1 M α- d -mannopyrannoside ( Sigma Chemical Co. ). Laminins are typically recovered in the latter two fractions; each fraction was independently concentrated to 10 ml with an Amicon concentrator (30-kD membrane) and applied to a 2.5 × 100 cm Sephacryl S-500 column in 0.5 M NaCl, 50 mM Tris-HCl, pH 7.8. The fractions of interest were pooled, dialyzed against Mono-Q buffer (0.1 M NaCl and 25 mM Tris-HCl, pH 7.8), and applied to a 1 × 5 cm Mono-Q column ( Pharmacia ). Elution was achieved with a 60-ml 0.1–0.5-M NaCl gradient. The fraction eluted at 250 mM NaCl was taken for further study. Protein sequencing was performed with minor modifications of published methods . In brief, laminin 12 was resolved on a polyacrylamide gel in the presence of 2-mercaptoethanol. The bands at 205, 185, and 170 kD were excised separately, digested with trypsin, and then separated by HPLC and sequenced on an Applied Biosystem sequenator. Analysis of a trypsin digest of laminin α2 isolated from laminin 12 was performed with matrix-assisted laser desorption time-of-flight mass spectrometry performed on a Finnigan Lasermat 2000 . By comparison of the laminin γ1 amino acid sequence with the dbEST database using the program BLAST one clone was chosen as a possible candidate for a new laminin γ chain. To extend the cDNA, specific primers for 5′ or 3′ extension were deduced from a previously published expressed sequence tag . Nested PCR on placental Marathon-Ready cDNA ( Clontech ) were performed following the manufacturer's instructions using the supplied nonspecific primers with the following gene-specific primers: for the 5′ extension; in the first round, (5′-dCGCATGTGCCGTTCTCGTGGCACTGG); in the second round, (5′-dGCGGCAGGTGCACTGTCCAGTCTTGG); for the 3′ extension, in the first round, (5′-dTGCACGGGACTGCAGCCGCTGCTACCC); in the second round (5′-dGCTGCTACCCTGGCTTCTTCGACCTCC). For PCR, the Long Expand PCR Kit ( Boehringer Mannheim ) was used with the following conditions: denaturation, 94°C for 3 min; 10 cycles of 94°C for 30 s, 63°C (−0.5°C per cycle) for 30 s, 68°C for 4 min; 25 cycles of 94°C for 30 s, 58°C for 30 s, 68°C for 4 min (+10 s per cycle); a final extension period at 68°C of 8 min. The PCR samples from the first round were purified (PCR Purification Kit; Qiagen) and 2% of the sample volume was used in the second round of PCR using the same PCR protocol. These PCR products were purified from an agarose gel (Gel Purification Kit; Qiagen) and either subcloned (into PCR II or PCR 2.1 vectors; Invitrogen) or directly used for sequencing. To reconfirm the nucleotide sequence and control for PCR-induced nucleotide substitutions, gene-specific primers were used to reamplify the entire γ3 cDNA. A first strand cDNA synthesis kit ( Clontech ) was used to synthesize cDNA from total placental RNA using oligo dT, random, or specific human laminin γ3 antisense primers following the manufacturer's protocol; PCR was used to generate overlapping clones complementary to the entire human laminin γ3 chain. Sequencing of all the obtained PCR products revealed the nucleotide sequence of laminin γ3 from what we eventually inferred as nt 297 to nt 5020. However, all further 5′ extensions failed to extend the sequence further toward the 5′ end. The sequence of the 5′ end of the cDNA was determined from the human genomic P1 clone DMPC-HFF#1-1461F2, which was obtained from a PCR-based library screen performed at Genome Systems, Inc. The oligonucleotide primers that were provided to Genome Systems specifically amplify exon 2 of the human γ3 gene (sense primer, 5′-dCCCCGCAGGGGAAGGCGGGTCCTG; antisense primer, 5′-dGGCTTATGAGATCACGTATGTGAG). To obtain the sequence of the missing 5′ end, the genomic clone was sequenced (in 4% DMSO) with gene-specific antisense primers. The sequence of the laminin γ3 5′ untranslated region was confirmed by RT-PCR from placental RNA in 4% DMSO, (sense primer, 5′-dCGCGCGGCGTCGGTGCCCTTGACC; antisense primer 5′-dGCTTGTAGATGGCAAAGCTCTCAGG). Nucleotide sequences were determined with a Thermo Sequenase cycle sequencing kit and 33 P-ddNTP ( Amersham Pharmacia ) using either the M13 forward or reverse primers or gene-specific primers synthesized in our laboratory. A 1:1 ratio of inosine to guanosine was included in the sequencing mix. Sequence data were assembled and manipulated using Genetyx-Max 8.0 and Genestream-1 at http://genome.eerie.fr/home.html (Software Development Co., Ltd.). The signal peptide cleavage site was predicted using http://genome.cbs.dtu.dk/services/SignalP/ . A 956-bp PCR product was generated (Long Expand PCR Kit; Boehringer Mannheim ) from placental cDNA, purified (PCR purification kit; Qiagen), and labeled with [ 33 P]dCTP (NEN) using the rediprime DNA labeling system ( Amersham ). Without further purification, the probe was denatured in the same buffer containing 1/10 (vol/vol) human Cot-1 DNA ( Boehringer Mannheim ), and 1/10 (vol/vol) sheared salmon testes DNA ( GIBCO BRL ) at 94°C for 5 min then chilled before use. Northern blots ( Clontech ) were prehybridized in 50% formamide, 5× SSPE, 1× Denhardt's, 1% SDS, 10% Dextran-sulfate, 0.1 mg/ml salmon sperm DNA ( GIBCO BRL ) at 42°C for 2 h the probe was added and hybridized for 20 h. The blot was washed three times in 2× SSC, 1% SDS at 42°C and two times in 0.1× SSC, 1% SDS at 42°C. Blots were placed on a BioMax MR film ( Kodak ) with a BioMax TranScreen-LE intensifying screen ( Kodak ) for 20 h at −70°C. A cDNA encoding the COOH terminus of human γ3 was cloned into the HisTrx and pPEP-T vectors (kindly provided by Richard Kammerer, Biozentrum, Basel, Switzerland; based on the pET system; Novagen). The HisTrx vector has a histidine-tagged bacterial thioredoxin cDNA as a carrier in front of the cloning site; pPEP-T has a piece of the coiled-coil domain of mouse β1 in front of the cloning site. The γ3 cDNA fragment used was amplified by PCR from human placenta cDNA (see cDNA cloning) using primers that include the EcoRI adapters: forward, 5′-GC GGATC C GAGGAAGCTGAGCGGGTGGGTGCTG-3′; reverse, 5′-GC GAA TTC TTACTGCCAGCTGGCACAGTTCTCGGG-3′. The resultant plasmids were transformed into BL21(DE3) pLysS bacteria (Novagen) and fusion proteins were isolated according to the pET System manual (Novagen). A recombinant fragment containing only histidine-tagged thioredoxin was similarly expressed and purified. The 170-kD band (i.e., γ3 chain) was excised from the reducing SDS-PAGE gel described above and injected into a rabbit for antibody production following standard procedures . The resulting serum (R16) was evaluated by Western analysis and shown to react with the 170-kD γ3 chain, and showed minor cross-reactivity with other laminin chains at high antibody concentrations. All antibody-related studies presented in this communication were conducted at concentrations well below those where cross-reactivity was observed. The histidine-tagged, thioredoxin-γ3 fusion protein was used for the production of a second rabbit antiserum (R21) which reacted with a single band in Western blots of placental extracts. The R16 antiserum was affinity-purified by binding to gel-purified γ3 that had been transferred to nitrocellulose and then eluted with 1 M acetic acid followed by immediate neutralization. The R21 serum was purified by binding to the histidine-tagged, mouse β1-human γ3 fusion protein coupled to activated CNBr-Sepharose; the bound antibodies were eluted with 2 M urea in PBS, or with 1 M acetic acid which was immediately neutralized. The immunofluorescent patterns produced by these two affinity-purified antibody pools were indistinguishable, and were similar to whole R21 serum with reduced background staining. Only affinity-purified preparations of R21 serum were used for these studies. Antibodies made against histidine-tagged thioredoxin were similarly isolated by affinity chromatography from R21 serum; immunofluorescent patterns with these controls were blank. Most tissues were obtained from various colleagues using specimens for other purposes: these include tissues from male and female rats; from normal human tissues discarded after surgery; and from rhesus monkey, Macaca mulatta . Bovine tissues were purchased from a local slaughterhouse. Dissected and blocked tissues were placed directly in embedding compound (O.C.T.; Sakura Finetek) and frozen by immersion in liquid nitrogen-cooled isopentane. 10-μm sections were made on a Leica CM 3000 or 3050 and collected on Superfrost slides ( Fisher Scientific ). Sections were air dried and stored at −20°C until use. Just before use, sections were immersed in acetone at −20°C and then rinsed three times in PBS at room temperature. Sections were incubated with primary antibodies diluted in PBS containing: 2% normal goat serum, 0.25% sodium azide, and 0.1% Triton X-100. Sections were incubated overnight at 4°C; they were washed in three changes of PBS (5 min per wash) and then incubated for 45–60 min with secondary antibody coupled to either Cy3, FITC, or Texas red. After incubation, sections were washed and coverslipped in Prolong (Molecular Probes). The sections were imaged on a Leica confocal laser scanning microscope (Leica TCS-NT). The gain was adjusted in each channel of the confocal to assure that there was no bleeding across the channels; this adjustment is performed at the outset of each confocal session. Images were transferred to Adobe Photoshop and cropped for reproduction. The brightness and contrast were adjusted to make printed images similar to that obtained on the microscope monitor. Other primary laminin reactive primaries used were: polyclonal anti-EHS-laminin-1 ( Sigma Chemical Co. ); monoclonal anti-laminin α2 chain ; polyclonal anti-laminin α4 ; polyclonal anti-laminin α5 chain ; two monoclonal anti-laminin β1 chain ; monoclonal anti-laminin γ2 chain . Monoclonal anti-PGP 9.5 (Ultraclone, Ltd.) was used to identify nerves in skin. Secondary antibodies used were: goat anti-rabbit FITC (ICN Pharmaceuticals); goat anti–rabbit-Cy3 (Jackson ImmunoResearch Laboratories). Paraffin sections were processed for in situ hybridizations as previously described in detail . In brief, cRNA probes for the laminin γ3 chain were generated from human γ3 clones; cRNAs were labeled during transcription by the incorporation of digoxigenin-UTP ( Boehringer Mannheim ); ∼1 μg/ml of cRNA was used for hybridization; hybridizations were performed at high stringency . After overnight hybridization, sections were washed (50% formamide, 1× SSC, for 30 min at 60°C) and the unhybridized probe was destroyed by RNase A. The hybrids were detected with an anti-digoxigenin antibody coupled to alkaline phosphatase ( Boehringer Mannheim ). Sections were incubated overnight with anti-digoxigenin diluted 1:1,000 in blocking solution ( Boehringer Mannheim ). After washing to remove unbound antibody, endogenous alkaline phosphatase activity was blocked by washing in levamisole for 10 min; the alkaline phosphatase reaction was carried out overnight at room temperature. SDS-PAGE and electrophoretic transfer of proteins to nitrocellulose with immunoblot analysis were performed essentially as previously described . For the FISH analysis, a 1217 bp cDNA fragment was generated by RT-PCR from placental RNA, using the sense primer 5′-dAGTGCCACTATAACGGCACATGCG and antisense primer 5′-dCTCGTGTCTGCAAGGAGTCTGTCA. The gel band was purified and subcloned (PCR II vector; Invitrogen). After the sequence of the fragment was verified, the resultant plasmid was used for the fluorescent in situ localization of the LAMC3 gene (SeeDNA Biotech, Inc.). Laminin 12 was extracted from human chorionic villi using EDTA and partially purified by a combination of DEAE-cellulose, Concanavalin A, Sephacryl S-500, and Mono-Q chromatography (see Materials and Methods). The final fraction of interest resulting from the above protocol contains multiple laminins. Laminin 12 was resolved from this mixture by SDS-PAGE (3–5% polyacrylamide) under nonreducing conditions. Six bands were resolved . Only the bands at ∼560 kD and at the top of the gel were reactive with a polyclonal anti-laminin antiserum ( Sigma Chemical Co. ; not shown). Therefore, the resolved band at 560 kD was excised, reduced in 10% 2-mercaptoethanol SDS-PAGE sample buffer, and resolved by 5% SDS-PAGE. Three bands were observed with masses of ∼205, 185, and 170 kD . The band at 185 kD reacted with a monoclonal antibody specific to the laminin β1 chain . Each of the three bands was digested with trypsin, the peptides were resolved by HPLC, and selected resolved peptides were sequenced. The sequences obtained are shown in Table I . The 205-kD chain contained three peptides sequence identical to human laminin α2 . On that basis, the band was identified as human laminin α2, despite our observation that the 205-kD band did not react with anti-α2 mAb . The band at 185 kD produced two peptides identical to human β1, and was thereby confirmed as human β1. In contrast to the easy identification of the other two bands, the band at 170 kD contained three sequences not contained within any known laminin chain. The NH 2 -terminal sequence of the 170-kD chain was determined, and it also was novel; i.e., nonidentical to known laminin sequences. As these four sequences from the 170-kD band were derived from an unknown laminin and we had identified the laminin α and β chains, we assumed these sequences were derived from a novel laminin γ chain that we call γ3. The apparent molecular masses for the 205- and 185-kD bands are not consistent with the literature values published for the α2 and β1 chains, respectively. Thus, these bands are indicated in Fig. 1 as α2t, β1t, and γ3t to indicate that they have been processed (truncated). Laminin 2 and laminin 4 were also present in these preparations; when characterized by similar procedures (not described here in detail) they showed molecular masses consistent with literature predictions, suggesting that our preparations were not extensively and nonspecifically degraded. Together these observations suggest that the truncations observed for the γ3-containing molecules may be physiologically relevant. The cDNA sequences of human γ1 and γ2 were used to probe the National Center for Biomedical Information (NCBI) expressed-sequence-tag database (dbEST), and a clone was identified that was homologous, but not identical, to γ1 and γ2. The sequence of this clone was used to design PCR primers for extensions at 3′ and 5′ ends (see Materials and Methods) using human placental cDNA, and additional sequence information was obtained by a combination of genomic DNA and placental cDNA sequencing. The resulting sequence is shown in Fig. 2 . The deduced amino acid sequence contains regions with 100% identity to all three of the peptide sequences obtained from the 170-kD band . The nucleotide sequence reported in this paper has been submitted to GenBank/EMBL Data Bank with the accession number AF041835 . The DNA sequence contains an open reading frame predicting 1620 amino acids, including a 19–amino acid-long putative signal peptide that closely meets the criteria described by Nielsen et al. . The predicted cleavage site was confirmed by protein sequencing of the γ3 NH 2 terminus; this sequence exactly matched the predicted amino acid sequence following the signal peptide. The overall sequence of γ3 is most similar to that of γ1, sharing 52% amino acid similarity with human γ1 . In addition, the amino acid sequence predicted by the γ3 cDNA contains a domain distribution most like that of the γ1 chain. All six domains are represented. Overall, the γ3 chain has 43.6% amino acid identity with the γ1 chain and 34% identity with the γ2 chain. The highest conservation is seen between domains γ1VI and γ3VI . Domains γ3V and III also show considerable similarity to domains γ1V and III and γ2V and III. The predicted γ3 sequence contains nine potential glycosylation sites , only two of which are conserved in both human and mouse γ1. As these conserved sites are contained within the globular domains IV and VI, it is likely that these sites are used physiologically. There is a single RGD sequence (boxed, hatched) within domain II, but this site is not conserved in either human or mouse γ1 and γ2 proteins. The sequence NVDPNAV occurs within the fourth EGF-like repeat of domain III and is a homologue of the nidogen binding site (NIDPNAV) within the same domain of γ1. These sequences differ by only a single conservative amino acid substitution. The γ3 chromosomal location was determined by searching the NCBI Human Genomic Sequencing Index data base with the γ3 cDNA sequence. The sequence is identical to a database, Sequence Tagged Sites , that has been localized to chromosome 9q33-q34. A 1.2-kb γ3 cDNA probe within domains I and II of the predicted protein, the regions of least homology among the γ chains, was used to localize LAMC3 by fluorescent in situ hybridization (FISH) analysis (SeeDNA Biotech, Inc.). The results confirm the localization to chromosome 9q31-q34 . To determine the domains present within α2t, the 205-kD gel band, purified from placenta, was fragmented with trypsin and the resulting peptides were fractionated by HPLC; the masses of the eluted peptides were determined by mass spectroscopy. The ion chromatograms were then evaluated relative to the masses predicted from the published amino acid sequence for α2 in order to determine the NH 2 - and COOH-terminal peptides present within the digest. The results identified a number of tryptic peptides; among these, the peptide LVEHVPGQP(VR), beginning at residue 70 within domain VI, was the most NH 2 -terminal; the peptide GTTMTPPADLIEK, beginning at residue 1367 within domain III, was the most COOH-terminal. These results indicate that α2t is a fragment containing the short arm of the laminin α2 chain. This conclusion is consistent with the observation that the initial peptide sequence identified from α2t was within the short arm domains (above). β1t and γ3t are also short arm fragments, as all the peptide sequences determined for both species are present within the short arm domains. However, the masses of α2t, β1t, and γ3t are greater than predicted for the short arms alone. In addition, α2t, β1t, and γ3t are not separable by gel electrophoresis without the reduction of disulfide bonds. Therefore, this truncated laminin 12 molecule is very likely to contain portions of domain II of all three chains, as the interchain disulfide bonds should lie between these domains. It is of interest to note that domain II of γ3 contains three cysteinyl residues whose bonding partners are not readily identified and are not present in domain II of other laminin chains. These three cysteinyl residues are conserved in mouse γ3 (Albus, A., and R.E. Burgeson, unpublished observation). Whether these cysteinyl residues could form intrachain, interchain, or intermolecular disulfide bonds that in some way contribute to the cleavage of γ3 chain-containing laminins is unknown. Tissue RNA blots and Master Blot dot blots ( Clontech ) were probed with a γ3 nucleotide probe . A single major transcript of ∼5 kb, consistent in size with other laminin γ chains, is present in several of the tissues examined . A small amount of a second larger transcript can also be detected. This larger transcript is most likely due to differences in polyadelylation or due to inefficient splicing. The γ3 chain RNA is abundant in spleen, testis, placenta, lung, and liver; lesser amounts are seen in kidney and ovary . The predominance of a single transcript allowed use of the RNA Master Blot ( Clontech ) to determine expression in a large number of other tissues. On this dot blot, tissue RNA concentrations have been normalized to housekeeping genes. The Master Blot confirms the abundant presence of γ3 transcripts in placenta, adrenal gland, testis, lung, and fetal kidney, but also shows detectable levels of γ3 transcripts in numerous additional tissues, including brain and skeletal muscle. A polyclonal antiserum, R16, was made in a rabbit to the γ3 chain excised from a reduced SDS-PAGE gel similar to that shown in Fig. 1 . Another, R21, was made to recombinant γ3 protein (see Methods). The R16 antiserum recognizes the γ3 chain on immunoblots of placental extracts, but at very high antibody concentrations, it shows some reactivity with the β1 and γ1 chains as well. Thus, as a control, human neonatal foreskin was immunostained with anti-laminin γ1 (polyclonal anti-laminin 1; Sigma Chemical Co. ), anti-laminin γ2 , and with anti-laminin γ3 (R16). Crisp, brilliant fluorescence was observed along the dermal-epidermal junction, and around capillaries with the anti-γ1 antibodies (data not shown), and in the basement membrane at the dermal-epidermal junction with anti-γ2 (data not shown); in contrast, no signal above background was detected using the anti-γ3 reagent (R16) when it was applied at dilutions of 1:250 or more (data not shown). The antigen could not be unmasked by treatment of the cryosections with 2, 4, or 6 M urea, or with 2 M guanidinium-HCl (data not shown). As all known laminin chains have been detected in skin within either the epithelial basement membranes or the vascular basement membranes, these results indicate that the cross-reactivity detected by Western blot analyses using the polyclonal anti-γ3 (R16) antibody was either not apparent by immunohistochemistry, or was below detection at the antibody concentrations used. For the subsequent anatomical experiments (below), R16 was diluted 1:250 or greater to assure no cross-reactivity was occurring. R21, the affinity-purified antiserum to recombinant γ3, was also tested on sections of neonatal foreskin. As with R16, no immunoreactivity was seen (data not shown); thus we conclude that this antiserum has no cross-reactivity with other known γ chains. Neither R21 nor R16 antiserums label the blood vessel basement membranes (see below) consistent with a lack of cross-reactivity to other γ chains. Unlike the lack of anti-laminin γ3 chain immunoreactivity seen in neonatal foreskin, laminin γ3 chain immunoreactivity was detected in human leg skin. As shown in Fig. 6 A, and consistent with published results, laminin γ1 chain reactivity is seen at the dermal-epidermal junction and within the basement membranes of the vasculature, while laminin γ2 chain immunoreactivity is restricted to the dermal-epidermal junction . The laminin γ3 chain immunoreactivity is further restricted to distinct patches widely spaced along the dermal-epidermal junction . In experiments not shown, the immunoreactivity did not correlate positively or negatively with sites of cell proliferation, nor did it correlate with fixed positions relative to the rete ridges. However, there is a direct correlation of the laminin γ3 chain immunoreactivity with sites where nerves cross the dermal-epidermal junction as detected by an antibody to the neuronal marker PGP9.5 , which reacts with ubiquitin COOH-terminal hydrolase . The results in skin suggest that γ3-containing laminins may be deposited into the dermal– epidermal junction by nerve or nerve associated cells, or that its expression by epithelial cells is induced by the adjacent nerve. Laminin γ3 is also expressed in the neural retina at the apical surface of the retina and in the outer synaptic layer (Libby, R.T., Y. Xu, E.P. Gibbons, M.-F. Champliaud, M. Koch, R.E. Burgeson, D.D. Hunter, and W.J. Brunken, manuscript submitted for publication); in the retina, the γ3 chain is coexpressed with the α4, α3, and β2 chains. Native γ3-containing laminins have not been isolated as yet from the retina; however, they have from another region of the central nervous system, the cerebellum, from which we have obtained two novel laminins, α3β2γ3 and α4β2γ3 (Champliaud, M.-F., unpublished observations). In addition, anatomical methods (immunohistochemistry and in situ hybridization), demonstrate the expression of γ3 in cerebellum and forebrain structures (Brunken, W.J., unpublished observations). It seems likely that γ3-containing laminins will be a general feature of the matrix in the CNS. The Northern analysis indicated that the laminin γ3 chain was most strongly expressed in placenta, testis, lung, liver, spleen, and ovary. Therefore, we examined the localization of γ3 chains within testis, lung, and ovary. The reactivity within the epididymis and the fallopian tube were particularly striking. Thus, the distribution of γ3 in these tissues was extensively studied. In the female reproductive system, the oviduct was strongly reactive. Cryosections of the bovine or rat ampulla reacted for γ3 using R16 , or R21 showed brilliant immunoreactivity at the apical surfaces of the tubal mucosa. Double immunofluorescent studies performed with laminin γ3 and either laminin α2 or laminin α5 antibodies demonstrated that both of these α chains are restricted to the basement membranes of the tubal epithelial and the subjacent endothelium whereas γ3 is expressed at the apical surface. The pre-immune serum from rabbit 21 was negative, as was the reactivity of anti-thioredoxin antibodies purified from the R21 serum by immunoaffinity . The pattern of immunoreactivity for R16 in the rat oviduct was identical to that seen in bovine tissue . Higher magnification micrographs of the epithelial apical surface of the rat ampulla show the γ3 chain to be localized to the apical surface of the epithelial cells at the base of the cilia. It should be noted that the labeling pattern of R21 differed somewhat from that of R16. In general, the pattern with R21 was somewhat punctate, showing large deposits of immunoreactivity at the apical surface, and increased cytoplasmic labeling of the tubal epithelium, whereas the R16 immunoreactivity was more restricted to the apical extracellular surface. These observations suggest that the R21 antiserum, made to recombinant domain I, may recognize the unfolded γ3 chain better than the R16 antiserum, which should recognize primarily short arm domains. The male monkey reproductive tract was examined also. Like the fallopian tube, the epithelium in the epididymis is a single columnar epithelium . In situ hybridization performed on adjacent sections of the monkey epididymis localized transcripts for the γ3 chain to the apical region of the epithelial cells . R16 (data not shown) and R21 sera gave similar patterns, reacting with both the basal and apical surfaces of the epithelial cells. The R21 antiserum reacted with apparently intracellular stores of γ3, as was seen in the bovine fallopian tube. The preimmune control serum from R21 showed only punctate autofluorescence . Potential chains partners were explored by examination of the same tissue with antibodies specific for a variety of other laminin chains: α2 , α4 (G), β1 (H), and β2 (I). We used two monoclonal antibodies to test for the presence of β1 at the apical surface (clones 545; and C21) both gave the same pattern of immunolabeling; only the results with clone 545 are shown. As can be seen readily, α2 and β2 were restricted to the basal surface of the epithelial cells, while staining for α4 and β1 were also seen at the apical surface. Thus, in contrast to the results from placental extracts, α4 (and not α2) appears to be a candidate chain partner for γ3 in the epididymis. These observations suggest that a wide variety of γ3-containing laminins will be expressed in a tissue-specific pattern. Expression of laminin γ3 chain was examined in the rat as well and the tissue distribution of γ3 in the rat epididymis was similar to that described for the monkey (data not shown); namely, γ3 immunoreactivity was localized to the apical surface of the epithelium. We also studied other regions of the rat reproductive system. Unlike laminin-1 immunoreactivity, which is localized to the basement membrane of the seminiferous tubules , γ3 immunoreactivity is not present within the basement membrane of the seminiferous tubules nor is it found around the interstitial cells . Within the seminiferous tubules, only the occasional tubule reacted strongly with the laminin γ3 reactive serum ; it was our impression that those tubules identified by the antibody contained nearly mature spermatids. Further along the male reproductive system, in the ductus deferens, laminin-1 immunoreactivity was seen along the epithelial basement membrane, in the lamina propria and ensheathing the smooth muscle cells of the muscular layer. In contrast, γ3 immunoreactivity (R16) was found at the apical and basal surfaces of the epithelial cells, as well as intracellularly . The apical distribution of the γ3 chain is not confined to the reproductive system; in rat lung, the ciliated epithelial cells lining the bronchi were also strongly reactive with the anti-laminin γ3 antiserum, R16 . Again, the fluorescence was apparent along the apical surface, as determined by differential interference contrast microscopy . No γ3-immunoreactivity was seen in respiratory epithelium nor in the pulmonary capillary bed (not shown). The laminin γ3 chain described here is the eleventh laminin subunit to be identified. The predicted primary and secondary structure of this chain suggests that γ3 is more closely related to human γ1 than γ2. Unlike γ2, the γ3 cDNA sequence predicts a laminin subunit without the short-arm truncations predicted for γ2. Perhaps more significantly, γ3 contains a γ1-like nidogen binding motif with only a single conservative amino acid substitution, suggesting that γ3-containing laminins should be capable of associating with other basement membrane molecules through nidogen interactions . In addition, domain VI of γ3 shares the highest sequence identity with domain VI of the γ1 chain. As this latter domain has been shown to support laminin self-assembly , it seems reasonable to suggest that domain VI of the γ3 chain may also support self-assembly. Two of the predicted glycosylation sites in the γ3 chain are also found in human and in mouse γ1; these are within short-arm globular domains, i.e., VI and IV. Interestingly, the glycosylation site in domain IV is also found in human and in mouse γ2. This remarkable conservation of glycosylation sites among these three chains and between these species suggests that these sites are indeed important and glycosylated; they are likely to be critical in the folding of this region or may play another important function. The RGD sequence within domain II of the γ3 chain is found in neither γ1 nor in γ2. Moreover, it seems likely that this sequence is not functional within native γ3-containing laminins as it is located within the coiled-coil region of γ3. However, it very well may promote integrin-mediated recognition of non-native molecules or of protein fragments. In placenta, the γ3 chain can combine with the laminin α2 and β1 chains. This observation suggests that, unlike γ2 which pairs preferentially with β3, γ3 may pair with any β chain, with the possible exception of β3, and with any of the known α chains. This prediction suggests the existence of an additional 10 laminins with the following chain compositions: α1β1γ3, α1β2γ3, α2β1γ3, α2β2γ3, α3β1γ3, α3β2γ3, α4β1γ3, α4β2γ3, α5β1γ3, and α5β2γ3. In both the epididymis and the fallopian tube, γ3 is not combined with α2. In the epididymis, the α4 and β1 chains appear to be potential partners. Given that the total number of human laminins is not known, at least one additional β chain has been identified in chicken and in mammals (Olson, P.F., unpublished observations), assigning a final laminin numerical identifier to these laminins is premature. However, as we have shown α2β1γ3 to be the twelfth laminin to be identified, we provisionally call this heterotrimer, laminin 12. The masses of the chains of laminin 12 as approximated by electrophoretic mobility are considerably less than predicted by the amino acid sequences and from prior experience with the α2 and β1 chains. They are also less than the α2, β1, and β2 chains present in laminins 2 and 4 obtained from the same preparations. The reason for these more rapid electrophoretic migration rates appears to be proteolysis within the domains II of the chains comprising this molecule. In placenta, this proteolysis may be physiological, since laminins 2 and 4 isolated from the same preparations are apparently intact. The significance of this observation awaits considerable additional experimentation before it is understood. However, we have observed three cysteinyl residues within domain II of the γ3 chain that are not present in other human α, β, or γ chains. It is possible that a disulfide bond between two of these residues distorts the coiled-coil conformation making molecules containing this chain more susceptible to proteolysis. At this time, we do not know if truncation of laminins containing the γ3 chain can be generalized to tissues other than placenta; however, this seems unlikely in that our isolation of γ3-containing laminins from the CNS do not show the same truncation (Champliaud, unpublished observations). The COOH-terminal truncation of α2t explains its lack of reactivity with an anti-α2 antibody, mAb 1922, which is specific for the α2 G domain . The antiserum to the recombinant γ3 domain I fusion protein (R21) was originally made to evaluate this potential processing, since epitopes contained within this domain should be absent from the processed molecule. In this regard, the immunohistochemical data is not definitive. Like R16, R21 immunoreactivity is seen at the apical surface; however, the apical reactivity is distinctly different than that for R16. Specifically, R21 immunoreactivity appears as a plaque-like structure at the cell surface, with some reactivity within the cells. Thus, it seems possible that R21 epitopes are entirely intracellular but it is also possible that some of the R21 epitopes are present at the apical surface of these epithelial cells. Further experimentation beyond the scope of this report is required to address this question. However, laminins containing γ3 chains have been immunoisolated from the medium of A204 cells derived from a human rhabdosarcoma (Champliaud, M.-F., unpublished data), indicating that γ3-containing laminins are capable of being secreted in vitro. These γ3-containing laminins from A204 are a mixture of processed (truncated) and unprocessed (untruncated) molecules. Reports of laminins in tissue locations not identified as basement membranes are increasingly frequent. In the brain, laminins have been observed not only within the basement membrane of capillaries, but also at other sites not conceptualized as basement membranes . In the eye, the laminin β2 chain has been identified in both basement membrane and non-basement membrane locations . Laminins have also been observed in cartilage . Intriguingly, the laminin γ3 chain appears most commonly to be associated with non-basement membrane structures. In the cerebellum, γ3 chains are detected in the pericellular nets surrounding both neurons and glia (Brunken, W.J., unpublished observations). Reported elsewhere (Libby et al., manuscript submitted for publication, see above), γ3 is present within the neural retina at two extracellular sites: between the outer segments of the photoreceptors, and at the synapses of the photoreceptors with the bipolar and horizontal cells. In the retina, these γ3-containing molecules are the products of the Müller glial cells which, like the tubal epithelium, contain a considerable store of intracellular γ3 chain. The laminin α3 and β2 chains are also present at these sites, whereas the γ1 and γ2 chains are absent. The functions fulfilled by these laminins are unclear, but possibilities include: stabilization of neural architecture; induction and stabilization of differentiated neural phenotypes ; and stabilization of synaptic junctions. However, the most abundant expression of γ3 as detected by Northern analyses is not within neural tissues, but rather is in the testis, the placenta, the spleen, the lung, and the ovary. γ3 immunoreactivity is present at the bases of the epithelial cilia of the epididymis, the trachea, the bronchi, and the oviduct. There are no structures resembling basement membrane at these sites. However, γ3 chains may be present within the basement membranes along the basolateral surfaces of some of these epithelia. The chain partners for the γ3 chain in these apical laminins are not yet known with certainty. However, in epididymis, the laminin α2 chain does not colocalize with γ3 at the apical epithelial surface; rather, the α4 and β1 chains are present at that location and, thereby, are potential chain partners. Thus, it seems likely that γ3 will be as promiscuous as γ1 with respect to partner choice during laminin assembly. The presence of laminins along ciliated epithelial surfaces was unexpected and their functions there are unknown. Perhaps a modified basement membrane containing at least laminin helps organize or stabilize the specialized cytoskeleton of the cilia. Laminins at these apical surfaces may also participate in the anchorage of mucins to the surface. Alternatively, laminins might stabilize the outfoldings of the plasma membranes of the cilia. Similar functions for laminins have been postulated to stabilize the junctional folds beneath synapses at neuromuscular junctions , and contribute to the organization of epithelial hemidesmosomes . Laminins expressed at the apical surface of the retina are thought to play a role in photoreceptor morphogenesis, specifically outer segment formation and synapse development . Consistent with the above speculations regarding an essential function for γ3 containing laminins in neural tissues, the chromosomal locus of LAMC3 at 9q31-q34 is shared with four diseases having various degrees of neural dysfunction in common: Fukuyama congenital muscular dystrophy (FCMD); muscle-eye-brain syndrome; Walker-Warburg syndrome; and retinitis pigmentosa-21 with deafness (RP-21). The genetic cause of the latter three of these conditions is unclear. While γ3 expression may be affected in FCMD, the LAMC3 cannot be the genetic cause of the problem as a retrotransposal insertion in a different gene has recently been identified in 87% of the FCMD alleles . However, LAMC3 is an excellent candidate for mutations underlying one or more of remaining syndromes in this cluster of human diseases, particularly RP-21. We have identified the laminin γ3 chain together with the α2 and β1 chains within laminin 12 from human placenta, but multiple other combinations are possible in other tissues. It would be of particular interest if γ3 were to associate with the β2 chain and well as with the α2 chain, as both these chains have been reported to show neural and muscle-associated expression and function. The γ3-containing laminins are likely to be the subject of considerable interest as they constitute a novel class of laminin molecules distributed outside of the traditional basement membrane. The identification of the function of this diverse family of laminins remains to be elucidated by future experiments. | Study | biomedical | en | 0.999997 |
10225961 | C2C12 myoblasts (American Type Culture Collection) were maintained in DME ( Gibco Laboratories ) containing 10% FBS in a 37°C incubator with 5% CO 2 . Subconfluent myoblasts were trypsinized, transferred to 35 × 10 mm tissue culture dishes, and switched to fusion medium upon confluency (DME, 5% horse serum). Myotubes were analyzed 3–5 d after fusion. Cytochalasin D ( Sigma Chemical Co. ) was used at 0.4 μM and genistein ( Calbiochem-Novabiochem Corp. ) was used at 25 μM; both inhibitors were added to cultures 2 h before the addition of ligand. Fused myotubes were incubated at 37°C with laminins or other proteins suspended in DME containing 0.5% BSA. Unattached protein was removed by washing four times with PBS containing 1 mM CaCl 2 . Cells were fixed with 3% paraformaldehyde for 10 min at room temperature and, when using antibodies against intracellular epitopes, permeabilized with PBS containing 0.5% Triton X-100 at 0°C. Cells were blocked for 30 min in PBS containing 0.5% BSA and 5% normal goat serum and incubated with primary antibodies diluted in wash buffer (PBS, 0.5% BSA, 0.5% normal goat serum) for 1 h at room temperature. After several washes, cells were incubated with FITC- or rhodamine-conjugated secondary antibodies for 1 h at room temperature. Cells were washed, coverslipped in 1,4 diazabicyclo[2.2.2]octane ( Sigma Chemical Co. ), and imaged using an Olympus IX-70 inverted fluorescent microscope and a cooled CCD camera ( Princeton Instruments Micromax). Rabbit polyclonal antibodies against mouse laminin-1, human laminin-2, laminin-1 proteolytic fragments E4, E8, and E3, and recombinant laminin α2-G domain were generated as described previously ( 13 ). Anti-E4, anti-E8, anti-E3, and anti-α2(G) were found to be highly specific to these regions of laminins after affinity purification and extensive cross-absorption. CY8 mouse monoclonal IgG ascites fluid raised against the α7 integrin subunit was provided by Dr. Randy Kramer (University of California, San Francisco, CA) and was diluted 1:500 for both blocking experiments and indirect immunofluorescence. IIH6 mouse monoclonal IgM raised against rabbit dystroglycan, and used at a 1:2 dilution of hybridoma medium, was provided by Drs. Hiroki Yamada and Kevin Campbell (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA). The following antibodies were obtained commercially. A mouse mAb specific for the COOH-terminal region of β-dystroglycan was used at 1:25 (Novocastra Laboratories). A hamster monoclonal IgM specific for the β1 integrin subunit was used at 10–20 μg/ml for blocking experiments, and at 5 μg/ml for indirect immunofluorescence ( PharMingen ). Mouse monoclonal IgM specific for the central domain of dystrophin was used at 1:50 dilution (Upstate Biotechnology). Mouse monoclonal IgG specific for sarcomeric α-actinin was used at 1:800 dilution ( Sigma Chemical Co. ). Rabbit polyclonal IgG specific for human fibronectin was used at 1:400 dilution ( Sigma Chemical Co. ). Mouse monoclonal IgG specific for vinculin was used at 1:100 dilution ( Sigma Chemical Co. ). Rhodamine- and fluorescein-conjugated secondary antibodies specific for mouse IgG, mouse IgM, hamster IgM, and rabbit IgG were used at the recommended dilutions (Jackson Immunochemicals and Sigma Chemical Co. ). Mouse laminin-1 was extracted from lathyritic EHS tumor and purified as described ( 63 ). A preparation of laminin-2 and -4 (both containing the α2 chain subunit) was prepared from human placenta as described ( 7 ), using a modification of a procedure developed previously ( 4 ). Defined fragments of laminin were prepared after digestion of laminin-1 with elastase or cathepsin G ( 63 ). Elastase fragments include: E1′, a partial complex of all three NH 2 -terminal short arms associated with a nidogen fragment; E4, β-chain short arm domains V and VI; E8, distal long arm coiled-coil region and proximal G domain (G repeats 1–3); and E3, distal G domain (G repeats 4 and 5). The cathepsin G fragment C8-9 is similar to the elastase fragment E8 but is larger, including most of the coiled coil. Recombinant laminin α-chain proteins α1(VI-IVb), α2(VI-IVb), and α2(G) were generated and purified as described previously ( 12 , 13 , 45 ). Human fibronectin was purchased from Sigma Chemical Co. 100 mM serine protease inhibitor p -aminoethylbenzenesulfonyl fluoride (AEBSF, 1 HCl) was incubated overnight on ice with laminin and used to inactivate laminin self-assembly . AEBSF-treated laminin was dialyzed to remove free AEBSF and taken through two rounds of polymerization conditions (37°C, 3 h) to remove any potentially active laminins. [ 14 C]AEBSF (American Radiolabeled Chemicals Inc.) had a specific activity of 55 mCi/mmol. For binding studies, [ 14 C]AEBSF was incubated overnight on ice with laminin and dialysis was performed to remove unbound material. Laminin was digested with elastase ( Boehringer Mannheim ) using an enzyme/substrate ratio of 1:100 for 24 h at 25°C. Bound [ 14 C]AEBSF was determined using PhosphoImaging (Bio-Rad GS-250; Bio-Rad Laboratories) on fragments separated by SDS-PAGE. Assays to evaluate laminin polymer formation and laminin cell adhesion were performed as previously described ( 7 , 12 ). To understand how laminin interacts with muscle cells, we evaluated laminins on C2C12 cells, a mouse myogenic cell line ( 60 ). When deprived of mitogens, these cells fuse and form long, multinucleated myotubes that spontaneously beat in culture . Fortunately, these cells appear to produce only low levels of endogenous laminin, so added laminin was monitored easily. We incubated fused myotubes with laminin-1 (α1β1γ1) and found that laminin bound to the dorsal myotube surface . Initially (15 min), laminin binding was diffuse , but later (1 h) appeared aggregated into a reticular pattern on the surface . By 4 h, laminin organization on the cell surface appeared predominantly in a repeating, polygonal network with dimensions generally between 1 and 3 μm (see higher magnification in c′ for greater detail). In addition, these longer incubations revealed a transition from widespread coverage of laminin networks to more focal regions of laminin (d shows another myotube at 4 h). This transition was accompanied by the appearance of compact networks of laminin surrounded by zones with little or no laminin. These regions maintained the repeating substructure seen in more widespread laminin networks, but the entire architecture was somewhat more densely packed. Constant replenishment of the incubation medium with fresh laminin did not alter this progression from ubiquitous to more focal laminin coverage, suggesting that a change was occurring in the accessibility, composition, or activation state of laminin receptors. In addition, myotubes incubated in the presence of laminin for up to 24 h remained healthy and maintained focal laminin networks. First, we sought to determine which regions of the large multisubunit laminin molecule interacted directly with the myotube cell surface . A battery of laminin proteolytic fragments and recombinant laminin domains was evaluated to determine which domains bound directly to myotube cell surface receptors. Here we considered the binding properties of both α1- and α2-chain–containing laminins, shown together in a composite model . This model depicts the boundaries of laminin fragments and some of the sites known to interact with receptors and extracellular matrix molecules. We found that laminin proteins composed of distal COOH-terminal domains, but not those from NH 2 -terminal domains, were detected on the cell surface using indirect immunofluorescence . The NH 2 -terminal short arm region from both the α1 and α2 laminin subunits (α1[VI-IVb] and α2[VI-IVb]) have been shown previously to possess active α1β1 and α2β1 integrin binding sites using PC12, HT1080, and primary rat Schwann cells ( 12 ). In contrast, we did not detect any significant level of binding between C2C12 myotubes and the α1 and α2 laminin short arms, indicating that fused muscle cells do not directly interact with these NH 2 -terminal domains. Also, we did not detect binding of short arm proteolytic fragments E1′ and E4 (data not shown). Proteolytic fragments E8 and E3, derived from the COOH-terminal long arm of mouse laminin-1 ( 34 ), bound to the cell surface, remained diffusely distributed, and had a punctate staining appearance. A recombinant laminin protein consisting of the entire α2 subunit G-domain ( 45 ) also bound to the surface and had a staining pattern similar to that of E3 and E8. Fragment E8 has been shown to mediate laminin binding to the α7β1 integrin ( 57 ), whereas fragment E3 has been shown to mediate binding to α-dystroglycan ( 22 ). Although both fragments bound to the cell surface, neither formed the large aggregates and networks seen with intact laminin-1 or laminin-2. One explanation for this difference could be that integrin and dystroglycan binding need to occur simultaneously, acting as coreceptors to stimulate surface aggregation. To test this possibility, we incubated cells with a mixture of E3 and E8. However, the staining pattern remained similar to that seen with individual fragments (not shown), indicating that occupancy of both receptor types was not sufficient to produce larger surface aggregates. Next, we tested the ability of surface binding proteins or antibodies to block or alter the organized pattern of intact laminin on myotube surfaces . First, cells were incubated for 1 h with COOH-terminal long arm fragments E3 or E8, or a combination of E3 and E8, or with blocking antibodies to each of the two principal receptors for laminin in mature muscle fibers, integrin and dystroglycan. Treatment with these reagents was followed by addition of intact laminin for 1 h. The cells were probed with antibodies specific for the NH 2 -terminal region of laminin, thereby only detecting intact laminin bound to the surface (i.e., not fragments E3 or E8). Surprisingly, function-blocking antibodies against the β1 integrin subunit or the α7 integrin subunit (not shown) had almost no effect on the ability of laminin to bind to and aggregate on myotube surfaces. These same antibodies completely blocked fragments E8 and C8-9 (larger than E8, fragment C8-9 also contains the agrin binding site) from binding to the myotube surface, demonstrating that these antibodies prevent α7β1 integrin binding interactions (not shown). In contrast, treatment with antibody that interferes with laminin– dystroglycan interactions (mAb IIH6) had a clear effect on laminin surface aggregation and network formation. Furthermore, fragment E3, which contains the dystroglycan binding site, significantly reduced laminin binding and aggregation on the myotube surface. Fragment E3 consistently had a greater blocking effect than antidystroglycan. One explanation may be that the E3 fragment contains additional, less prominent receptor binding sites. It is also possible that a partial or weak blocking effect exerted by this monoclonal IgM may be responsible for the difference. Nevertheless, a more prominent role in both direct binding and subsequent aggregation can be attributed to the E3-mediated interaction(s), compared with those mediated by E8. Finally, incubations combining fragment E3 with fragment E8 now completely blocked laminin from binding to the myotube surface. The extent of inhibition seen with single or multiple reagents persisted during longer laminin incubations: after treatment with laminin for 4 h, laminin network and cluster formation was significantly blocked by E3, very little by E8, and completely by a combination of the two (not shown). To determine whether laminin–laminin polymer bonds were required to mediate laminin network formation on a cell surface, we used several methods to selectively analyze polymer formation. A nonpolymerizing laminin was generated by treating laminin with AEBSF, a serine protease inhibitor that we fortuitously found has the ability to bind covalently to laminin . Binding specificity was determined by incubating laminin with [ 14 C]AEBSF, followed by the standard elastase proteolysis conditions used to generate laminin fragments ( 34 ). Fig. 4 A shows the relative amount of isotope bound to various laminin elastase proteolytic fragments. We found that nearly all of the label was associated with E1′, a fragment consisting mostly of the α– and γ–NH 2 -terminal short arms and containing sites essential for laminin polymer formation ( 48 , 63 ). In contrast, the COOH-terminal long arm fragments E3 and E8 bound little, if any, [ 14 C]AEBSF. Although treatment with AEBSF abolished laminin's ability to form a polymer in solution , its ability to interact with surface receptors (including α1β1, α2β1, α3β1, α6β1, and α7β1 integrins) in the cell types we have tested so far remained unchanged. Adhesion of C2C12 myoblasts to both untreated and AEBSF-treated laminin-1 is shown in Fig. 4 C. In addition, we found that the dystroglycan-binding fragment E3 retained its ability to interact with the muscle cell surface after treatment with AEBSF (not shown) and that the affinity of laminin for dystroglycan remained unchanged after AEBSF treatment (Combs, A., and J. Ervasti, personal communication). Essentially, this reagent produced a nonpolymerizing but otherwise functional laminin molecule, and allowed us to test directly the role of laminin polymer formation independent of its cell adhesive functions. We found that nonpolymerizing AEBSF–laminin was unable to form aggregates and networks on the cell surface . Conditions used to disrupt laminin polymer bonds selectively in solution ( 48 ) also were found to disrupt laminin cell surface organization, but had no effect on the binding of surface receptors . Specifically, molar excess of either laminin short arm proteolytic fragments E1′or E4 prevented formation of laminin surface aggregates that appear after 1 h. Inhibition of polymerization also prevented development of laminin networks and clusters seen after longer incubations. After 4 h, polymerization-competent laminin appeared in both widespread networks and more compact clusters. However, AEBSF-treated laminin, or laminin blocked with fragment E1′ did not form these structures. The organization of receptors and cortical cytoskeleton in the presence or absence of laminin was evaluated. Treatment of myotubes for 4 h with polymer-competent laminin resulted in the rearrangement of α7β1 integrin, dystroglycan, dystrophin, and cortical vinculin, but not α-actinin . Laminin-treated myotubes were double-labeled to visualize the laminin network, sarcolemmal proteins (α7 and β1 integrin subunits, dystroglycan, vinculin, dystrophin, and α-actinin) and their overlapping distributions . Integrin (α7 and β1 subunits), dystroglycan, vinculin, and dystrophin formed repeating polygonal structures similar in appearance and location to those formed by laminin. Beneath the laminin network, both receptor types also codistributed . Myotubes incubated with BSA and those incubated with nonpolymerizing laminin are shown in the bottom two rows of Fig. 6 . The organization of β1 integrin, dystroglycan, vinculin, and dystrophin was very different in these myotubes, appearing diffusely distributed with regions of small clusters. Therefore, laminin that could engage receptors, but could not form surface polymers, was insufficient to induce receptor and cytoskeleton rearrangements. The architecture of the laminin polymer, with its ∼35-nm strut meshwork, can be appreciated only at the level of electron microscopy. While triggered by polymerization, the organized semi-regular appearance of the laminin surface network suggested that creation of this architecture might be an active process perhaps requiring remodeling of the cortical actin network, or even signaling events. We investigated the role of actin by comparing the organization of laminin on myotubes incubated in the presence or absence of cytochalasin, an agent that prevents assembly and disassembly of filamentous actin . In contrast to control myotubes, cytochalasin-treated myotubes did not show prominent rearrangement of surface laminin into complex networks, even after 4 h. On cytochalasin-treated myotubes, laminin coverage remained widespread and had a near continuous, almost mattelike quality. Inspection at higher magnification revealed a delicate substructure to regions of this laminin covering, one that was very different from the larger, more sharply delineated polygonal units seen in control cultures. In addition, the clearance of laminin from large regions of the myotube surface was inhibited. Next, we evaluated whether energy-dependent signaling cascades might be required to promote laminin network formation on the cell surface. We found that genistein, a general inhibitor of protein tyrosine kinases, had a large effect on laminin's ability to form a surface network . On genistein-treated myotubes, laminin coverage appeared ubiquitous and without much complexity, similar to that seen on cytochalasin-treated cells. In contrast, several other inhibitors of signaling molecules had virtually no effect on laminin network formation (not shown). These were wortmannin, an irreversible inhibitor of phophatidylinositol 3-kinase, and ML-7 hydrochloride, a selective inhibitor of myosin light chain kinase that is often used to interfere with actin–myosin contractility. The observed requirement for actin remodeling suggested a role for the Rho family of small GTPases ( 27 ). To address this possibility, we used C3 transferase, a selective inhibitor of Rho, but this treatment was found to disrupt myotube adhesion and structure grossly. Therefore, it could not be used to evaluate the role of Rho-mediated signaling in laminin surface network formation. Mice homozygous for the dy 2J LAMA2 allele were found to have a point mutation within the 5′ coding region that causes an abnormal splicing event ( 55 , 59 ). This genetic defect causes a severe and progressive muscular dystrophy thought to be analogous to the many human congenital muscular dystrophies caused by LAMA2 mutations. Because of irregular splicing, dy 2J / dy 2J mice express a mutated laminin α2 chain lacking 57 amino acids within domain VI, the most NH 2 -terminal short arm domain . Nonetheless, this aberrant laminin subunit forms a heterotrimer with the β and γ chains, is secreted, and is localized to the sarcolemmal basement membrane. Subsequently, it has been found that the α2-chain–containing laminins (a combination of laminins-2 and -4) from the skeletal muscle of dy 2J / dy 2J mice is defective in its ability to form a polymer in solution (Colognato, H., and P.D. Yurchenco, manuscript in preparation). This polymerization defect most likely accounts for the dystrophy seen in these mice since receptor recognition sites in the laminin α2 domain VI do not seem to be used in mature skeletal muscle. In vivo, this mutant laminin appears to be held within the basement membrane solely through linkage to the collagen IV network, since we find that digestion of muscle tissue with collagenase liberates dy 2J -laminin into solution. Normally, muscle laminin requires an additional treatment with EDTA to disrupt the calcium-dependent laminin polymer. We purified α2-laminin extracted from wild-type and dy 2J / dy 2J muscle and evaluated the ability of these laminins to attach to muscle cell receptors and organize into a surface network . Myotubes incubated for 4 h with wild-type mouse α2-laminin showed extensive formation of laminin surface networks. The inset at higher magnification (3×) clearly shows the characteristic laminin network architecture seen with laminin-1. On average, the spacing of repeating polygonal units was often more compact when compared with that of laminin-1, but the unit size range was similar for both. In contrast, dy 2J –α2-laminin bound to the myotube surface but did not form these organized structures, remaining in a diffusely distributed punctate pattern. This pattern was similar to that seen with nonpolymerizing laminin-1 . Myotubes were incubated with either wild-type or dy 2J – α2-laminin and visualized by indirect immunofluorescence. Double staining revealed the location and relationship of laminin and the following sarcolemmal proteins: β1 integrin subunit, dystroglycan, and vinculin . Induction of corresponding integrin, dystroglycan, and vinculin networks was observed in the presence of wild-type α2-laminin, but dy 2J –α2-laminin failed to induce this reorganization. As in the presence of nonpolymerizing laminin-1, laminin receptors remained in a diffusely distributed, punctate pattern, as did vinculin. In addition, the intensity of vinculin staining at the cortical region appeared to be greater in myotubes incubated with polymerizing laminin-1 or -2, suggesting that vinculin was being recruited into the cortex and reorganized. The extracellular matrix molecule laminin has been described previously as having two major, though independent, functions: ( 1 ) a ligand for cell surface receptors, mediating such processes as neurite outgrowth and prevention of apoptosis, and ( 2 ) a structural molecule that forms a polymer and is required for basement membrane architecture, providing mechanical support to adjacent cells. In this study, we have presented evidence that these two types of functions are in fact integrated, acting synergistically to reorganize the cell surface and adjacent cortical cytoskeleton . It seems likely that this laminin-induced process represents a specific mechanism for the transmission of complex cellular signals. For these cells, this mechanism appears to be laminin-specific, not induced by the addition of fibronectin, collagen IV, or even by the addition of divalent antibodies to artificially cluster nonpolymerizing laminin (not shown). We have shown that laminin attaches to the cell surface while bound to dystroglycan, integrin α7β1, and possibly other receptors for the laminin long arm. The concentration of receptor-engaged laminin is thereby selectively increased, now exceeding the critical concentration for laminin self-assembly and driving polymerization preferentially on cell surfaces (receptor-facilitated self-assembly). Furthermore, through cooperative polymer and receptor interactions, laminin organizes into complex polygonal arrays on the cell surface. This appears to be an active process that requires remodeling of actin filaments and tyrosine kinase signaling. Laminin polymerization induces reorganization of laminin receptors, both integrin and dystroglycan, and elements of the cortical cytoskeleton, vinculin and dystrophin. Moreover, we find that a mutated laminin causing muscular dystrophy and dysmyelination in the dy 2J / dy 2J mouse is defective in its ability to form a surface network and to induce these specific changes in the sarcolemmal architecture. In the past few years, numerous mutations in the LAMA2 gene have been identified as the cause of congenital muscular dystrophy (CMD, ref. 53). Furthermore, the hallmark of this emerging class of disorders is the accompaniment of peripheral and central nervous system abnormalities, indicating a requirement for this laminin beyond the skeletal musculature. Although the isolation of these laminin mutations offers a significant breakthrough in the understanding of these congenital disease processes, insight into the molecular mechanism has been elusive. Many identified mutations result in absence of the laminin α2 protein altogether, but may be accompanied to varying degrees by upregulation of additional laminin chains such as α4 and α5, whose degree of overlapping function remains unclear ( 43 ). Mutations resulting in expressed protein with specific functional deletions, a smaller group, may lead to a more selective analysis of the contributions of specific laminin functions. Since laminin isoforms have many overlapping functions, structure/function correlation may permit identification of laminin candidates that could be upregulated or expressed to correct α2-deficient CMDs, similar to the rescue of dystrophin-negative dystrophic mdx mice using the dystrophin homologue, utrophin ( 44 , 56 ). The dystrophic dy 2J mouse has an abnormally spliced laminin α2 transcript resulting from a point mutation in a splice donor site ( 55 , 59 ). The resulting α2 protein is expressed, incorporated into a laminin heterotrimer, and localized to the sarcolemmal basement membrane. This α2-chain short arm defect in domain VI is analogous to several human CMDs that express partially functional α2 proteins with defects in the NH 2 -terminal short arm ( 1 , 11 , 42 ). In this study, we find that fused muscle cells bind to laminin through receptor-binding sites in the COOH-terminal long arm, ∼110 nm away from the short arm region that is defective in polymer formation (Colognato, H., and P.D. Yurchenco, manuscript in preparation). This laminin remains within the basement membrane through interactions with cell surface receptors and through linkage to the type IV collagen network, the latter mediated by entactin/ nidogen. In this context, it seemed unlikely that the prime role of laminin polymerization is simply to dock laminin into the basement membrane and provide a mechanical tether. Therefore, we sought to understand how laminin polymerization might alter the muscle sarcolemma in such a way that could promote muscle function and, ultimately, survival. The novel laminin function that we report here seems a likely candidate: the induction of dramatic changes in organization of matrix, receptors, and cortical cytoskeletal components in response to laminin polymerization. This induction may underlie a key requirement for laminin polymerization in maintenance of proper cellular architecture and function. Signaling pathways mediated by some growth factors not only have been found to be dependent on ligand occupancy of receptors, but also on dimerization of these receptors ( 3 , 9 , 62 ). In a related concept, receptor aggregation, mediated by fibronectin fibrils, is thought to recruit a large collection of cytoskeletal components and signaling molecules to these sites ( 38 , 61 ). For this process, it has been shown that ligand occupancy alone (provided by RGD peptide) activates only a small subset of the responses mediated by a fibrillar fibronectin network. Similar or related mechanisms might play a role in laminin signaling, although laminin differs significantly from fibronectin in its distribution, receptor partners, and mode of supramolecular assembly. For instance, fibronectin forms a fibrillar rather than a meshlike polymer and its assembly process does not occur in the absence of receptor interactions. This difference turned out to be helpful to our study, as we were able to evaluate laminin's polymerization process independent of receptor occupancy and assess the extent to which laminin-induced changes were polymer-induced. Traditionally, basement membranes have been thought of as supportive substrates for adjacent cells that enable cell adhesion or migration. The data presented here suggest a more dynamic, instructive role for the supramolecular organization of laminin in the basement membrane. Evidence has been building in recent years for the idea that cellular architecture has the ability to transmit information. At the forefront of this paradigm has been the concept of tensegrity, a model for mechanotransduction in which cells respond to mechanical stresses and changes in the physical environment through a matrix receptor–cytoskeleton nuclear continuum ( 30 ). In support of this model, spatial arrangement of matrix molecules has been shown to mediate processes such as proliferation, apoptosis, and arrangement of cytoskeletal architecture ( 6 , 31 , 33 , 40 ). In this study we have shown that a change in laminin architecture is able to instruct muscle cells, leading to changes in receptor and cytoskeletal architecture. Interestingly, preliminary data suggest that laminin polymerization may activate tyrosine phosphorylation above and beyond that seen with nonpolymerizing laminin (Hanus, C., and P.D. Yurchenco, unpublished observations). Studies in which certain laminin domains have been masked or proteolytically altered also support the notion that laminin architecture has the ability to influence cellular responses ( 5 , 25 ). In these studies, the modified laminin domains were completely distinct from those regions interacting with the cell receptor(s) in question. In a study done by Giannelli and colleagues, it was found that proteolytic cleavage in the short arm of the γ2 subunit transformed cell interactions mediated by the G-domain of laminin-5, converting a static adhesion activity to an invasive, migratory activity used by cancer cells. The mechanism for this functional switching remains unclear, but information transmitted through the architectural arrangement of these ligands may very well contribute. The relationship between laminin–receptor interactions and basement membrane formation has been examined in recent years. Targeted gene disruptions of the β1 integrin subunit and dystroglycan, both lethal to embryonic development, have indicated a requirement for receptors in the proper assembly and subsequent function of embryonic basement membranes ( 19 , 52 , 58 ). Furthermore, mutations in the α3, α6, and β4 integrin subunits cause defects in the epidermal basement membrane of the skin, often leading to epidermolysis bullosa ( 17 , 24 , 41 ). Teratomas formed by β1-null embryonic stem cells injected into normal mice formed aberrant basement membranes that appeared to develop and slough off of adjacent cells, often in multiple and abnormally thickened layers ( 2 , 47 ). Dystroglycan-null embryos die after implantation after failing to develop a functional extraembryonic Reichert's membrane, allowing maternal blood into the yolk sac cavity. Examination of this specialized basement membrane revealed only occasional patchy regions of laminin and collagen IV staining instead of the continuous staining observed normally. Furthermore, recent work has shown that dystroglycan −/− embryonic stem cells grown in culture to form embryoid bodies also have defects in basement membrane assembly ( 29 ). How does the removal of dystroglycan, or other laminin receptors, lead to such profoundly defective basement membranes? Other molecular bonds, such as laminin–integrin or laminin–nidogen-collagen IV, could presumably be sufficient to retain laminin within the basement membrane of dystroglycan-null cells. This suggests that the laminin– dystroglycan interaction may provide an inductive role to cells in addition to providing a simple mechanical linkage. The ability of dystroglycan binding fragment E3 to disrupt markedly not only laminin surface binding but also its surface assembly is consistent with this hypothesis. The large effect seen by inhibiting dystroglycan, versus integrin, interaction sites may be due to a larger reduction in laminin accumulation on the surface, or a greater mobility of dystroglycan through the lipid bilayer compared with integrin, but it could also indicate an inductive role specific to the laminin–dystroglycan interaction. The laminin–dystroglycan relationship also has been examined in the context of neuromuscular junction formation. It has been found that AChR receptor clustering is mediated in part by neural agrin through a mechanism dependent on the tyrosine kinase receptor MuSK ( 15 , 21 ). Agrin, which has laminin-G domainlike repeats, has been shown to bind to both laminin and dystroglycan ( 16 , 23 ). It has been suggested that laminin and dystroglycan may each serve as reservoirs for agrin at the neuromuscular endplate, perhaps facilitating agrin's ability to induce AChR clustering. Moreover, recent work has illustrated a MuSK-independent AChR clustering activity that is activated by the addition of exogenous laminin ( 54 ). In fact, the laminin–dystroglycan interaction specifically was required for this laminin-induced AChR clustering to occur ( 39 ). However, an earlier study by Cohen and colleagues found that by adding laminin to Xenopus muscle cultures, small dystroglycan clusters are converted to larger more focal clusters but that these laminin-induced clusters were not accompanied by accumulation of AChR ( 10 ). Using the C2C12 muscle culture system, we do observe an increase in AChR clusters upon addition of laminin, but only a subset of these clusters colocalizes with laminin (micrographs not shown). It should be interesting to learn whether the receptor and cytoskeletal rearrangements induced by laminin polymerization play a role in signaling this MuSK-independent pathway for AChR clustering. In summary, we present evidence that laminin polymerization induces a cortical cellular architecture comprised of matrix, receptor, and cytoskeletal elements. This process requires a unique synergism between laminin architecture–forming and receptor–ligating functions, mediated by distal NH 2 -terminal and COOH-terminal epitopes, respectively. Furthermore, we demonstrate that a mutant laminin responsible for CMD and nervous system defects is defective in its ability to form surface networks and subsequently induce receptor–cytoskeleton rearrangement. This organizational signaling induced by laminin introduces a novel mechanism by which the supramolecular configuration of basement membranes instructs adjacent cells and tissues. | Study | biomedical | en | 0.999998 |
10225962 | To overexpress myogenin and Id-1 in differentiated muscle cells the myogenin ( MMg ) and Id-1 ( MId ) transgenes were inserted into the genome of mice by conventional transgenic techniques described previously . In brief, the MMg transgene contains rat myogenin cDNA and the MId transgene contains mouse Id-1 cDNA . Both transgenes are driven by the myosin light chain 1 promoter and 3′ 1/3 enhancer, which have been shown to confer expression only in differentiated post-mitotic muscle fibers . Moreover, the expression is fiber type-dependent, such that the level of expression declines in the order 2B > 2X > 2A > 1 . Animals containing the MMg transgene have a high neonatal lethality, whereas bearers of the MId transgene have the same survival rate as nontransgenic littermates. The MId transgene can overcome the deleterious effect of the MMg transgene such that bearers of both transgenes ( MMg + MId ) have normal survival rates . Thus, we propagate the MMg transgene in double transgenic mice ( MMg + MId ), which are mated to wild-type mice to yield offspring of the following four genotypes identified by PCR analysis : MMg , MId , MMg + MId , and wild-type. The wild-type littermates served as controls in the present study . In all cases the analyzed muscles were from mice that were 2–6-mo old. RNA was extracted as described by Chomczynski and Sacchi , and the level of rat myogenin mRNA from the transgene was determined by RNase protection assays as described by Melton et al. . The specificities of the protected bands were determined by hybridization against equal amounts of yeast tRNA. For further details see Gundersen et al. . Protein extracts were prepared in 50 mM Tris-HCl, pH 7.4, 0.6 M NaCl, 5 mM EDTA, 10% SDS, 1 μg/ml pepstatin A, 0.1% PMSF, 1% β-mercaptoethanol by homogenization in volumes normalized for wet weight of tissue. After separation on 12.5% SDS-PAGE, electroblotting to Amersham Hybond-C super filters, and blocking with 3% Tween 20, 5% horse serum in PBS, proteins were reacted with 1:70 diluted primary monoclonal tissue culture supernatants in 0.5% Tween 20, PBS. Detection was with 1:1,500 HRP-conjugated rabbit anti–mouse Ig (Dako Corp.) followed by the ECL kit. Mice were anesthetized by ether, single muscles were dissected out, and snap frozen in liquid nitrogen. Muscle samples were homogenized directly from frozen in a stabilizing medium containing 50% glycerol, 20 mM phosphate buffer, pH 7.4, 5 mM β-mercaptoethanol, 0.5 mM EDTA, 0.02% BSA at a dilution of 1:100 based on wet weight. All assays for a particular enzyme were performed on the same day, over a 60-min incubation period at 25°C. Activities are given as mol · kg wet wt −1 h −1 . Final readings were made fluorometrically in a vol of 1 ml. Standards were carried through the entire procedure. Detailed protocols for analyzing the activity of each of the enzymes were published previously . Individual mouse muscles were dissected from hindlimbs, mounted in OCT, frozen in freezing isopentane, and 10–15 μm cryostat sections were cut from the midbelly region, mounted on gelatin coated glass slides, and stained as described by Dubowitz for either: periodic acid-Schiff to reveal glycogen content, NADH-tetrazolium reductase to show mitochondrial complex I content, or succinate dehydrogenase to determine mitochondrial complex II content. For immunohistochemistry, sections were stained as in Hughes et al. using Vector biotin-conjugated class-specific secondary antibodies and the Vectastain ABC kit. HRP was detected with diaminobenzidine and cobalt enhancement. Serial cryosections were exposed to mAbs that recognize epitopes on distinct MyHC isoforms. A4.951 and A4.840 were used to detect slow MyHC, A4.74 to detect type 2A MyHC, and N2.261 to detect both slow and 2A MyHC . To distinguish developmental isoforms of fast MyHCs F1.652 (embryonic), N3.36, A4.1519 and N1.551 (staining subsets of neonatal and type 2), BF-F3 (adult 2B) and BF35 (all non-type 2X) were employed . Analysis and photography were performed on a Zeiss Axiophot using Normarski optics. Fiber numbers (see Table II ) were determined by scoring each fiber in the full cross section of the muscle as either positive or negative for each antibody. Thus, 300–500 soleus fibers and ∼1,000 extensor digitorum longus (EDL) fibers were scored in each leg. Cross section images close to the midbelly of EDL muscles that had been stained for NADH-tetrazolium reductase were captured on a Macintosh computer and analyzed with NIH Image. Sections were only included in the analysis if fiber major axes showed near random orientation, indicating they were cut close to transversely. Several hundred fibers in each muscle were outlined with the mouse and their parameters (including staining pattern, area, and axis orientation) were recorded. Mean fiber areas were calculated for each fiber type in each muscle. Average fiber size from groups of the same genotype was determined by a weighted average of these means. However, to test accurately for statistically significant differences, an ANOVA test was performed, using Mathematica . Mouse strains have been shown to differ in fiber type content, so we first determined the activity of ten enzymes in three different muscles, plantaris, tibialis anterior, and soleus, in wild-type mice of similar genetic background to the transgenic animal. AK (see Table I for full enzyme names) is a mitochondrial enzyme involved in high energy phosphate transfer and we found no significant difference between fast and slow muscles (Table I ). HK, PHRL, GOPDH, PK, and LDH are enzymes catalyzing individual steps in glycolysis. With the exception of HK, these enzymes had highest activity in the fast glycolytic plantaris muscle, and lowest in the slow oxidative soleus (Table I ). HK is known to be high in slow muscles and this was confirmed in the present study (Table I ). In contrast, Thiol, BOAC, Cit Syn, and MDH are all enzymes that take part in oxidative energy production, and these were all highest in the oxidative soleus and lowest in the glycolytic plantaris muscles (Table I ). The ratio of the activity of each enzyme in the slow soleus to the fast plantaris varied between 0.2 and 0.5 for the glycolytic enzymes (excluding HK), and between 2 and 3 for the oxidative enzymes. These values are illustrated in Fig. 1 which shows the enzyme profile of the slow soleus compared with the fast plantaris. It is evident, and this is in agreement with previous studies, that glycolytic enzyme activities in our nontransgenic mice are higher in fast muscles than in slow muscles, while the opposite is the case for oxidative enzymes. To determine the role of myogenin in adult muscle fibers, we examined MMg transgenic mice in which myogenin cDNA is expressed from the myosin light chain 1 promoter and 3′ 1/3 enhancer . The level of transgenic rat mRNA in the mice was previously reported to be 100-fold of the endogenous form . We confirm by RNase protection that muscle RNA from the MMg mice and MMg + MId mice hybridized strongly with rat mRNA sequence, while RNA from MId and wild-type mice was below the detection limit . To demonstrate that myogenin protein is accumulated from the transgene, we performed Western analysis on extracts of adult transgenic and wild-type littermate thigh muscle . While no myogenin was detectable in wild-type muscle extract, in MMg muscle extract a band was readily detected of ∼32,000 M r , the reported size of myogenin protein . No aberrant sized bands were observed to differ between MMg and wild-type animals. We confirmed the transgene-specificity of the 32,000- M r band by analyzing mice that carried MMg on the MId transgenic background. Whereas the MId mouse did not contain detectable myogenin immunoreactivity, the MMg + MId double transgenic animal showed higher myogenin immunoreactivity than the MMg animal . Attempts to visualize myogenin protein in tissue sections of wild-type, MMg , MId , or MMg + MId mice were not successful, despite use of three distinct mAbs to different epitopes, including the F5D reagent employed in the Western analysis. It is possible that in vivo adult muscle myogenin may be present in an immunologically unreactive form. Nevertheless, the MMg transgene causes accumulation of a protein with immunoreactivity and size typical of myogenin in the predominantly fast thigh muscle of adult mice. As the MMg transgene is expected to be expressed predominantly in fast muscles , we first investigated the fast white plantaris muscle of adult MMg mice. When this and other fast muscles were inspected macroscopically, they appeared more red than in wild-type mice, suggesting a higher myoglobin content, a classical sign of a change in fiber type . The levels of the oxidative enzymes Thiol, BOAC, Cit Syn, and MDH were 6.1 ± 0.1, 25.0 ± 1.9, 4.3 ± 0.1, and 40.2 ± 0.1 (two muscles, mean ± SEM, mol · kg −1 h −1 ), respectively. This represents a two- to threefold increase compared with levels found in plantaris muscles of wild-type littermate mice . The activity levels of the glycolytic enzymes HK, PHRL, GOPDH, PK, and LDH were 2.8 ± 0.0, 7.8 ± 0.26, 3.4 ± 0.4, 55.9 ± 1.5, and 85.5 ± 0.9, respectively. This represents reductions to levels 0.3– 0.6 times the levels in wild-type, with the exception that the glycolytic HK was not significantly changed . Thus, the plantaris muscle of the MMg mice had a changed enzyme profile from one typical of a fast muscle to one typical of a slow muscle. This is most evident by comparing the profiles in Fig. 1 and the upper panel of Fig. 3 , which are remarkably similar. In quantitative terms, MMg plantaris muscles had even higher oxidative enzyme activities than those found in the soleus, but the glycolytic enzymes were not reduced to levels quite as low as in the wild-type soleus. Similar comparisons were made for the fast tibialis anterior muscle . Again, all the oxidative enzymes were significantly increased. The effect on glycolytic enzymes was less pronounced, but the tibialis anterior showed a tendency towards an increase in glycolytic activity. The mouse soleus contains a mixture of type 1 and 2A fibers with no 2B fibers, and very few 2X fibers . Although the MMg transgene expression would be expected to be relatively low in this muscle, several of the oxidative enzymes showed significantly increased activity . There were no significant changes in glycolytic enzymes , but these enzymes are already low in the wild-type soleus. We have previously reported that ∼90% of the myogenin overexpressing mice died during the first few days after birth . The effect was probably not insertion specific. A pathological evaluation revealed nothing unusual in the neonates, but since the regulatory regions in the transgene confer expression only in muscle, it was suggested that the deaths were caused by some subtle muscle pathology . As we are dealing with outbred mice, this raised the possibility that the different enzyme levels observed in surviving adult MMg mice were caused by selection rather than specific effects of overexpressing myogenin. Therefore, we investigated the effects of overexpressing myogenin in animals that had no increased lethality. This was achieved by crossbreeding the MMg animals with transgenic MId animals overexpressing the negative regulator Id-1 . After determining that the MId transgene itself had minimal effects on enzyme levels , enzyme activity was investigated in animals having both transgenes ( MMg + MId ). These mice had enzyme levels similar to those found in muscles containing only the MMg transgene . In the MMg + MId mice, the effect of myogenin overexpression cannot be attributed to selection since the doubly transgenic animals had no increased neonatal lethality . Thus, in all muscles examined, the MMg transgene caused a shift towards oxidative character. To analyze the changes in metabolic properties of MMg and MMg + MId muscles in greater detail, we employed enzyme histochemistry on tissue sections. Although this approach is nonquantitative, it has the advantage of revealing which muscle fibers are altered by the transgene. We examined the well-defined fast EDL muscle, and observed dramatic changes in metabolic properties of a subpopulation of fibers in the presence of the MMg transgene . Fast glycolytic type 2B fibers became oxidative, whereas no change was detected in oxidative type 2A fibers. In wild-type mice, the smallest type 2A oxidative fibers generally contain little cytoplasmic glycogen . In MMg mice, glycogen content of such fibers remained low, consistent with the predicted low level of expression of the transgene in these fibers. In contrast, in wild-type mice the large fast fibers, almost all of which express type 2B or 2X MyHC, fell into two groups: one group that had low levels of glycogen, and another group of slightly smaller size that contained higher levels of glycogen . In MMg mice, the overall number of glycogen-rich fibers was greatly increased, so that fibers in both groups must have raised glycogen content, compared with wild-type littermates . In parallel with the alteration in cytoplasmic glycogen content of the fastest fibers, there was a change in mitochondrial enzyme activities, which are normally highest in mitochondria-rich oxidative fiber types. NADH-tetrazolium reductase and SDH are part of the mitochondrial electron transport chain. The activities of both enzymes were markedly increased in type 2B/2X fibers of MMg EDL, compared with the wild-type . In contrast, the high mitochondrial enzyme activity of type 2A fibers remained unchanged. Thus, the change in mitochondrial enzyme activity detected in whole muscle homogenates could also be detected at the single fiber level. Therefore, fibers expressing distinct MyHC isoforms and with distinct glycogen content could not be distinguished by mitochondrial enzymes in the MMg EDL. The fastest classes of fibers showed the greatest change in MMg mice. Similar changes were also observed in MMg + MId mice, but not in control animals carrying the MId transgene alone, in which fiber type heterogeneity in metabolic properties was clearly detectable and similar to wild-type mice . Analysis of several animals of each genotype gave similar results. However, the phenotype in MMg + MId mice appeared more variable than that of MMg mice. The vast majority of the 2B fibers of all pure MMg animals reacted more strongly than did normal 2B fibers when stained for glycogen, SDH, or NADH-tetrazolium reductase. Two out of the four MMg + MId animals analyzed displayed a similar histochemical picture, but in two other animals the 2B fibers were less affected, and a significant number of the fibers were no different from 2B fibers in wild-type mice. In wild-type mice, type 1 and 2A oxidative fibers normally have lower cross sectional area than glycolytic 2B fibers. Small fiber size in oxidative fibers aids rapid diffusion of oxygen from the surrounding capillaries into the fiber interior core. In MMg mice, in which all fibers are essentially oxidative, the size was much more uniform, and in particular, the 2B fibers were much smaller than in the wild-type . The mean area of this fiber type was depressed almost 50%, and the maximal 2B fiber cross sectional area was reduced from 2,500 μm 2 in wild-type to 1,300 μm 2 in MMg mice. In wild-type animals, the ∼50% of fibers with low mitochondrial content tended to have areas of ∼1,500 μm 2 , and the other 50% of mitochondria-rich fibers had areas of ∼800 μm 2 . In contrast, in MMg mice the average area of all fibers (essentially all of which are mitochondria-rich) was significantly reduced to ∼800 μm 2 . Massive disappearance of mitochondria-poor fibers in wild-type animals could have contributed to the observed results. However, the total number of fibers in four EDL muscles from wild-type mice was 1,071 ± 250 (SD), whereas four EDLs from MMg mice contained 844 ± 151 fibers. This potential small difference was not significant ( t test) and could not account for the wholesale metabolic fiber type conversion observed in the MMg mice. Fiber area was also reduced in MMg + MId mice. Moreover, even the residual 25% of mitochondria-poor fibers in some of these animals was significantly reduced in size to ∼840 μm 2 . However, as we reported previously, MId transgenic mice also show alterations in muscle fiber area , so it is impossible to determine whether the reduction in area of the residual glycolytic fibers in MMg + MId mice is due to the MMg or the MId transgene. Nonetheless, we conclude that when the MMg transgene acts alone it causes the large glycolytic 2B fibers normally present in EDL to acquire a size comparable to the smaller oxidative fiber types. This change could be an adaptation to the increased oxidative metabolism, but we cannot exclude that it might be related to impaired growth during development. Having observed changes in the size of fibers and activity of glycolytic and oxidative metabolic enzymes typical of fast muscles, we examined the expression of fast and slow MyHC isoforms to determine whether these muscle properties also responded to the presence of the MMg transgene. Slow soleus and fast gastrocnemius, plantaris, tibialis anterior, and EDL muscles were dissected from MMg transgenic and control wild-type littermates, and the proportion and distribution of distinct fiber types analyzed with antibodies to specific MyHC isoforms. Despite normal animal to animal variation, none of the investigated muscles showed any consistent difference in the number or distribution of MyHC examined , although the sizes of type 2B MyHC-containing fibers were reduced, as described above . In particular, no increase in the number of A4.840 reactive slow fibers was observed in either fast or slow muscle, nor was any shift in the expression of distinct fast MyHC isoforms detected. For example, there was no increase in the number of fibers reactive with the A4.74 antibody for 2A MyHC usually present in oxidative fast fibers , and no decrease in the number of fibers reactive with BF-F3 antibody for 2B MyHC normally characteristic of fast glycolytic fibers . Analysis with antibodies to embryonic MyHC (F1.652), neonatal fast MyHC (N1.551, N3.36), and antibody BF35, thought to detect MyHCs in all fibers except type 2X , showed no difference in MMg mice compared with wild-type littermates (data not shown). Thus, MMg mice show no alteration in fiber type composition based on expression of a wide variety of MyHC isoforms. To rule out the possibility that selective mortality among MMg pups could hide differences in MyHC, we also examined MId and MMg + MId for changes in fibers expressing MyHC isoforms. Again, no differences from control animals were observed in the proportions of fibers of particular MyHC types (Table III ). Thus, despite showing a shift in the metabolic character of both fast and slow muscles, we were unable to detect a change in MyHC expression induced by the MMg transgene. Overexpression of myogenin influenced the activity of metabolic enzymes, inducing a shift from glycolytic metabolism to oxidative fat-using metabolism in muscles that are normally glycolytic. This shift was accompanied by a reduction in glycolytic fiber size to a diameter typical of oxidative fibers, an increased level of glycogen, and a reddening color of white muscles which expressed the transgene at high levels. This complex change in phenotype essentially mimics the effects of endurance training on muscle , and such changes are thought to be a prerequisite for the increased muscular fatigue-resistance obtained by training. It has been observed previously that myogenin is more highly expressed in regions with high oxidative capacity and mitochondrial content than in glycolytic muscles, in both rodents and fish . Hence, the present findings suggest that myogenin might somehow be involved in gene regulation during fiber type differentiation or adaptation to different training states. Although mechanical stretch and hormones also seem to play a role, it is generally thought that physical activity regulates muscle gene expression through electrical activity . Myogenin expression is strongly regulated by activity , and thought to be involved in the development of denervation supersensitivity for acetylcholine by directly transactivating the genes for the subunits of the acetylcholine receptor . Although it is tempting to draw parallels between activity regulation of acetylcholine receptor and activity regulation of metabolic properties, there are important differences. For example, denervation only has small and variable effects on metabolic enzymes despite leading to a strong upregulation of all myogenic factors, including myogenin, and having dramatic effects on acetylcholine receptor expression. Upregulation of myogenin alone in MMg mice, on the other hand, leads to decline of MyoD and MRF4 mRNAs, and only a mild upregulation of the acetylcholine receptor , but has strong effects on metabolic enzymes. It adds to the complexity of this system that myogenic factors of the helix-loop-helix family are thought to work as heterodimers with E proteins , that may themselves vary between tissues . On the basis of correlations between myogenic factors and MyHC expression, it was previously suggested that the myogenin/MyoD ratio might regulate fiber phenotype . The present work seems to support this idea since manipulation of myogenin expression, which leads to a reciprocal downregulation of MyoD , causes an increase in oxidative metabolism in muscle fibers. The correlations observed previously between MyHC and myogenic factors could be accounted for by a relationship to metabolic properties, because slow MyHC expressing fibers are oxidative and fast 2B fibers are glycolytic. Our data support the conclusion drawn from studies on effects of moderate endurance training: oxidative enzyme activity and MyHC type can be regulated independently . On the other hand, the finding that myogenin also had an effect on glycolytic enzymes without accompanying change in MyHC contrasts with training studies in which glycolytic enzyme changes were observed only after more extensive training or electrical stimulation, and so were thought to parallel changes in MyHC fiber type . Our data suggest that even changes in glycolytic enzymes can be uncoupled from alterations in MyHC fiber type. Nevertheless, we cannot exclude the possibility that myogenin is involved in MyHC fiber type conversions under different conditions than those existing in surviving MMg mice. The absence of MyHC changes could be related to the level of myogenin expressed, or perhaps to a mechanism compensating for persistent artificial high levels of myogenin in MMg animals. A compensatory mechanism might be suspected because, as described previously, only a minority of the mice overexpressing myogenin survive . It is notable that myoD null mice also show increased neonatal death , and that the surviving myoD mutants have only small changes in MyHC phenotype . While we prefer a model where myogenin is a link in the pathway between activity and muscle phenotype, we cannot exclude that the overexpression of myogenin in itself influences activity. Although the animals appeared to move normally, one can speculate that myogenin might have impaired neuromuscular transmission. Due to the high safety factor in neuromuscular synapses, impairment of transmission would have to be major, and more importantly, reduced levels of activity would tend to induce shifts in metabolism to the opposite direction of the one observed, i.e., from oxidative to glycolytic. On the other hand, the shift towards oxidative metabolism could be explained if the myogenin overexpression animals had higher activity levels than wild-type animals. Increased cell autonomous fibrillatory activity such as observed in denervated muscles cannot be excluded. Alternatively, increased activity could be triggered from the CNS, e.g., caused by ectopic transgene expression. However, when the same expression system was coupled to the reporter chloramphenicol acetyltransferase (CAT), which provides a sensitive assay for detection of expression, CAT activity was not found in the brain or any other nonmuscle tissue . All the oxidative enzymes measured are mitochondrial enzymes, and all were increased in MMg mice. However, AK activity did not change, even though two of the three forms of AK are nuclearly encoded proteins located in mitochondria . The distinct changes, in particular mitochondrial enzymes, are similar to the changes observed after endurance training, and suggest not only an increase in mitochondria, but also an alteration in mitochondrial composition . NADH tetrazolium reductase and SDH activities of the electron transport chain increased, indicating coordinated upregulation of mitochondrial proteins encoded by both nuclear and mitochondrial genes. Moreover, NADH-TR and SDH staining patterns gave the impression of an increase in number and distribution of mitochondria, supporting the conclusion that an alteration in number and makeup of mitochondria is promoted by myogenin overexpression. The pathway connecting myogenin to changes in mitochondrial content in the MMg mice is unknown. In other systems, mitochondrial activity is thought to be at least partly mediated through transcriptional regulation, but the mechanisms governing the concerted expression of several nuclear and mitochondrial encoded genes for mitochondrial proteins are still not well understood . A possible effect of myogenin on the genes for metabolic enzymes could be direct, by binding to the promoter of such genes, or indirect, through altering processes that normally regulate mitochondrial biogenesis and cell metabolic status. In a variety of mammalian cell types, recent studies implicate nuclear respiratory factor 1 (NRF-1) in nuclear synthesis of both RNAs encoding several mitochondrial proteins and in genes involved in mitochondrial DNA replication . In cardiac myocytes, NRF-1 expression is induced by electrical activity by an unknown mechanism . Although myogenic factors are not expressed in heart, our data raise the possibility that myogenin could serve as a link between activity and NRF-1–regulated mitochondrial biogenesis in skeletal muscle. Several promoters of metabolic enzyme genes contain the consensus sequence for the binding site of helix-loop-helix transcription factors (E-box), and in genes encoding muscle/heart-specific forms of the cytochrome c oxidase subunits VIII and VIa, it has been shown that an intact E-box is required for efficient tissue-specific transcription . For the VIII subunit, negative effects of Id-1 were also demonstrated, strengthening the idea that helix-loop-helix proteins are important in regulating this gene. The VIa subunit required a MEF-2 site, in addition to the E-box, so myogenic factors might also interact indirectly through this sequence . The promoters for several glycolytic enzymes also contain E-boxes, but in these cases the E-box was found unnecessary for muscle-specific expression. It is, however, still possible that myogenic factors act indirectly on these promoters , since MEF-2 sites are important for muscle-specific expression . Besides, even if myogenic factors might not be required for tissue-specific expression, such factors may still regulate fiber type- or activity-dependent expression. Moreover, few studies have addressed the possibility that myogenic factors could act as transcriptional suppressors, rather than activators (e.g., of glycolytic enzymes). In conclusion, our data raise the possibility that skeletal muscle myogenin, regulated by electrical activity, brings about metabolic changes either through direct effects on genes coding for muscle-specific mitochondrial and cytoplasmic enzymes, and/or indirectly on more ubiquitous mitochondrial genes by enhancing expression of master regulators of mitochondrial biogenesis, like NRF-1. Recently, it has been reported that cyclosporin A can alter muscle fiber type, possibly through effects of the calcium-activated protein phosphatase calcineurin on the NFAT transcription factors . The relationship of the results presented here to the effect of cyclosporin A is currently unclear. However, it would not be surprising if several distinct groups of transcription factors were capable of modulating different aspects of muscle fiber type. | Study | biomedical | en | 0.999996 |
10228178 | Cyclic nucleotide–gated (CNG) 1 channels are present at very high density in the plasma membranes of retinal rod photoreceptor cells, where they generate the electrical response to light . They are activated by the direct binding of cGMP , which occurs at nearly the diffusion-limited rate . With four sites for cooperative binding, low affinity for cyclic nucleotide, and a lack of desensitization in the continued presence of cyclic nucleotide, CNG channels are ideally suited for their role as fast, exquisitely sensitive, molecular switches . Despite their weak voltage dependence , the primary amino acid sequence of CNG channels is similar to that of voltage-dependent channels . Like the other members of the voltage-dependent channel superfamily, CNG channel subunits are thought to contain six transmembrane domains, including an S4 region . CNG channels also contain a pore-lining P region linking the S5 and S6 transmembrane domains, which exhibits sequence similarity to the P region of voltage-gated channels . CNG channels are formed as a tetramer of four subunits around a centrally located pore . The intracellular carboxyl terminal domain of CNG channels contains a highly conserved stretch of ∼120 amino acids that forms the binding site for cyclic nucleotides. This region has significant sequence similarity to the cyclic nucleotide–binding domains of other cyclic nucleotide– binding proteins, including cGMP- and cAMP-dependent protein kinases and Escherichia coli catabolite gene activator protein . The activation of CNG channels is thought to involve an allosteric mechanism whereby ligand binding enhances channel opening . In support of this mechanism, Karpen et al. observed a voltage-dependent closed–open equilibrium of native channels at saturating concentrations of cGMP, indicating the presence of a closed–open equilibrium after the last cGMP molecule had bound. In addition, spontaneous open probabilities have been measured for CNG channels . Thus it appears that ligand binding is not an obligatory step that must precede channel opening. Rather, the opening conformational change can occur in the absence of cyclic nucleotide and is simply made more favorable by the bound cyclic nucleotide. The divalent cation Ni 2+ has been shown to have a potentiating effect on channel activity when applied to the cytoplasmic side . In particular, Ni 2+ causes an increase in the maximal current, especially for weak agonists, and an increase in the apparent affinity for cyclic nucleotide. The mechanism of action of Ni 2+ is thought to involve the coordination of Ni 2+ when the channel is in the open conformation by the histidines at position H420 on adjacent subunits of the channel . This mechanism suggests that Ni 2+ may be acting as an agonist in that, when bound, it shifts the equilibrium toward the activated conformation. The goal of this investigation was to determine how the energetics of the allosteric transition are changed by allosteric modulators, including cyclic nucleotides and Ni 2+ . These experiments provide insights into the mechanism of action of allosteric ligands and the molecular mechanism of the allosteric transition. Our approach was to record steady state single-channel currents from bovine rod (BROD) CNG channels at saturating concentrations of cGMP, cIMP, and cAMP in the presence and absence of Ni 2+ . We analyzed the stochastic sequence of openings and closings of the channel using a signal processing method based on hidden Markov models to determine the number of states and their conductances and to obtain unbiased estimates of the rate constants. From the rate constants, we determined the energetic effects of the allosteric modulators on the allosteric transition. We argue that the interactions of these allosteric modulators with the channel stabilize the open conformation and are partially formed at the time of the transition state for the allosteric transition. Xenopus oocytes were injected with cRNA coding for the α subunit (subunit 1 or CNG1) of the bovine rod channel . Oocyte preparation and cRNA transcription and expression were carried out as previously described . Recordings were typically made 1–10 d after the injection. Initially, the oocytes were stored at 16°C, but once the level of expression was determined to be appropriate for obtaining single-channel recordings, the oocytes were moved to 4°C. The patch-clamp technique was used to record single CNG channel currents from inside-out patches. The patch pipettes, fabricated from borosilicate glass, were coated with Sticky Wax (sds Kerr) and were polished to an initial pipette resistance of 5–20 MΩ. The experiments were carried out at room temperature (20–22°C). The patch pipettes were filled with 130 mM NaCl, 3 mM HEPES, 0.2 mM EDTA, and 500 μM niflumic acid, pH 7.2. The intracellular solution contained 130 mM NaCl, 3 mM HEPES, pH 7.2, and the indicated concentration of cyclic nucleotide (cGMP, cIMP, or cAMP) with either 0.2 mM EDTA or 1 μM Ni 2+ as indicated. Control solutions contained no cyclic nucleotide and either 0.2 mM EDTA or 1 μM Ni 2+ as indicated. Intracellular solutions containing cyclic nucleotides were changed using a DAD-12 Superfusion System ( ALA Scientific Instruments Inc.) controlled by an MRI MB-8000 PC and modified such that each solution had a separate exit port. The patch was then positioned at the mouth of an exit port when recording the currents in the presence of each solution. All reagents were obtained from Sigma Chemical Co. The single-channel currents were recorded using an Axopatch 200B patch-clamp amplifier ( Axon Instruments ). The output of the patch-clamp amplifier was low-pass filtered at 5 kHz through an eight-pole Bessel filter (Frequency Devices Inc.) and digitized at 25 kHz using an ITC-16 computer interface ( Instrutech Corp .). The data were acquired using a Quadra 800 Macintosh computer running HEKA Pulse software ( Instrutech Corp .). For an initial analysis, the data were idealized using the half-amplitude threshold detection technique implemented using TAC single-channel analysis software (Bruxton Corp.). In this method, a transition is detected every time the half-amplitude current level was crossed. The amplitude histogram for the cIMP or cGMP trace of a particular experiment was used to set the full-amplitude current level. Minor adjustments to the baseline level were made by eye to correct for baseline drift. From the idealized current reconstruction, the closed and open durations were measured, and closed and open duration histograms were constructed. Dwell-time distributions were plotted with the Sigworth-Sine transformation, which plots the square root of the number of intervals per bin without correcting for the logarithmic increase in bin width with time . With this transform, the peaks in the duration histograms fall at the time constants of the major exponential components. The dwell-time histograms were fitted using TacFit software (Bruxton Corp.) to the sums of exponential probability density functions using the maximum likelihood method. The histograms were corrected for the distorting effect of the half-amplitude threshold technique on the durations of events between one and two dead times . The half-amplitude threshold method has been the standard for single-channel analysis. However, more rigorous methods for analysis, which use signal processing methods based on hidden Markov models (HMMs), have recently become available for single-channel analysis . The method we used was developed by Lalitha Venkataramanan and Fred Sigworth and is implemented as part of TAC v. 4.0X software (Bruxton Corp.). Unlike previous HMM methods, the HMM approach we used models the observed current as the sum of two components: (a) a noiseless discrete signal that represents the current levels of conducting states generated as the ion channel makes transitions from one state to another and (b) Gaussian noise. The method distinguishes between actual events and noise in a more sophisticated fashion than is possible with the half-amplitude threshold method, which assumes that every time the half-threshold level has been crossed an event has occurred. The algorithm is an extension of the forward–backward equations and the Baum-Welch method . The output of the hidden Markov model is “hidden” because the current observed in an experiment does not directly specify the state of the channel because of additive noise and because multiple closed or open states may share the same conductance. The algorithm uses iterative methods to directly estimate the maximum likelihood set of rate constants for a given specified model. The HMM approach uses inverse filtering to substitute a sharp cut-off filter with corner frequency fixed at 0.4× the sampling frequency for the gradual eight-pole Bessel filter that was used to record the data. Thus, for our experiments recorded with the Bessel filter set at 5 kHz and a sampling frequency of 25 kHz, the effect of inverse filtering was to effectively remove the 5 kHz filter and impose a sharp cutoff filter with a corner frequency of 10 kHz. Because of inverse filtering, the effective bandwidth doubles, making it possible to detect short duration events and obtain estimates for fast rate constants, which were previously missed . This improved frequency response is particularly helpful for measuring the short duration openings that were observed with cAMP and for measuring flicker closings. The inverse filtering is based on the step response of the system. We measured the step response of the system by configuring HEKA Pulse ( Instrutech Corp .) to output voltage steps, converting the voltage steps into current steps using a voltage-to-current converter ( Instrutech Corp .), and directly inputting the current steps into the head stage of the patch-clamp amplifier. The HMM program was run on a Macintosh PPC 8100 computer (100 MHz) configured with 176 MB of RAM. The program was run in the continuous time mode with four auto-regressive coefficients and a 10 −8 level of precision in the log likelihood. A typical data segment was 150,000 points or 6 s of data, which required about 150 iteration cycles or ∼30 min of computation time. The accuracy of the HMM analysis was confirmed by simulating current records using a QS-1 electronic channel simulator ( Instrutech Corp .). The output of the channel simulator was passed through a voltage-to-current converter and fed into the headstage of the patch-clamp amplifier. The simulated recordings were analyzed in a manner identical to the patch-clamp recordings. The simulations were calculated for a C 0 ↔ O 1 ↔ C 2 model with rate constants set to approximately the values determined for the BROD channel when activated by cGMP, cIMP, or cAMP. For simulations with a duration of 10 s, the rate constants determined from the HMM analysis were generally within 10% of the values used in the simulation. The precision of the HMM analysis was determined by simulating 20 different 1-s long segments of data and analyzing each segment individually. The standard deviation of the rate constants due to stochastic variation was between 5 and 30% of the mean value. To obtain the set of rate constants for the C 0 ↔ O 1 ↔ C 2 scheme , the rate constants determined using HMM for the C 0′ ↔ C 1′ ↔ O 2′ scheme were converted to the equivalent set of rate constants for the C 0 ↔ O 1 ↔ C 2 scheme. An exact conversion is possible because the two schemes share the same eigen values. Using primes to designate the rate constants for the C 0′ ↔ C 1′ ↔ O 2′ scheme, the equations we used were: \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_{01}=\frac{(k_{01}^{\prime}+k_{10}^{\prime}+k_{12}^{\prime})-\sqrt{(k_{01}^{\prime}+k_{10}^{\prime}+k_{12}^{\prime})^{2}-4k_{01}^{\prime}k_{12}^{\prime}}}{2},\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{21}=\frac{(k_{01}^{\prime}+k_{10}^{\prime}+k_{12}^{\prime})+\sqrt{(k_{01}^{\prime}+k_{10}^{\prime}+k_{12}^{\prime})^{2}-4k_{01}^{\prime}k_{12}^{\prime}}}{2},\end{equation*}\end{document} \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{10}=\frac{k_{01} \left[ \frac{k_{21}^{\prime}}{k_{12}^{\prime}} \left( 1+\frac{k_{10}^{\prime}}{k_{01}^{\prime}} \right) -\frac{k_{21}^{\prime}}{k_{21}} \right] }{1-\frac{k_{01}}{k_{21}}},\end{equation*}\end{document} and \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_{12}=k_{21}^{\prime}-k_{10}.\end{equation*}\end{document} The rate constants determined from fitting the COC scheme directly were <1% different from the equivalent rate constants converted from the CCO scheme, and the likelihood values for the two schemes were identical (within our precision level of 10 −8 ). We injected Xenopus oocytes with cRNA encoding the α subunit of the BROD channel and recorded currents through the expressed channels using the inside-out configuration of the patch-clamp technique. By adjusting the amount of cRNA injected, the time after injection, and the diameter of the tip of the pipette, we obtained patches containing only a single CNG channel. A long continuous recording at +80 mV of a typical BROD single-channel patch is shown in Fig. 1 . For the duration of the trace, the cytoplasmic face of the patch was bathed continuously with a saturating concentration of cGMP (16 mM), the physiological agonist of BROD CNG channels. This channel showed bimodal behavior, with alternating long-lived quiescent and bursting periods. During the quiescent mode, there were occasional short-lived openings. The quiescent periods were difficult to characterize because their durations varied from patch-to-patch and, within any given patch, only a few were observed. For the following analyses, we focused on the bursting periods by omitting all quiescent periods of duration longer than 200 ms. During the bursting periods, the channels were very highly activated in the presence of 16 mM cGMP, a saturating concentration for BROD channels . Since the binding of cGMP to the channel is thought to occur at 5 × 10 7 M −1 s −1 , binding would be expected to occur with a time constant of ∼1 μs at 16 mM cGMP. Since the sample interval in our experiments was 40 μs, the kinetics at saturating cyclic nucleotide concentrations do not reflect the rate constants of binding or unbinding of the cyclic nucleotide. Rather, they reflect gating events occurring after the full complement of ligands have bound to the channel. BROD CNG channels can be activated not only by the physiological agonist cGMP but also by cIMP and cAMP. These agonists are similar in structure and bind to the same binding site with similar initial binding affinities but differing abilities to promote channel activation . Since these cyclic nucleotides differ in only the most distal portion of their purine ring, interactions between the purine ring and the binding domain must be involved in the allosteric transition. In Fig. 3 , current families elicited by voltage steps from 0 mV to between −80 and +80 mV are shown for activation by saturating concentrations of cGMP, cIMP, and cAMP in the absence (A) and presence (B) of 1 μM Ni 2+ . Since the effect of Ni 2+ was not instantaneous, the currents in the presence of Ni 2+ were recorded after Ni 2+ had been applied for several minutes when the currents were stable. The leak currents in the absence of cyclic nucleotide were subtracted, and all currents were normalized to the current obtained at +80 mV in the presence of 16 mM cGMP + 1 μM Ni 2+ . In Fig. 3 A, we see that cGMP activated the most current (I cGMP /I cGMP+Ni = 0.96 ± 0.03, mean ± SEM, n = 6), cIMP was intermediate (I cIMP / I cGMP+Ni = 0.60 ± 0.05, n = 6), and cAMP activated the least (I cAMP /I cGMP+Ni = 0.012 ± 0.005, n = 6). When 1 μM Ni 2+ was added, the cGMP-induced currents were largely unaffected, suggesting that the currents were already nearly maximally activated before Ni 2+ was applied. The cIMP-induced currents in the presence of Ni 2+ became comparable in size to those of cGMP (I cIMP+Ni /I cGMP+Ni = 0.94 ± 0.04, n = 6), and the cAMP currents increased dramatically in size (I cAMP+Ni / I cGMP+Ni = 0.42 ± 0.11, n = 6). We interpret these results to indicate that cyclic nucleotides and Ni 2+ are noncompetitive allosteric modulators and that cIMP and cAMP are partial agonists. To investigate the kinetic basis for the differences in the amounts of current elicited by the three cyclic nucleotides in the presence and absence of Ni 2+ , we recorded the currents through single CNG channels. Examples of traces and amplitude histograms for a representative single-channel patch are shown in Figs. 4 and 5 . The currents were recorded at +80 mV in the continuous presence of the indicated cyclic nucleotide in the absence and presence of 1 μM Ni 2+ . In the control traces, there was no evidence for openings in the absence or presence of 1 μM Ni 2+ , although open probabilities <2 × 10 −3 would have been missed by the method. Based on the absence of distinguishable intermediate current levels in the traces and the absence of intermediate level peaks in the amplitude histograms, it appears that CNG channels gate primarily between only two conductance levels, open and closed, at saturating concentrations of cyclic nucleotide. From the amplitude histograms, it is apparent that there were differences in the open probabilities elicited by the three cyclic nucleotides and that Ni 2+ increased the open probability for each ligand without affecting the single-channel conductance. Thus we conclude that cyclic nucleotides and Ni 2+ behave as allosteric modulators and that the open states were indistinguishable based on open channel current level. To investigate this effect quantitatively, we recorded currents from a set of single-channel patches and calculated the open probability from fits of the amplitude histograms with the sums of two Gaussians. Across this set of experiments, the open probability averaged 0.93 ± 0.01 (mean ± SEM, n = 14) for cGMP, 0.49 ± 0.05 ( n = 13) for cIMP, 0.008 ± 0.002 ( n = 13) for cAMP. In the presence of 1 μM Ni 2+ , the open probability increased for all three agonists: the open probabilities were 0.94 ± 0.01 ( n = 5) for cGMP + Ni 2+ , 0.95 ± 0.01 ( n = 3) for cIMP + Ni 2+ , and 0.55 ± 0.10 ( n = 3) for cAMP + Ni 2+ . These open probabilities were very similar to the fractional activations measured in the macroscopic current experiments , indicating that the differences in fractional activations measured in macroscopic current experiments could be entirely accounted for by differences in open probability . Many of the cAMP-induced openings in the absence of Ni 2+ were comparable in duration to the dead time (40 μs) and thus were missed altogether or appeared as transitions of less than the expected full amplitude level . In addition, the open channel peak in the amplitude histogram was not resolved, thus making it difficult to quantify the open probability or the single-channel conductance. Fig. 6 A illustrates that it was possible to resolve an open level peak for cAMP by plotting the amplitude histogram data for a single-channel patch activated by cAMP on log-linear axes. The fit is to the sum of two Gaussians, which appear as parabolas on log-linear axes. The open probability was 0.006, and the peak of the open histogram was centered at 1.5 or 0.7 pA less than the 2.2 pA level measured for cGMP and cIMP in the same experiment. To improve the frequency response, the data were inverse filtered (see methods ), and the effect on representative cAMP openings and on the amplitude histogram is shown in Fig. 6 B. After inverse filtering, the data appeared noisier but with faster response time, and the apparent current for the openings was larger. The effect on the amplitude histogram was to increase the apparent single-channel current by ∼0.2–0.6 pA. The combination of inverse filtering and log-linear axes for amplitude histograms is useful for studying rare activity modes, such as spontaneous openings and subconductance states. In Fig. 7 is the set of amplitude histograms after inverse filtering for the experiment shown in Figs. 3 and 4 . As can be seen in this figure, the single-channel amplitude was approximately the same in each of the conditions, and the amplitude histograms were well fit by the sum of two Gaussians. There was no evidence for substate activity at these saturating concentrations of cyclic nucleotides. In addition, there was no evidence for a resolved spontaneous opening peak in the absence or presence of Ni 2+ without cyclic nucleotides. Thus, we conclude that the absence of an observed peak places an upper limit on the spontaneous open probability of 2 × 10 −3 in the presence or absence of Ni 2+ . This result is in no way inconsistent with estimates for the spontaneous open probability of 1.25 × 10 −4 and 1.5 × 10 −5 , as the approximate resolution of our method for measuring small open or closed probabilities is 2 × 10 −3 . To further characterize the single-channel current amplitude, we tabulated the single-channel amplitude across experiments. The results are plotted in Fig. 8 . For each experiment, the single-channel current was measured by constructing an amplitude histogram over a short segment of data (to avoid error due to slow baseline drift) and fitting the sum of two Gaussians to the inverse-filtered data. Some variation in the single-channel amplitude across experiments was observed. There are several possible sources for this variation: (a) small voltage offsets, (b) small amounts of baseline drift, and (c) the limited frequency response of the system. For the case of cAMP in the absence of Ni 2+ , the major source of error was the limited frequency response of the system, which prevented many of the openings from reaching full amplitude, thereby broadening and distorting the open-channel distribution and shifting the open-channel peak toward a smaller amplitude. This error was alleviated for cAMP + Ni 2+ . For the case of cGMP + Ni 2+ , the limited frequency response of the system was again the major source of error, but in this case the effect was on the closed-channel peak, as many of the closed durations failed to reach the closed-amplitude level. Despite variation, it is clear that the large differences in the fractional activations measured in macroscopic current experiments are due to difference in open probability, not single-channel conductance. To obtain a preliminary analysis of the single-channel kinetics, the half-amplitude threshold method was used to measure the open and closed times (see methods ). The half-amplitude method requires a high signal-to-noise ratio to avoid noise crossings of the half-threshold level. Thus this analysis was done on noninverse filtered data. Shown in Figs. 9 (without Ni 2+ ) and 10 (with Ni 2+ ) are the duration histograms for the patch illustrated in Figs. 4 and 5 . The histograms were corrected for the distorting effect of the half-amplitude technique on event durations between one and two dead times. In Fig. 9 , the open duration histograms were generally well fit by single exponential distributions while the closed duration histograms were fit by the sum of two exponentials. The time constants of the short duration component of the closed duration histograms appeared to be independent of cyclic nucleotide. The longer duration component was shortest for cGMP, longer for cIMP, and much longer for cAMP. The open duration was longest for cGMP, intermediate in duration for cIMP, and very short for cAMP. On application of Ni 2+ , the open durations became longer, with the most dramatic effect on cAMP. We have also analyzed a number of records in the absence of cyclic nucleotide (control records) using the half-amplitude threshold-crossing method. In each case, only a handful of threshold-crossing events were obtained over 5–10 s of data, suggesting that unliganded openings are rare (data not shown). From the half-amplitude analysis of the data, it thus appears that the kinetics at saturating ligand concentrations can be described by two closed and one open states. It is also clear that many of the open events in the presence of cAMP without Ni 2+ and many of the short duration closed events in the presence of all three ligands both with and without Ni 2+ are missed because of the limited frequency response of the recording system. For a more rigorous analysis of the kinetics, we used a signal processing method based on hidden Markov model methods to estimate the most likely transition rate constants for a set of kinetic schemes (see methods ). The HMM approach we used has a number of useful features for the analysis of single-channel data: (a) it extracts the rate constants from single-channel data, even with a poor signal-to-noise ratio; (b) it automatically corrects for baseline drift and periodic noise; (c) it does not require idealization of the single-channel data; (d) it naturally takes into account missed open and closed events due to the limited frequency response of the recording system; (e) it considers the sequence of events that occurs (information that is lost in binned duration histograms); (f) it provides a maximum likelihood value for discriminating among models; and (g) it extends, through the use of inverse filtering, the frequency response of the recorded system, enabling fast rate constants to be estimated more accurately . Many of these features, in various combinations, are offered by other single-channel analysis methods. The main disadvantage of the HMM analysis is that it is restricted (at present) to relatively small amounts of data because of the extensive computer time required. The HMM approach directly optimizes the rate constants for a specified scheme without idealizing the data. It considers all possible state sequences to account for the data, not just the most likely sequence. However, a current and level reconstruction that represents the most likely state sequence can be helpful for comparing the HMM method to the half-amplitude threshold method. Fig. 11 A shows a short segment of data recorded in the presence of 16 mM cIMP and an analysis using the two methods. With the half-amplitude threshold technique, an event is detected every time the half-amplitude level is crossed . The HMM method provides a current reconstruction and a predicted state sequence. Shown are the predicted current and state sequence (D) for the C 0 ↔ O 1 ↔ C 2 scheme. This comparison reveals that the HMM method predicts events that are missed by the half-amplitude method. The ability of the HMM method to extend the effective frequency response is a result of inverse filtering and the HMM algorithm. The HMM approach provides two outputs: (a) the most likely set of rate constants for a particular gating scheme and (b) the maximum likelihood of the data given the scheme. By comparing the maximum likelihood values for each of a number of different schemes, the HMM approach can be used to determine the minimal scheme that captures the major features of the gating kinetics. To minimize the number of models that we needed to test, we used generic uncoupled models . These schemes are considered uncoupled because every closed state is connected directly to every open state. Such uncoupled schemes were selected because, theoretically, they provide the maximum likelihood of the data given the model for any scheme with the same number of closed and open states . A table of log likelihood ratios relative to the likelihood of the C ↔ O model for channels activated by cGMP, cIMP, and cAMP is shown in Fig. 12 . As can been seen in this figure, the log likelihood ratio increased with increasing model complexity for each of the cyclic nucleotides. The addition of a second open state caused only a moderate increase in log likelihood. However, the addition of a second closed state caused a large increase in the log likelihood, suggesting that two closed and one open states are absolutely required to describe the gating kinetics. Similar results were seen for two other patches. Based on the Asymptotic Information Criterion , the increase in likelihood observed with the models containing two closed and one open states is significant for all cyclic nucleotides for all patches analyzed. For models more complicated than two closed and one open states, there were small improvements in the maximum likelihood. These much smaller increases in likelihood were not consistently significant for all cyclic nucleotides or for all patches. In addition, the rate constants for these more complex models were poorly determined and inconsistent between patches. These small increases may signify that the underlying gating is more complicated than two closed and one open states, or may arise from slight differences in noise or nonstationary behavior between recordings. Thus, we focused our analysis on schemes containing two closed and one open states. With two closed and one open states, there is the possibility of two linear schemes (C 0′ ↔ C 1′ ↔ O 2′ and C 0 ↔ O 1 ↔ C 2 ) and of a cyclic three-state scheme. Since the cyclic three-state scheme has an additional free parameter but is computationally no more likely, we focused on the two linear schemes. Despite the computational equivalence of the C 0′ ↔ C 1′ ↔ O 2′ and C 0 ↔ O 1 ↔ C 2 schemes, the physical interpretations of these two schemes are quite different. In particular, the C 0′ ↔ C 1′ ↔ O 2′ scheme could, for example, describe the activation of a channel composed of two functional dimers, both of which would have to enter the activated conformation for the channel to open. Such a model has recently been proposed for the BROD channel . In contrast, in the C 0 ↔ O 1 ↔ C 2 scheme, a single concerted conformational change could underlie the C 0 ↔ O 1 transition, while open-channel block or the closing of a secondary gate could underlie the O 1 ↔ C 2 transition. We analyzed the kinetics for a number of different experiments in terms of the C 0′ ↔ C 1′ ↔ O 2′ and C 0 ↔ O 1 ↔ C 2 schemes. The experiments analyzed included eight single-channel experiments and six multichannel (two or three channels) with significant (>0.5 s) periods during which only one channel was activated at a time. As before, we performed a burst analysis by rejecting any quiescent periods of duration exceeding 200 ms from analysis. For the multichannel experiments, we viewed the records by eye and rejected any periods during which there were two or more simultaneous openings. For cAMP, this may have occasionally resulted in our analyzing short segments of data containing two channels. However, no systematic differences between the rate constants for cAMP for single-channel and multichannel patches were observed. The median duration of data selected for each cyclic nucleotide in an experiment was 6 s, ranging from 0.66 to 10 s. A summary of the rate constants for the C 0′ ↔ C 1′ ↔ O 2′ scheme is shown in Fig. 13 . As can be seen in this figure, there was cyclic nucleotide dependence in the rate constants for the C 0′ ↔ C 1′ transition ( k 01′ and k 10′ ) and for the C 1′ ↔ O 2′ transition ( k 12′ and k 21′ ). For both transitions, the forward rate constants ( k 01′ and k 12′ ) were fastest for cGMP, intermediate for cIMP, and slowest for cAMP. Conversely, the reverse rate constants ( k 10′ and k 21′ ) were fastest for cAMP, intermediate for cIMP, and slowest for cGMP. Based on a Student's t test, all four rate constants were significantly different between cGMP and cAMP ( P < 0.05). Our observation that the rate constants for both transitions were cyclic nucleotide dependent indicates that both conformational changes involve interactions of the channel with the cyclic nucleotide. Mechanistically, the C 0′ ↔ C 1′ ↔ O 2′ scheme could describe two coupled conformational changes occurring during channel activation. Recently, it has been proposed that BROD channels exist as functional dimers and that the activation process could involve independent conformational changes in each of the two dimer pairs . If these conformational changes are independent, then the k 01′ rate constant would be expected to be 2 × k 12′ . Similarly, the k 21′ rate constant would be expected to be 2 × k 10′ . Comparing the median values for these rate constants for each of the cyclic nucleotides, we found that k 01′ was 2–10-fold slower than k 12′ . The k 21′ rate constant ranged from 2-fold faster to 17-fold slower than k 10′ . Thus, our results are not quantitatively consistent with a mechanism involving two independent conformational changes during activation. Rather, a mechanism involving cooperative interactions between the dimers would be predicted. Shown in Fig. 14 are the rate constants for the C 0 ↔ O 1 ↔ C 2 scheme. These rate constants were calculated by converting the rate constants we obtained for the C 0′ ↔ C 1′ ↔ O 2′ scheme to the equivalent set of rate constants for the C 0 ↔ O 1 ↔ C 2 scheme (see methods ). For the C 0 ↔ O 1 ↔ C 2 scheme, we observed cyclic nucleotide dependence in both rate constants for the C 0 ↔ O 1 transition ( k 01 and k 10 ). Based on a Student's t test, both rate constants associated with the first transition were significantly different for all three cyclic nucleotides ( P < 0.05). Thus, interactions between the places where the three cyclic nucleotides differ (the purine rings) and the channel are formed during the first transition. Since there was cyclic nucleotide dependence in both rate constants, these interactions were partially formed at the time of the transition state for the transition. In contrast, aside from the large range in values for the k 12 rate constant for cAMP, the O 1 ↔ C 2 transition was cyclic nucleotide independent. The large range in values for k 12 for cAMP reflects the fact that this rate constant was not well determined since, when activated by cAMP, the channels spent only a small fraction of the time open and thus made very few transitions to the C 2 state. The lack of cyclic nucleotide dependence in the second transition suggests that interactions between the cyclic nucleotide and the channel are not involved in this transition. Rather it appears that activation involves interactions between the cyclic nucleotide and the channels but that, once activated, the channels are capable of undergoing a second closed–open transition outside of the activation process. This second transition could involve the closing of a secondary gate or the block of the channel pore, but at the present time we have no direct evidence in support of either mechanism. To investigate the errors in the determination of the rate constants by the HMM approach, we mapped the curvatures of the likelihood surfaces for each of the rate constants and the amplitude. The results are shown in Fig. 15 for the C 0′ ↔ C 1′ ↔ O 2′ (A) and C 0 ↔ O 1 ↔ C 2 (B) schemes. These maps show how sensitive the log likelihood is to the exact value of each parameter. This analysis was performed on a short segment of data (0.73 s or 18,198 sample points) from an experiment in which a single channel was observed in the patch and for which currents were elicited by cGMP. The maximum likelihood estimate was determined with all parameters allowed to vary. The curvature of the likelihood surface for each parameter was determined by calculating the variation in the log likelihood with small deviations in the value of the parameter away from its maximum likelihood value. Specifically, while holding a parameter constant at values a few percent above or below the optimal value, the log likelihood was maximized again, allowing all the parameters except the parameter under investigation to vary freely. The resulting log likelihood values were plotted as the difference in log likelihood from the maximum likelihood versus the percent change from optimal value, as shown. The curves were fit by the 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*}{\Delta}LL=-\frac{1}{2} \left( \frac{x}{s} \right) ^{2},\end{equation*}\end{document} where Δ LL is the difference in the log likelihood from its maximum likelihood value, x is the percent change in the parameter under investigation, and s is the 1-SD confidence limit on the estimated rate constant in units of percent. The values for s were 0.4% for the single-channel amplitude, 35% for k 01′ , 37% for k 10′ , 8.3% for k 12′ , 9.7% for k 21′ , 36% for k 01 , 42% for k 10 , 9.2% for k 12 , and 8.3% for k 21 . For this analysis, we see that the errors in the two rate constants for a particular transition were similar. In addition, because there were more transitions to the C 2 state than to the C 0 state for the case of cGMP activation, the errors in the measurements of the rate constants for the O 1 ↔ C 2 transition were smaller than for the C 0 ↔ O 1 transition. In general, we expect that the errors estimated here are likely to be larger than typical, as errors decrease with duration of record, and the segment of data selected for this analysis was shorter than for most experiments. Even so, in all cases for cGMP, the rate constants for the O 1 ↔ C 2 transition would be expected to be better determined than for the C 0 ↔ O 1 . For cIMP, the rate constants for both equilibria would be expected to be well determined. For cAMP, the rate constants for the first transition of C 0 ↔ O 1 ↔ C 2 would be expected to be better determined than for the second transition because the majority of events would be between C 0 and O 1 . For cAMP, we had the additional problem of not being certain as to the number of activated channels in the patch. We believe that any error in the determination of the number of channels present in a cAMP trace could be responsible for at most a threefold (reflecting the maximum number of channels in a patch that was analyzed) effect on the k 01 rate and would have a negligible effect on the k 10 rate. To compare the variation across experiments with our confidence limits on the measurement, we compared the standard deviations in the rate constants for the 14 experiments with the confidence intervals determined above. The results for cGMP are shown in Table I . For every rate constant, there is considerably more variation across experiments than our confidence limits on the measurement can explain. In addition, we have analyzed the amount of stochastic variation expected by analyzing 20 different 1-s long segments of simulated data (see methods ). The standard deviation of the rate constants due to stochastic variation was between 5 and 30% of the mean value, once again suggesting that there is considerably more variability across experiments than can be explained by stochastic variation. This result indicates that there is heterogeneity in the channels. There are several possible sources of this heterogeneity: (a) tyrosine dephosphorylation ; (b) serine/threonine phosphorylation ; (c) possible differences across oocytes, such as the level of glycosylation, lipid composition, or changes in the levels of accessory proteins that interact with the channel; and (d) small temperature differences. The transition metal divalent Ni 2+ has been shown to potentiate rod CNG channel currents when applied in the presence of cyclic nucleotides . Ni 2+ potentiation has been used previously to estimate the equilibrium constant L for the allosteric transition from macroscopic current experiments . To test the validity of this method, we compared the values for Δ G 0 = − RT ln ( k 01 / k 10 ) determined for the allosteric transition of the C 0 ↔ O 1 ↔ C 2 scheme from the set of single-channel experiments to the values for Δ G 0 = − RT ln L measured from a set of macroscopic current experiments, where L = I /( I max − I ), I is the current measured in a macroscopic current experiment, and I max was estimated using Ni 2+ potentiation, R is the universal gas constant, and T is the absolute temperative. While we could not discriminate between the C 0 ↔ O 1 ↔ C 2 and C 0′ ↔ C 1′ ↔ O 2′ schemes based on likelihood, we prefer the C 0 ↔ O 1 ↔ C 2 scheme because there was cyclic nucleotide dependence in only the first transition of the C 0 ↔ O 1 ↔ C 2 scheme. Also, the C 0 ↔ O 1 ↔ C 2 scheme has the simple physical interpretation of the first transition being the allosteric transition and the second transition being to a flicker closed state out of the activation pathway. Fig. 16 shows that there were excellent correspondences between the values for Δ G 0 for the two methods, partially validating our assumption of a C 0 ↔ O 1 ↔ C 2 scheme and indicating that the single-channel behavior is representative of what is observed in macroscopic current experiments. To determine the effects of Ni 2+ on the gating at saturating concentrations of cyclic nucleotide, we analyzed the gating in the presence of 1 μM Ni 2+ in terms of the C 0 ↔ O 1 ↔ C 2 scheme and compared the rate constants we obtained to the rate constants we obtained in the absence of Ni 2+ . The effects of Ni 2+ on the single-channel kinetics at saturating cIMP are summarized in Fig. 17 . As can be seen in this figure, the k 10 rate constant was 25-fold slower in the presence of Ni 2+ . The median k 01 rate constant was 1.6× faster, although this is not a significant effect. The k 12 and k 21 rate constants were largely unchanged. The overall effect on the C 0 ↔ O 1 transition was a 2.1-kcal/mol stabilization of the allosteric transition by Ni 2+ . Fig. 17 B shows a comparison of the values for Δ G 0 obtained from single-channel experiments to the values for Δ G 0 obtained from macroscopic experiments. This comparison reveals that there was a fairly good correspondence between the macroscopic and single-channel experiments. For cAMP, the effect of Ni 2+ is summarized in Fig. 18 . In this case, Ni 2+ sped up the k 01 rate 5-fold and slowed down the k 10 rate 30-fold. The combination of these two effects translates into a 2.9-kcal/mol more favorable allosteric transition. Comparing the values for Δ G 0 obtained from single-channel experiments to the values for Δ G 0 obtained from macroscopic experiments, again the correspondence was good. For cGMP with Ni 2+ , the k 12 and k 21 rate constants were largely unchanged, but we obtained considerable variation in the values for the k 01 and k 10 rate constants (data not shown). The most likely source of this variation was the fact that the open probability in the presence of cGMP and Ni 2+ was so high that there were so few transitions to the C 0 level and the HMM algorithm was not able to measure the k 01 and k 10 rate constants with any precision. Quantitatively, this was an expected finding as, for cGMP without Ni 2+ , the median value for the k 10 rate was 26/s. Assuming that Ni 2+ slowed this rate 25–30-fold as it did for cIMP and cAMP, then the expected value of the k 10 rate constant for cGMP plus Ni 2 would be 1/s, which is too slow a rate for us to determine in our experiments. Focusing on the results for cAMP and cIMP with Ni 2+ , we conclude that Ni 2+ acts like the cyclic nucleotides in that its primary effect is on the C 0 ↔ O 1 allosteric transition and not on the O 1 ↔ C 2 transition. This finding suggests that Ni 2+ is a weak noncompetitive agonist of BROD channels. Here we have investigated the gating kinetics of single CNG channels at saturating concentrations of different cyclic nucleotides in the presence and absence of the transition metal divalent Ni 2+ . We found that a simple two-state C ↔ O scheme is not sufficient to explain the kinetics. Rather, an additional closed state is required. Adding this additional closed state to the C ↔ O model, either on the opposite side of the open state (as in C ↔ O ↔ C) or on the same side of the open state (as in C ↔ C ↔ O) improved the description of the kinetics at saturating ligand concentrations. Although these two schemes are equivalent on the basis of their likelihood values, their interpretations are quite dif-ferent. Monod et al. proposed a mechanism for the activation of allosteric proteins known as the Monod- Wyman-Changeux (MWC) model. The major tenet of their hypothesis was that activation involves a single concerted conformational change. Previously it has been suggested that CNG channels might conform to a MWC model . However, recently it has been suggested that the activation of CNG channels is inconsistent with models in which the four subunits activate in a single concerted step (MWC model) . Multiple open states with different conductance levels have been observed in channels containing covalently attached ligands . However no substate activity was apparent in our recordings at saturating concentrations of either full or partial agonists . In addition, it has been suggested that the four subunits may associate and activate as two independent dimers . It has also been shown that the underlying gating at saturating cyclic nucleotide concentrations is consistent not only with a single concerted step but also with two or three consecutive conformational changes . Based on these precedents, the C ↔ C ↔ O scheme, which could describe two coupled conformational changes in different subunits or dimers, seems plausible. However, the difference in the cyclic nucleotide dependence of k 01′ and k 12′ argues against this possibility. Alternatively, we have found that the C 0 ↔ O 1 ↔ C 2 scheme has significant cyclic nucleotide dependence in only one of the two transitions. We interpret the first transition of C 0 ↔ O 1 ↔ C 2 to be the allosteric transition. Because the rate constants for the second transition of C 0 ↔ O 1 ↔ C 2 were cyclic nucleotide independent, this transition is unlikely to be a part of the allosteric transition. Rather, it may represent the closing of a secondary gate or a pore blocking state. A rapid flickery proton block has been described in CNG channels . While the underlying mechanism is undoubtedly more complex than two closed and one open states, this mechanism adequately describes our data and provides a physical interpretation of the results. The qualitative conclusions concerning the effects of cyclic nucleotide and Ni 2+ on the stability of the open and transition states should still be valid in more complex models. These conclusions are based on the effects of these allosteric modulators on the open and closed durations and are not dependent on any particular model. As shown in Fig. 16 , the overall Δ G 0 for the allosteric transition ranges was between −2 and 3 kcal/mol for cGMP and cAMP, respectively. Since these cyclic nucleotides differ in only the most distal portion of their purine ring, we conclude that the cyclic nucleotide–binding domain interacts with the purine ring of the cyclic nucleotides differently during the allosteric transition. The energetics for these favorable cyclic nucleotide– binding domain interactions must therefore be determinants of the stability of the open state. Since the cyclic nucleotides affected both the opening and closing rate constants, these interactions can affect the energetic determinants of both the opening and closing conformational changes. If there is a single high energy transition state that determines both the opening and closing rates, these results suggest the interactions between the purine ring and the cyclic nucleotide– binding domain are partially formed at the time of the transition state and serve to reduce the energetic barrier for activation. Alternatively, if the opening and closing rates are determined by a number of low energy transitions, these results suggest that these interactions can be formed before channel opening, and broken before channel closing. The effects of Ni 2+ on the free energy of the allosteric transition was 2–3 kcal/mol . The mechanism of action of Ni 2+ has previously been shown to involve the coordination of Ni 2+ when the channel is in the open conformation by histidines at position H420 on adjacent subunits of the channel . This mechanism suggests that, during the allosteric transition, there is a rearrangement of H420 from neighboring subunits permitting Ni 2+ to bind with 2–3 kcal/mol greater affinity to open than to closed channels. Here we show that Ni 2+ affected both the opening and closing rate constants , suggesting that the interactions between Ni 2+ and H420 are partially formed at the time of the transition state for the allosteric transition or can be formed before channel opening, and broken before channel closing. Interpreted in terms of the Monod-Wyman-Changeux model, this result suggests that Ni 2+ should be considered as an allosteric ligand of BROD channels. Ni 2+ does not merely hold the channel open, rather it induces and stabilizes the allosteric transition. If Ni 2+ is an agonist, then Ni 2+ would be expected to be capable of activating BROD channels in the absence of cyclic nucleotide. Yet, we were unable to observe a peak in the amplitude histogram for our control traces in the presence of Ni 2+ . Would we have expected to see an open-channel peak in this histogram? The spontaneous opening probability has been reported to be 1.25 × 10 −4 or 1.5 × 10 −5 . If Ni 2+ promotes the allosteric transition by the same amount in the absence as in the presence of cyclic nucleotide (40–150-fold), we would expect that the open probability in the presence of Ni 2+ would be between 6 × 10 −4 and 1.9 × 10 −2 based on these estimates for the spontaneous opening probability. Since an open probability of 1.9 × 10 −2 is similar to the value for cAMP activation, we believe that we would have observed openings in the presence of Ni 2+ if the open probability were that high. Our inability to do so down to a resolution of 2 × 10 −3 suggests that Ni 2+ is a weaker agonist than cAMP. Alternatively, the lack of apparent spontaneous openings by Ni 2+ could indicate that Ni 2+ only promotes the opening of liganded channels. This would suggest that the conformational change involved in the spontaneous openings is different from the conformational change involved in the ligand-induced openings, at least with reference to the movement of H420. Perhaps, due to some compliance in the gating machinery, the gate can spontaneously open without the movement of the entire C-linker region between the S6 transmembrane segment and the cyclic nucleotide–binding domain. This mechanism might also explain the recent finding of other alterations in the C-linker that do not affect spontaneous openings . The kinetics at saturating cyclic nucleotide concentrations are described by a model with two closed and one open states. The allosteric transition involves the formation of stabilizing interactions between the cyclic nucleotide and the channel, and these interactions are partially formed at the time of the transition state for the allosteric transition. In the presence of cyclic nucleotides, Ni 2+ acts like an agonist of the BROD channel, suggesting that Ni 2+ , like cyclic nucleotides, should be considered an allosteric modulator of the channel. | Study | biomedical | en | 0.999998 |
10228179 | Cyclic nucleotide-gated (CNG) 1 ion channels of retinal rod photoreceptors are exquisitely sensitive molecular detectors of the cyclic nucleotide concentration. The binding of cyclic nucleotide triggers an allosteric conformational change in the channel protein that opens the channel pore. In the preceding paper , we showed that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition. These interactions serve to reduce the transition-state energy and stabilize the activated conformation of the channel relative to the closed state. In this paper, we extend our analysis of the kinetics of the allosteric transition of bovine rod (BROD) CNG channels by determining the effects on the allosteric transition of mutations at position 604 in the binding domain and of substitution of the olfactory amino terminal region. These experiments provide insight into the sequence of molecular events that occur during the conformational change involved in channel activation. CNG channels contain, in their intracellular carboxyl terminal region, a domain with significant sequence similarity to the cyclic nucleotide–binding domain of a number of other cyclic nucleotide–binding proteins, including cGMP- and cAMP-dependent protein kinases and Escherichia coli catabolite gene activator protein (CAP) . CAP is a cAMP-activated transcription factor whose structure, while bound to cAMP, has been determined by x-ray crystallography to 2.5 Å resolution . The structure of the cyclic nucleotide– binding site of CAP consists of eight β strands that form a β roll structure, followed by two α helices, designated the B helix and C helix. Each cAMP molecule binds in the anti configuration with the ribose and cyclic phosphate binding to the pocket formed by the β roll and with the N6 hydrogen of adenine hydrogen bonding with a threonine at position T127 and a serine at position S128 on the opposite subunit . Rod CNG channels differ markedly in their apparent affinities for cGMP, cIMP, and cAMP . To account for the selectivity for cGMP over cAMP in the rod CNG channel, Varnum et al. proposed that the cyclic nucleotides bind in the anti configuration . For the case of cGMP, a pair of hydrogen bonds could then form between the N1 and N2 hydrogens of the guanine ring of cGMP and the aspartate residue at position 604 in the binding site . Cyclic AMP, which has an unshared pair of electrons at the N1 position, would form an unfavorable electrostatic interaction with the aspartate at position 604 and thus bind with lower affinity. Alternatively, it has also been proposed for the cGMP-dependent protein kinases and for the cGMP-gated channels that the cyclic nucleotides bind in the syn configuration, with the N2 hydrogen of guanine hydrogen bonded to a threonine residue in the β roll found primarily in cGMP-selective proteins . The amino terminal region of CNG channels has been shown to affect the free energy of the allosteric transition . In particular, the amino terminal region of olfactory CNG channels has been shown to promote the allosteric transition. This effect of the olfactory amino terminal region is transferable to rod channels, as chimeric rod channels with the olfactory amino terminal region open more favorably, and, conversely, chimeric olfactory channels with the rod amino terminal region exhibit much less favorable opening . Using in vitro protein interaction assays, specific interactions have been observed between the amino and carboxyl terminal regions of the olfactory CNG channel and between the olfactory amino terminal region and the rod carboxyl terminal region . Similar results were seen with the rod amino terminal region . These results indicate that the amino and carboxyl terminal regions of CNG channels bind with high affinity. Thus, conformational changes in the cyclic nucleotide– binding domain in the carboxyl terminal region could be transduced to the amino terminal region because of its close proximity and high affinity interaction. In addition, these results provide a molecular mechanism for how the gating of olfactory CNG channels can be modulated by Ca 2+ -calmodulin . In this paper, we analyze the kinetic behavior of single BROD CNG channels in which mutations at amino acid position 604 in the binding domain have been introduced and/or the rat olfactory amino terminal region has been substituted for the BROD amino terminal region. These experiments test the hypothesis that a pair of hydrogen bonds forms between cGMP and D604 during the allosteric transition and probe the mechanism by which the amino terminal region affects the energetics of the allosteric transition. Using a hidden Markov model approach, we analyzed the single-channel records to determine the underlying rate constants. We used thermodynamic mutant cycle formalism to determine the coupling energies for these interactions. By comparing the free energies of the transition state for the allosteric transition relative to the closed and open state energies across mutants and cyclic nucleotides, we postulate a sequence for the molecular events that occur during the allosteric transition. Oocyte preparation, cRNA transcription, and expression were carried out as described previously . Site-specific mutations were generated using oligonucleotide- directed mutagenesis and PCR and were confirmed by sequencing as described previously . Patch-clamp experiments and analysis of data were carried out as described in the preceding paper . In brief, patch-clamp experiments were performed in the inside-out conformation using an Axopatch 200B amplifier ( Axon Instruments ). Currents were low-pass filtered at 5 kHz (eight-pole Bessel) and sampled at 25 kHz. Recordings were made at 20–22°C. Initial pipette resistances were 5–20 MΩ. Intracellular and extracellular solutions contained 130 mM NaCl, 3 mM HEPES, and 0.2 mM EDTA, pH 7.2. For the experiments with Ni 2+ , 1 μM Ni 2+ was substituted for the EDTA. 500 μM niflumic acid was included in the patch pipette to reduce endogenous calcium-activated chloride currents. Intracellular solutions containing cyclic nucleotides and/or 1 μM Ni 2+ were changed using a DAD-12 Superfusion System (Adams and List Associates Ltd.) controlled by a MRI MB-8000 PC and modified such that each solution had a separate exit port. All reagents were obtained from Sigma Chemical Co. Fractional activations ( I / I max ) for a particular cyclic nucleotide were calculated by dividing the current ( I ) in the presence of the cyclic nucleotide by the maximum current obtained in the presence of 1 μM Ni 2+ plus the best agonist for that channel ( I max ). The fractional activation was used to estimate the free energy change of the allosteric transition by assuming that the equilibrium constant ( L ) for this transition is given by: I / I max = L /( L + 1). Thus the standard free energy for the allosteric transition is Δ G 0 − RT in L , where R is the universal gas constant and T is the temperature. Conversions of rate constants to transition state energies were made according to Eyring rate theory , which assumes that there is a quasi-equilibrium between the transition state and the ground state and that the rate of break down to product of the high energy intermediate depends on the vibrational energy of a covalent bond at room temperature. While originally proposed for chemical reactions, this theory has been applied to conformational changes in proteins and is useful because it provides an estimate of the transition state energies . Thus Δ G ‡ = RT ln( k B T / k obs h ) = (17.3 − 1.35 log k obs ) kcal/mol at 22°C, where Δ G ‡ is the transition state energy, R is the universal gas constant, T is the temperature, k B is the Boltzmann constant, k obs is the observed transition rate constant, and h is Planck's constant. To investigate the molecular interactions underlying the allosteric transition, we recorded macroscopic and single-channel currents from 10 BROD CNG channel constructs in which mutations were introduced at position 604 in the binding domain and/or the rat olfactory amino terminal region was substituted for the BROD amino terminal region (CHM15). The mutations at D604 included D604E (present in the mammalian olfactory α subunit), D604Q (present in the fish olfactory α subunit), D604N (present in the rod β subunit), and D604M (present in the olfactory β subunit). These constructs were expressed as homomultimers in Xenopus laevis oocytes, and macroscopic currents from inside-out patches at saturating concentrations of cGMP, cIMP, and cAMP are shown in Figs. 2 – 4 , respectively. In Fig. 2 are shown representative current families elicited by voltage steps from 0 mV to between −80 and +80 mV in the presence of 16 mM cGMP, a saturating concentration for each of the 10 constructs . The currents in the absence of cyclic nucleotide were subtracted from each trace. To compare the currents across experiments, each current family was normalized to the maximum current obtained at +80 mV in the presence of 1 μM Ni 2+ and the cyclic nucleotide that best activated the channel. As can be seen in Fig. 2 A, the fractional activation for BROD with cGMP was greatest for D604, averaging 0.96 ± 0.01 (mean ± SEM, n = 6). The fractional activation was slightly less (0.81 ± 0.05, n = 4) for the conservative D604E mutation. When the amino acid at position 604 was mutated to a polar uncharged residue (D604Q or D604N), there was a dramatic reduction in the fractional activation (D604Q, 0.12 ± 0.03, n = 4; D605N, 0.08 ± 0.02, n = 4). The fractional activation further decreased to 0.03 ± 0.01 ( n = 3) in D604M. Thus, the fractional activation decreased as the amino acid at position 604 became progressively less polar. Since saturating concentrations of cyclic nucleotide were used in all of these experiments, a reduction in fractional activation indicates that the allosteric conformational change for fully liganded channels was less favorable for the mutant channels than for the wild-type channel. The dramatic reduction in fractional activation from 0.96 for D604 down to 0.03 for D604M is a testament to the importance of D604 for the allosteric transition. This result is consistent with what has been previously reported and has been proposed to result from hydrogen bonding between the carbonyl group of D604 and the guanine ring of cGMP . For comparison, the same mutations at position 604 were also studied in the CHM15 background. CHM15 is identical to the BROD construct except that the rat olfactory amino terminal region has been substituted for the BROD amino terminal region . The olfactory amino terminal region produces a more favorable free energy change for the allosteric transition of chimeric channels , making the effects of mutations that dramatically decrease the ability of cyclic nucleotides to promote the allosteric transition easier to characterize at both the macroscopic and single-channel levels. In Fig. 2 B are illustrated the currents in the CHM15 background for the D604 mutants in the presence of cGMP. Again as the amino acid at position 604 was made progressively less polar, the fractional activation decreased. The fractional activities were 0.96 ± 0.02 ( n = 4) for CHM15-D604, 0.98 ± 0.01 ( n = 3) for CHM15-D604E, 0.90 ± 0.01 ( n = 3) for CHM15-D604Q, 0.82 ± 0.02 ( n = 5) for CHM15-D604N, and 0.52 ± 0.07 ( n = 3) for CHM15-D604M. We interpret these results to indicate that, compared with the BROD background, the trend across constructs was similar, although the differences in fractional activation were smaller. Despite smaller effects on the fractional activations, the effects of the D604 mutations on the free energy of the allosteric transition is the same in both the BROD and CHM15 backgrounds (see Table I ). Cyclic IMP is similar in chemical structure to cGMP except that the inosine moiety lacks a 2-amino group. Therefore, different abilities of cGMP and cIMP to promote the allosteric transition should reflect the contribution of interactions of the cyclic nucleotide–binding domain of the channel with the guanine 2-amino group of cGMP to the allosteric transition. We recorded macroscopic currents from the 10 constructs activated by cIMP, and these currents are illustrated in Fig. 3 , A and B, for the BROD and CHM15 backgrounds, respectively. As can be seen in this figure, the fractional activation was less for the D604Q, D604N, and D604M mutants than for D604 and D604E in both the BROD and CHM15 backgrounds. In BROD D604, the fractional activation by cIMP was only 0.60 ± 0.02 ( n = 6) and, for BROD D604E, the fractional activation was slightly lower (0.41 ± 0.05, n = 4). For D604Q and D604N, the fractional activations were 0.05 ± 0.01 ( n = 4) and 0.08 ± 0.02 ( n = 4), respectively. For D604M, the fractional activation was 0.06 ± 0.004 ( n = 4). For the CHM15 constructs, the fractional activations were 0.97 ± 0.01 ( n = 4) for D604, 0.98 ± 0.01 ( n = 3) for D604E, 0.73 ± 0.05 ( n = 5) for D604Q, 0.84 ± 0.03 ( n = 5) for D604N, and 0.71 ± 0.03 ( n = 3) for D604M. Thus, mutating D604 to a polar uncharged residue (Q or N) or nonpolar uncharged residue (M) decreased the fractional activation. Compared with the effects for cGMP, the differences in fractional activation were smaller, suggesting that cIMP is less sensitive to the identity of the amino acid at position 604. This finding likely reflects cIMP's lesser potential for hydrogen bonding interactions than cGMP's. Thus, the energetic effects of mutations at position 604 would be expected to be smaller for cIMP than for cGMP. Like cIMP, cAMP lacks a 2-amino group, but it has two other differences in the purine ring. cAMP has an amino group instead of a carbonyl group at the 6-position, and it has an unshared pair of electrons instead of a hydrogen at the 1-position. In Fig. 4 are illustrated currents activated by 16 mM cAMP. Here the pattern across the constructs was quite different from the pattern that was observed with cGMP and cIMP. For D604, the fractional activation was only 0.012 ± 0.002 ( n = 6) or almost two orders of magnitude less than for cGMP on the same construct. For D604E, the activation was almost twice as large but still small: 0.02 ± 0.01 ( n = 3). For D604Q and D604N, the activation increased to 0.05 ± 0.01 ( n = 4) and 0.05 ± 0.02 ( n = 4), respectively. For D604M, the fractional activation was significantly larger: 0.18 ± 0.01 ( n = 3). For the CHM15 constructs, the fractional activation was 0.19 ± 0.02 ( n = 4) and 0.53 ± 0.03 ( n = 3) for D604 and D604E. For D604Q and D604N, the fractional activations were 0.74 ± 0.04 ( n = 5) and 0.69 ± 0.03 ( n = 6), respectively. For D604M, the activation was 0.90 ± 0.03 ( n = 3). Thus, as the amino acid at position 604 was made progressively less polar, cAMP became a better agonist. The molecular interpretation of this result is that the reduction in the polarity of the residue at position 604 reduces the unfavorable interaction that would be expected to occur between the unshared pair of electrons at the N1 position of cAMP and a polar residue at 604. The slight improvements in the factional activations by cAMP of D604E over D604 may be related to differences in the local pKa's of aspartate and glutamate. Specifically, Gordon et al. showed that the fractional activation by cAMP on D604 improves as the pH is decreased. The cyclic nucleotide–specific pH effect disappeared when D604N was introduced, suggesting that cAMP is sensitive to the protonation state of D604. The pH dependence was proposed to result from neutralization of a negative electrostatic interaction between the negative charge on 604N and the unshared pair of electrons at the 1-position of the purine ring of cAMP. At pH 7.2, D604 would be unprotonated, but it is possible that D604E is partially protonated at pH 7.2, thereby improving the fractional activation by cAMP by removing some of the negative charge on the carboxylic acid. To summarize the results from the macroscopic experiments, we converted the fractional activations to free energies for the allosteric transition (see materials and methods ) and used these energies to construct thermodynamic mutant cycles. Thermodynamic mutant cycles provide a way of separating out indirect effects of mutations on the machinery of the allosteric transition from direct interactions of the ligand with the ligand-binding domain. Thermodynamic mutant cycles have been applied to interactions between the amino acids within proteins and between a toxin and a voltage-dependent K + channel . We wanted to determine if the effects of mutations at 604 reflect direct interactions between D604 and the places on the purine rings where the cyclic nucleotides differ. An example of the use of thermodynamic mutant cycles is shown in Fig. 5 . In Fig. 5 A, we tested for direct interaction of the amino terminal region with the purine ring of the cyclic nucleotide. The horizontal arrows reflect the effect of the amino terminal substitution on the free energy change of the allosteric transition, while the vertical arrows reflect the effect of changing cyclic nucleotide on the free energy change of the allosteric transition for the particular channel constructs. We would expect that the effect of the mutation might have two components: (a) a nonspecific component relating to indirect effects on the allosteric transition machinery and (b) a specific component due to direct effects on the interaction of the ligand with D604. As can be seen in Fig. 5 A, the ΔΔ G s for both of the horizontal arrows are negative, indicating that the olfactory amino terminal region makes the allosteric transition more favorable for both cGMP and cAMP. The difference between the values of the two ΔΔ G s should subtract out the nonspecific effect while leaving the direct effects. This difference is referred to as the coupling energy. Since CNG channels have four subunits , the coupling energies reflect the sum of the direct interactions occurring in each subunit. For this cycle, the coupling energy was only 0.49 kcal/mol, indicating that the effect of substituting the amino terminal region is relatively cyclic nucleotide independent. This result was an expected finding since it has been previously shown that the effect of the olfactory amino terminal region is cyclic nucleotide independent . Thus, the differences in the way cAMP and cGMP interact with the channel because of the differences in their purine ring structures are preserved on substituting the olfactory amino terminal region. That the effect of the olfactory amino terminal region was cyclic nucleotide independent was also reflected in the macroscopic traces; the trends across the D604 mutants were similar in both the CHM15 and BROD backgrounds. Illustrated in Fig. 5 B is a thermodynamic mutant cycle analysis of the interaction between D604 and the cyclic nucleotide. Here the D604M mutation makes cGMP a worse agonist (ΔΔ G = 4.09 kcal/mol), but makes cAMP a better agonist (ΔΔ G = −1.79 kcal/mol). Equivalently, the cyclic nucleotide selectivity was inverted by the D604M mutation, as evidenced by the negative value for ΔΔ G (ΔΔ G = −1.00 kcal/mol) between cGMP and cAMP on D604M and positive value for ΔΔ G (ΔΔ G = 4.88 kcal/mol) between cGMP and cAMP on D604. Here, the coupling energy was large and negative (coupling energy = −5.88 kcal/mol), indicating a high degree of interaction between the amino acid at position 604 and the purine ring of the cyclic nucleotide. Summarized in Table I are the coupling energies for each of the possible thermodynamic mutant cycle comparisons for the 10 different mutants with the three different cyclic nucleotides. All energies of magnitude ≥2.5 kcal/mol are double underlined, and all energies of magnitude between 2 and 2.5 kcal/mol are single underlined. As seen along the diagonal, the coupling energies of changes in the amino terminal region with changes in cyclic nucleotide were generally small. We interpret this to mean that the effect of the olfactory amino terminal region was cyclic nucleotide independent. Note that the coupling energies between CHM15 and BROD were slightly larger than for the other comparisons, probably because the fractional activations of cGMP and cIMP on CHM15 were so large that the method of using Ni 2+ potentiation to measure ΔG 0 loses resolution. In addition, all of the cells comparing the BROD-D604M and CHM15-D604M to the BROD, CHM15, BROD-D604E, and CHM15-D604E between cIMP and cAMP and between cGMP and cAMP are underlined. This result strongly indicates that cAMP is sensing the amino acid at position 604 quite differently from cIMP and cGMP. This result gives good support for the hypothesis proposed by Varnum et al. that an acidic residue at position 604 in BROD channels is critical for ligand discrimination and interacts directly with the purine ring of the cyclic nucleotide . This conclusion is also supported by the fact that the majority of the cells for the more conservative D604Q and D604N vs. D604 and D604E in the BROD and CHM15 backgrounds were also >2 kcal/mol for cIMP vs. cAMP and cAMP vs. cGMP. For the cGMP vs. cIMP comparisons, the coupling energies were smaller in magnitude, as expected since the chemical differences between cGMP and cIMP are smaller relative to their differences with cAMP. As can be seen in Table I , there was a ≥2 kcal/mol coupling energy in the BROD vs. BROD-D604N, CHM15-D604N, BROD-D604M, and CHM15-D604M. Since the only difference between cGMP and cIMP is at the 2-positions of their purine rings, this finding supports the hypothesis of Varnum et al. that the guanine 2-amino group of cGMP interacts with D604 . For CHM15, the same analysis did not reveal large coupling energies with the same four constructs vs. CHM15. However, we do not feel that this negative result indicates a lack of interaction. Rather, it is probable that we were not able to pick up a differential interaction because the CHM15 construct had such a favorable ΔG 0 for the allosteric transition so that the currents were nearly maximal without Ni 2+ in the presence of cGMP and cIMP. For BROD-D604E and CHM15-D604E, the coupling energies with BROD-D604M and CHM15-D604M ranged from −0.92 to −1.46 kcal/mol. These values are smaller than the values of −2.21 and −2.30 kcal/ mol between BROD and BROD-D604M and CHM15-D604M, possibly reflecting weaker interactions because of the larger side chain of glutamate over aspartate. Thus, overall, we conclude that the comparisons between the D604 and D604E constructs and the D604M constructs provide support for the hypothesis of Varnum et al. that cIMP forms one hydrogen bond with D604 while cGMP forms two. The coupling energies we measured are within the range of energies expected for such an interaction . To probe the effects on the opening and closing rate constants, not just the overall ΔG 0 , we obtained single-channel recordings at +80 mV for each of the 10 constructs in the presence of saturating concentrations of cGMP, cIMP, and cAMP. From each recording, we selected regions where a single channel was active, and we omitted from our analysis quiescent periods 200 ms or longer in duration . Representative 200-ms portions of our recordings are illustrated in Figs. 6 , 8 , and 10 for cGMP, cIMP, and cAMP, respectively. The amplitude histograms for the recordings shown in Figs. 6 , 8 , and 10 are shown in Figs. 7 , 9 , and 11 . Fig. 6 shows representative single-channel traces for the 10 constructs activated by 16 mM cGMP. As 16 mM cGMP is a saturating concentration, the kinetics that we observe do not reflect the rate constants of binding or unbinding of cGMP. Rather, they reflect transitions after the full complement of ligands has bound to the channel. For D604, the channel was highly activated in the BROD construct, but more so for CHM15. In both cases, the single-channel conductance was the same. We compare the open probabilities and single-channel current level in the amplitude distributions in Fig. 7 , which shows BROD and CHM15 distributions side-by-side. As expected given the slightly lower fractional activation of D604E compared with D604 in macroscopic experiments, the open probability of D604E was slightly lower than for D604. For BROD-D604Q and BROD-D604N, the open probability decreased substantially. For BROD-D604M, the open probability was very low but increased to ∼0.5 in CHM15-D604M. In each case, the single-channel conductance appears to be unaffected by mutations at position 604 or the presence of the olfactory amino terminal region, and openings appear to be to only the full amplitude level, not to subconductance states. Thus, the large differences in fractional activation determined from the macroscopic current experiments are due entirely to differences in the open probabilities produced by D604 mutations and the olfactory amino terminal region. In particular, the open probabilities in cGMP decreased as D604 became less polar and increased with the addition of the olfactory amino terminal region. For cIMP activation of the 10 constructs, representative traces and amplitude histograms are shown in Figs. 8 and 9 , respectively. Here, the trend across the constructs was quite similar to the trend for cGMP, although there were a few differences. With cIMP, the open probability was only 74% in D604. Like for cGMP, the open probability decreased as the amino acid at position 604 became less polar and increased with the addition of the olfactory amino terminal region. Interestingly, openings in BROD-D604M were slightly more numerous and longer-lived than for cGMP. For cAMP activation across the 10 constructs, representative traces and amplitude histograms are shown in Figs. 10 and 11 . As can be seen here, the open probability was low when a polar residue was present at position 604. For BROD-D604M, the openings were significantly longer in duration. The same trends were observed in the CHM15 constructs. We analyzed the single-channel kinetics for each of the 10 constructs using a hidden Markov modeling (HMM) approach. The HMM approach, described in detail in the accompanying paper , directly estimates the rate constants for a given specified kinetic scheme and uses iterative techniques to converge on the maximum likelihood set of rate constants. The HMM approach provides a maximum likelihood value that allows the number of closed and open states required to explain the data to be determined. In the previous paper, we found that a simple two-state C ↔ O scheme is not sufficient to explain the kinetics at saturating ligand concentrations. Rather, an additional closed state was required. Adding this additional closed state outside the activation pathway (as in C 0 ↔ O 1 ↔ C 2 ) or within the activation pathway (as in C 0′ ↔ C 1′ ↔ O 2′ ) significantly improved the description of the kinetics at saturating ligand concentrations. Since the likelihoods for the C 0 ↔ O 1 ↔ C 2 and C 0′ ↔ C 1′ ↔ O 2′ linear schemes are identical, we could not discriminate between these two schemes. However, we prefer the C 0 ↔ O 1 ↔ C 2 scheme because there was cyclic nucleotide dependence in only the first transition of the C 0 ↔ O 1 ↔ C 2 scheme. Also, the C 0 ↔ O 1 ↔ C 2 scheme has the simple physical interpretation of the first transition being the allosteric transition and the second transition being to a flicker closed state out of the activation pathway. While the underlying mechanism is undoubtedly more complex than two closed and one open states, this mechanism adequately describes our data and provides a physical interpretation of the results. The qualitative conclusions concerning the effects of mutations on the stability of the open state and transition state should still be valid for a C 0′ ↔ C 1′ ↔ O 2′ scheme and more complex models. These conclusions are based on effects of these mutations on the open and closed durations and are not dependent on any particular model. For the analysis of the 10 constructs described in this paper, we determined the rate constants for the C 0 ↔ O 1 ↔ C 2 scheme. Shown in Fig. 12 is a summary of the rate constants from multiple patches for the C 0 ↔ O 1 step for all 10 constructs and for all three cyclic nucleotides. The opening rate k 01 became progressively slower for cGMP as the amino acid at position 604 became less polar . This effect was observed in both the BROD and CHM15 constructs. For cIMP, the slowing of the k 01 rate was smaller but in the same direction. For cAMP, the k 01 rate constant was nearly independent of construct, perhaps increasing slightly. In Fig. 12 B, we see that the effects of the D604 mutations on the k 10 rate constant are larger than for the k 01 rate constant. For cGMP, there was a progressive speeding up of the closing rate ( k 10 ) constant as the amino acid at position 604 became less polar. For cIMP, the trend across constructs was generally the same but smaller in magnitude. For cAMP, the k 10 rate constant was relatively independent of the amino acid at position 604, thus indicating that the interactions of cAMP with D604 are unimportant for determining the closing rate constant. For the CHM15 constructs, the effect on the k 10 rate constant was remarkable. For each mutation at position 604 in the BROD construct, the corresponding mutation in the CHM15 construct was ∼20–100-fold slower. This result contrasts with the result for the opening rate constant, which appeared to be relatively independent of the presence or absence of the olfactory amino terminal region. The large effect on the closing rate constant indicates that the positive interactions incurred with the substitution of the olfactory amino terminal region are a major determinant of the closing rate constant. Shown in Fig. 13 are the rate constants for the second transition O 1 ↔ C 2 across constructs and for the three cyclic nucleotides. As can be seen in this figure, the reopening rate constant k 21 was generally independent of cyclic nucleotide and construct and was fast (∼5,000/s). The closing rate k 12 was also generally independent of cyclic nucleotide and construct but was much slower (∼100/s). Thus, the equilibrium for the O 1 ↔ C 2 step is strongly toward the open state, and sojourns in the C 2 state were short in duration, not unlike what would be expected for flickery open-channel block. More importantly, the effects of the D604 mutations and the olfactory amino terminal region are selective for the first transition of C 0 ↔ O 1 ↔ C 2 , as was previously shown for the effects of cyclic nucleotides and Ni 2+ . The observation that each of these modifications is affecting the same step provides further support for the hypothesis that the first transition of the C 0 ↔ O 1 ↔ C 2 scheme is the allosteric transition, and the second transition is not involved in the allosteric transition. A comparison between the values for Δ G 0 calculated as − RT ln( k 01 / k 10 ) from the single-channel experiments, relative to the values for Δ G 0 = − RT ln L , where L is the equilibrium constant for the allosteric transition and was determined using Ni 2+ potentiation of macroscopic experiments, is shown in Fig. 14 (see materials and methods ). Overall, the correspondence between the values is good. It may be noted that for BROD D604Q, D604N, and D604M, the median single-channel estimate for Δ G 0 was slightly larger than the macroscopic estimate in each case. The probable explanation for this observation is that currents obtained with Ni 2+ in macroscopic experiments slightly underestimate the maximum current. This underestimation is occurring because the Δ G 0 for the transition is so unfavorable for each of these constructs that, even with Ni 2+ , the currents do not approximate the theoretical maximum current that would be obtained if the fractional activation were 1. Note that this slight difference could not be explained by error in the determination of the number of channels in a single-channel patch, as the effect of having too many channels would be to increase the opening rate constant, thus making the Δ G 0 more favorable, not less. This figure illustrates that there was generally good correspondence between the values obtained for the macroscopic and single-channel experiments. This result supports the use of Ni 2+ for the estimation of Δ G 0 from macroscopic experiments and provides additional evidence that the C 0 ↔ O 1 transition represents the transition modulated by Ni 2+ . Using Eyring rate theory , we converted the median rate constants we obtained from our HMM computations to activation energies (see materials and methods ). Eyring rate theory assumes that there is a quasi-equilibrium between a high energy transition state and the ground state, and that the rate of break down to product of the high energy intermediate depends on the vibrational energy of a covalent bond at room temperature. Other theories have been proposed to explain the slow rate of protein conformational changes ; however, Eyring rate theory is generally accepted and provides a unique estimate of the transition state energies . While the absolute activation energy (Δ G ‡ ) is dependent on estimates of a preexponential factor, the changes in activation energy (ΔΔ G ‡ ) are less dependent on this estimate. Fig. 15 A illustrates the energetics for the allosteric transition for the three cyclic nucleotides. In this figure, the energy profiles were aligned at 0 kcal/mol when the channels were in the closed state (C 0 ). As can be seen in this figure, the overall Δ G 0 for the allosteric transition is between −2 and 3 kcal/mol for cGMP and cAMP, respectively. Since these cyclic nucleotides differ in only the most distal portion of their purine ring, we conclude that the cyclic nucleotide–binding domain interacts with the purine ring of the cyclic nucleotides differently during the allosteric transition. The energetics for these favorable cyclic nucleotide–binding domain interactions must therefore be determinants of the stability of the open state. Since the energy of the transition state (Δ G ‡ ) also differs among the cyclic nucleotides, we conclude that these interactions are partially formed at the time of the transition state and serve to reduce the energetic barrier for activation. The effects of Ni 2+ presented in the previous paper on the transition-state energies are diagrammed in Fig. 15 B. This figure illustrates that, like the cyclic nucleotides, Ni 2+ also affects the transition state energy and stability of the open state relative to the closed state. For both cIMP and cAMP, the presence of Ni 2+ stabilizes the open state and has a small effect on the energy of the transition state. This finding suggests that the movement of the H420 residues on each of the subunits of the channel into the Ni 2+ coordinating position is associated with channel opening. These interactions between Ni 2+ and the channel are partially formed at the time of the transition state for the allosteric transition. Fig. 15 C shows the energetics for activation of BROD and BROD-D604M channels by cGMP. As can be seen in this figure, D604M channels activated by cGMP have an allosteric transition energetically very similar to BROD channels activated by cAMP, both with regard to the standard free energy of the transition and the activation energy. This is a testament to the idea that the purine ring of cGMP interacts directly with D604. Disrupting this interaction with either cAMP or D604 mutations has a very similar effect. Fig. 15 D shows the energetics for activation of BROD and CHM15 channels by cIMP. As can be seen in this figure, the mechanism of action of the olfactory amino terminal region is a stabilization of the open state without affecting the transition state energy. This finding suggests that the stabilizing interactions between the autoexcitatory domain of the olfactory amino terminal region do not form until after the transition state for the allosteric transition. Thus, the interactions of the cyclic nucleotide and the binding site form at the time of the transition state, while the stabilizing effect of the olfactory amino terminal region arises after the transition state. To probe the sequence of events underlying the allosteric transition, we calculated the fraction of the energetic effect of the modifications studied that occurs after the allosteric transition and plotted the median values in Fig. 16 . These calculations were made by considering the effect on the k 01 and k 10 rate constants of switching cyclic nucleotide on BROD-D604 channels, switching from D604 to D604M in either the BROD or CHM15 backgrounds and for each of the cyclic nucleotides, applying Ni 2+ on BROD channels , and switching from BROD-D604 to CHM15-D604 for each of the cyclic nucleotides. As can be seen in Fig. 16 , in each case, the majority of the energetic effect of each modification occurred on the closing rate constant, meaning after the transition state for the allosteric conformational change. Intriguingly, the median fraction of the effect occurring after the transition state was 68% for the interactions of the channel with the portions of the cyclic nucleotides that differ and 70% for the effect of switching to D604M. The close correspondence between these two values gives further support for the hypothesis that the purine rings of the cyclic nucleotides and the amino acid at position 604 interact directly because it indicates that the D604M mutation and switching to a different cyclic nucleotide not only affect both rate constants, but they affect both rate constants commensurately. For Ni 2+ , there was more variation in the fraction of the energetic effect occurring after the transition state. For the olfactory amino terminal region, the median value was strongly skewed toward the right, indicating that only a small fraction of the energetic effect of the amino terminal region has occurred by the time of the transition state. Hence, we would say that the state-dependent stabilization of the allosteric transition occurs very late in the reaction coordinate. It is tantalizing to conclude that the interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 occur early in the reaction coordinate. These interactions are followed by the interactions of H420 in the C linker with Ni 2+ . Finally, the stabilizing interactions with the olfactory amino terminal region, when present, form late in the reaction coordinate, after the channel has switched to the active conformation. In this paper, we have investigated the effects of mutations at position 604 in the binding domain and of the olfactory amino terminal region on the kinetics at saturating concentrations of three different ligands. We have shown that direct interactions between the cyclic nucleotide and the amino acid at position 604 occur during both the opening and closing transitions of the allosteric conformational change. In contrast, the substitution of the olfactory for the rod amino terminal region appears to affect only the closing rate constant, suggesting that the favorable interdomain interactions that form when the auto-excitatory domain is present occur almost entirely after the channel has switched to the open conformation. A molecular mechanism for the binding of cyclic nucleotides to CNG channels has been proposed, based on the CAP structure . In this mechanism, the cyclic nucleotide–binding site is exposed when the channel is in the closed configuration because the C helix is rotated away from the β roll. The cyclic nucleotide then binds to the closed channel primarily through interactions between the β roll and the ribose and phosphate of the cyclic nucleotide. Activation involves a conformational change in the channel protein. At the level of the binding site, the conformational change is thought to involve movement of the β roll relative to the C helix, permitting residues in the C helix, in particular D604, to interact specifically with the purine ring of the bound cyclic nucleotide. For cGMP, it was proposed that the negatively charged carboxylic acid side chain of D604 in the C helix forms a pair of hydrogen bonds with the N1 and N2 hydrogen atoms on the guanine ring. This type of hydrogen bonding has been shown to occur in solution and in high affinity GTP binding proteins such as the α subunit of transducin , elongation factor EF-Tu , and H-Ras . This molecular mechanism is not unique, and we cannot completely rule out that substituents on the purine ring do not alter the chemical properties of the ring, or that mutations at D604 do not have indirect effects on the structure of the cyclic nucleotide–binding site. However, given the large body of data in support of a direct interaction between D604 and the purine ring, we have chosen to interpret our results in terms of this mechanism. Based on the results of this investigation, the kinetic and molecular aspects of this mechanism for the allosteric transition have been refined. In particular, we have determined the rate constants at which the interactions are forming and unforming, and we now know the relative effects on the opening and closing transitions for each of the modifications studied. For the case of cGMP, we conclude that the pair of hydrogen bonds between the guanine ring and the aspartate is partially formed at the time of the transition state for the allosteric transition. Thus, the activation of the channel involves the formation of these two hydrogen bonds, and the closing of the channel involves the dissolution of these hydrogen bonds. This observation indicates that the formation of these hydrogen bonds is intimately involved in the allosteric transition, not an event that occurs before or immediately after it. For cIMP, we conclude that the formation of a hydrogen bond between D604 and the inosine moiety is also intimately involved in the allosteric transition, but the energies are approximately half those of cGMP, indicating a molecular mechanism where only a single hydrogen bond forms per subunit. The open probability increased for activation by cAMP when D604 was mutated to a polar uncharged residue, suggesting that interactions between the adenine ring and D604 destabilize the allosteric transition. Thus, other portions of the cyclic nucleotide and interactions not investigated here may explain the fact that cAMP is an agonist, not an antagonist, of the BROD channel. Alternatively, D604 may interact weakly with the adenine ring and less polar residues may interact more strongly. Our analysis of the effect of the olfactory amino terminal region on channel activation indicates that the amino terminal region exerts its auto-excitatory effect late in the reaction coordinate and that the effects of the olfactory amino terminal region are cyclic nucleotide-independent. Previously, it has been shown that the rat olfactory CNG channel amino terminal region promotes channel activation by directly interacting with the carboxyl terminal gating machinery . This mechanism was proposed based on in vitro binding assays and occurred even in the absence of cyclic nucleotide. The results of this investigation indicate that the affinity of the two terminal regions is only 2 kcal/mol higher for the open than the closed state. Since this energy difference is small, the most likely interpretation is that the two regions interact with high affinity both when the channels are open and closed and that the 2 kcal/mol energetic stabilization reflects the formation of a few hydrogen bonds per subunit or the removal of a few unfavorable interactions and not the binding or unbinding of the two terminal regions with each transition. Thus, our results refine the molecular mechanism of action by suggesting that the stabilization exerted by the amino terminal region occurs primarily after the transition state for the allosteric transition and occurs later in the reaction coordinate than the interactions between the cyclic nucleotides and the amino acid at position 604. | Study | biomedical | en | 0.999996 |
10228180 | Voltage-gated K + channels (Kv channels) 1 activate and open upon membrane depolarization. Most Kv channels, however, do not remain open during a sustained depolarization. Instead, they adopt a nonconducting conformation by a process known as inactivation. Kv channels may exhibit multiple mechanisms of inactivation that involve fast and slow processes. A great deal has been learned about the mechanisms of inactivation of Shaker K + channels. These channels exhibit two clearly distinct forms of inactivation: N- and C-type . N-type inactivation is fast and involves a “ball and chain”–type mechanism , where approximately the first 20 amino acids at the NH 2 terminus of the Shaker subunit act as a tethered internal particle capable of blocking the open pore . The cytoplasmic loop between the S4 and S5 seqments (the S4–S5 loop) contributes to the putative receptor for the inactivation particle in the pore of the channel . C-type inactivation is typically slower than N-type and depends on residues in the pore region (the S5–S6 loop) and the external section of the S6 region . The rate of C-type inactivation is slower in the absence of N-type inactivation, suggesting that the two processes are coupled . A current hypothesis proposes that C-type inactivation involves a rearrangement of the extracellular mouth of the pore resulting from a cooperative conformational change of the channel subunits . As a result, C-type–inactivated channels become more permeable to Na + . Although it is clear that certain non- Shaker K + channels may also undergo inactivation by the N- and C-type mechanisms , in various instances the presence of N- or C-type inactivation has been difficult to recognize . Therefore, there is a distinct possibility that other Kv channels may undergo inactivation by processes that are not yet understood. In a previous study, we found that rapid inactivation in Kv4.1 K + channels (members of the Shal family) depends on the concerted action of the cytoplasmic NH 2 - and COOH-terminal domains of the channel protein . This process, however, has little impact on the slower processes that contribute to most of the time course of inactivation. To gain insights into the molecular mechanism of slow inactivation of Kv4.1 K + channels, we have focused on residues that may contribute to the inner vestibule of the channel. An important clue about the mechanism of inactivation of Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) originated from previous studies that examined the action of 4-aminopyridine (4-AP) on these channels. 4-AP blocks native Kv4 A-type K + channels from cardiac tissue and cloned Kv4 K + channels at an internal site with a keen state dependence. Blockade occurs almost exclusively in the closed state, and can be relieved by channel opening and inactivation . 4-AP binding and channel inactivation are mutually exclusive because inactivation cannot occur until 4-AP dissociates, and channel inactivation prevents 4-AP binding. These results suggested that residues contributing to the 4-AP binding might also be involved in controlling inactivation of Kv4 channels at an internal site. Inactivation may physically hinder 4-AP binding, and the sites that control the underlying conformational change may also interact with 4-AP. In the distal section of S6 (a putative component of the inner vestibule of the pore), two critical positions are occupied by valines in most Kv channels , and their presence is associated with moderate or high sensitivity to 4-AP. Shab K + channels (Kv2), which exhibit very low 4-AP sensitivity and inactivate very slowly, appear to be the exception . There, the equivalent positions are occupied by isoleucine. We asked whether V → I mutations at these conserved positions in Kv4.1 (VI) could simultaneously reduce 4-AP sensitivity and alter inactivation gating in the presence and absence of the cytoplasmic NH 2 - and COOH-terminal domains. In several Kv channels, mutations in the S4–S5 loop and the cytoplasmic regions of S5 and S6 affect binding of the inactivation particle, single channel conductance, K + selectivity, and blockade by internal tetraethylammonium, 4-AP, Ba 2+ , and Mg 2+ . Therefore, the S4–S5 loop and the cytoplasmic halves of S5 and S6 segments appear to contribute to the permeation pathway at the inner vestibule of Kv channels. We hypothesize that a Kv4-specific cysteine in the S4–S5 loop (C322) also contributes to inactivation gating directly or through an interaction with residues in the distal section of S6. The equivalent position is occupied by serine in other Kv channels . Thus, we also examined the biophysical properties of C322S. The main effects of the VI and C322S mutations in the Kv4.1 K + channels were: (a) to reduce the sensitivity to 4-AP, and (b) to drastically slow the development of macroscopic inactivation and current deactivation (with little effect on the recovery from inactivation). However, while VI also slowed the rate of closed state inactivation, C322S did not. The results suggest novel interactions between channel closing, closed-state inactivation, and blockade by 4-AP, which involve the inner vestibule of the pore. To test whether the distal section of S6 may have a similar function in other A-type channels, we also examined the effect of the homologous mutations in Kv1.4 (VI), a mammalian Shaker K + channel that exhibits open-channel blockade by 4-AP and features the N- and C-type inactivation mechanisms . The VI mutation in Kv1.4 had little or no effect on the development of macroscopic inactivation, but slowed current deactivation and recovery from inactivation, and eliminated blockade by 4-AP. These results further support the idea of a novel mechanism of inactivation at the inner vestibule of Kv4 K + channels. The main observations can be modeled by a kinetic scheme assuming that an important pathway of Kv4 inactivation originates from a closed state that precedes channel opening. Wild-type mouse Kv4.1 and human Kv1.4 were maintained in pBluescript II KS (Stratagene Inc.) and pRc/CMV (Invitrogen Corp.), respectively. Dr. Carol Deutsch (University of Pennsylvania, Philadelphia, PA) kindly provided Kv1.4. ΔN71/ΔC158 was generated as described previously . Point mutations were created using two methods. QuickChange (Stratagene Inc.) was used according to the manufacturer's specifications to obtain VI, VI, VI, ΔN71/ ΔC158/C322S, and ΔN71/ΔC158/VI. In brief, pairs of mutagenic oligonucleotide primers (Nucleic Acid Facility, Jefferson Cancer Institute) with complementary sequences were used to introduce the desired mutations. The reaction mixture consisted of reaction buffer, DNA template (50 ng), complementary primers (125 ng each), free deoxyribonucleotide mix (2.5 mM, each nucleotide), and Pyrococcus furiosus (pfu) DNA polymerase. This mixture was subjected to thermal cycling (95°C, 30 s; 55°C, 1 min; and 68°C, 12–17 min). Over 12–18 cycles (depending on the number of substitutions), the mutagenic oligonucleotide primers annealed to the melted DNA template and by a simple extension process pfu polymerase synthesized the new DNA strands with the intended mutation(s). DpnI ( Promega Corp. ) was then added to cleave the original DNA template (DpnI cleaves methylated DNA only). Subsequently, the mutated DNA was electroporated into DH5α cells ( GIBCO BRL ) and selected colonies were analyzed for the presence of the mutated plasmid. To confirm the phenotype of these mutants, at least two independent clones were examined. C322S and other mutations in the S4–S5 loop were obtained by oligonucleotide-directed mutagenesis using the Altered Sites II in vitro Mutagenesis System ( Promega Corp. ) as described before . All mutations were confirmed by automated sequencing (Nucleic Acid Facility). Capped cRNA for expression in Xenopus oocytes was produced by in vitro transcription using the Message Machine Kit (Ambion Inc.). Wild-type and mutant Kv4.1 or Kv1.4 cRNAs were injected into defolliculated Xenopus oocytes (∼50 ng/cell) using a Nanoject microinjector (Drummond Scientific Co.). Currents were recorded 1–7 d after injection. The two-microelectrode voltage-clamp technique (TEV-200; Dagan Corp.) was used to record whole-oocyte currents. Microelectrodes were filled with 3 M KCl (tip resistance was <1 MΩ). The bath solution contained (mM): 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.4, adjusted with NaOH. In some experiments with low-expressing oocytes, we supplemented the bath solution with 100–500 μM diisothiocyanatostilbene-2,2′-sulfonic acid (DIDS) to block endogenous Ca 2+ -activated Cl − conductance and phospholemman-like currents . Current traces (generally 900-ms depolarizations) were low-pass filtered at 1 kHz (−3 db) and digitized at 500 μs/point. The average voltage offset recorded at the end of an experiment was generally small −0.4 + 2.5 mV, n = 38) and was not subtracted from the command voltage. Correction was applied when offset appeared to be greater than 1 SD. The leak current was subtracted off line, assuming ohmic leak or using a P/4 procedure. 4-AP ( Sigma Chemical Co. ) was dissolved directly in the external bath solution with a reduced concentration of NaCl to maintain normal osmolarity and ionic strength (pH was adjusted to 7.4 with HCl). Patch-clamp recording was conducted as described before using an Axopatch 200A ( Axon Instruments ). Patch pipettes were constructed from Corning glass 7052 (Warner Instrument Corp.) and coated with Sylgard elastomer (Dow Corning Co.). Typically, the tip resistance of the recording pipettes in the bath solution (see below) was <1 and 5–30 MΩ for macropatch recording and single channel recording, respectively. The pipette solution (external) was as described above. The bath solution contained (mM): 130 K-aspartate, 10 KCl, 1.8 CaCl 2 , and 10 HEPES, pH 7.3, adjusted with KOH. Passive leak and capacitive transients from macropatch currents were subtracted on line using a P/4 procedure. For single channel recordings, the passive components of the currents were subtracted using an average of blank sweeps (no single channel currents). Depending on the speed of the current relaxations and the duration of single channel current transitions, the recordings were filtered at 0.5–8 kHz (−3 db, eight-pole Bessel filter; Frequency Devices Inc.) and digitized at 2–40 kHz. Whole-oocyte currents were recorded at 23°C using a temperature-controlled microscope stage (PDMI-2; Medical Systems Corp.). Patch-clamp experiments were recorded at room temperature (22 ± 1°C). Contrary to the wild-type channels, some biophysical properties of VI channels were sensitive to seasonal variation. Most of the experiments reported here were conducted during the Summer and Fall of 1997. Greater variability was apparent when examining the results of experiments conducted ∼3 mo later. Mainly, the parameters affected were the midpoints of the conductance–voltage (G/V) curve and the prepulse inactivation curve, which were more depolarized than the values observed earlier (∼10 mV in both cases). Consistent with a more depolarized midpoint of prepulse inactivation, closed-state inactivation appeared slower (at least 20 s were necessary to reach a steady state level). Also, the 50% rise time was approximately twofold slower but the tail current relaxations were not significantly affected (see results ). Voltage-clamp protocols and data acquisition were controlled by a 586 desktop computer interfaced to a 12-bit A/D converter . Data analysis was conducted using Clampfit (pClamp 6.0; Axon Instruments ), Sigmaplot (Jandel Scientific), or Origin (Microcal Software, Inc.). Current relaxations and other time-dependent processes were described assuming a simple exponential function or the sum of exponential terms . Unless indicated otherwise, all values are expressed as mean ± SD. Model simulations were conducted by determining the initial equilibrium probabilities of occupying a set of states and the characteristic differential equations of the model. For a particular pulse protocol and set of rate constants, this system of equations was solved numerically using Vjump (Fortran program created by user) or SCoP 3.51 (Simulation Resources, Inc.). The presence of common structural determinants of inactivation and 4-AP binding in Kv4 K + channels may explain the mutually exclusive interaction between 4-AP and inactivation gating (see introduction ). To test this hypothesis, we introduced point mutations that were expected to reduce 4-AP sensitivity and studied their effects on inactivation gating. A double mutant VI was investigated because previous findings reported that isoleucines at the equivalent positions in Kv2.1 conferred low 4-AP sensitivity . As expected, VI channels exhibited a significantly reduced blockade by 4-AP. At 20 mM, 4-AP blocked ∼17% of the mutant peak current, compared with ∼55% of the wild-type peak current (Table I ). In addition, the kinetic properties of the Kv4.1 current were dramatically affected . Cell-attached macropatch recordings of wild-type Kv4.1 macroscopic currents showed a rapid rising phase and a decaying phase, nearly identical to those observed from two-electrode voltage-clamp recordings . Inactivation of these currents was almost complete by the end of the 900-ms depolarization. By contrast, the currents expressed by VI decayed at a much slower rate at all voltages and failed to inactivate by the end of the pulse . The degree of inactivation was determined by the ratio I (450 ms) /I peak . Wild-type and mutant channels exhibited 10-fold difference in the degree of inactivation at +50 mV (wild type: 0.08 + 0.02, n = 5; VI: 0.79 + 0.04, n = 9). Over a prolonged depolarization, however, VI mutant channels continued to inactivate . A double exponential fit to the decay of these currents (at +50 mV) showed that, relative to the wild-type currents, the estimated steady state level of the current increased approximately fourfold (Table I ). This increase suggests the possibility that the inactivated state was destabilized at positive voltages, and, consistent with this idea, the VI channels exhibited moderately accelerated recovery from inactivation at −100 mV . Because the VI channels inactivate at a very slow rate, a larger fraction of channels will continue to open over a longer period of time after the onset of a depolarization. This fraction corresponds to those channels that were yet to open before inactivation occurred in wild-type channels. In agreement with this idea, we found that the rising phase of the VI currents appeared significantly slower when compared with that of wild type . The 50% rise time of current activation at +50 mV was increased by ∼50% (Table I ), but the activation delay was not significantly affected (0.97 ± 0.3 and 1.03 ± 0.13 ms for wild type and mutant, respectively, n = 6; see discussion ). Overall, these experiments demonstrated that VI simultaneously inhibits inactivation and closed-state block by 4-AP, suggesting that the mutated residues are involved in the mechanism that underlies the mutually exclusive interaction between inactivation and 4-AP binding in Kv4 K + channels. We also investigated whether both mutations were necessary to produce the changes described above. Qualitatively, the single mutant V406I exhibited reduced blockade by 4-AP and biophysical properties that were similar to those of the double mutant (Table I ). The main difference was that the V406I mutant channels exhibited a less dramatic effect on inactivation. The ratio I (450 ms) /I peak for V406I was 0.62 ± 0.04 ( n = 4), compared with 0.79 + 0.04 ( n = 9) for VI. Yet the inhibition of the peak current by 4-AP was comparable between the single and double mutants (Table I ). The V404I mutant produced nonfunctional channels (two independent clones and three separate batches of oocytes). Because the main difference between VI and V406I was the degree of inactivation, we inferred that V404 mainly contributes to inactivation or that the double mutation is necessary to affect inactivation (see below). The state dependence of 4-AP blockade indicates that channel gating may dictate 4-AP action. Therefore, we asked whether the reduced 4-AP sensitivity and slowed inactivation of the VI mutant were associated with changes in voltage-dependent gating . The peak G/V relationships for both wild type and mutant were well described assuming fourth-order Boltzmann distributions . Relative to the wild type, the peak G/V relationship of the VI mutant showed a hyperpolarizing shift of ∼6 mV in the midpoint potential for activation of one subunit and a reduced slope factor . The midpoint potential of prepulse inactivation exhibited a depolarizing shift of ∼10 mV and the slope factor was also reduced . These results suggested that the mutations had caused a relative stabilization of the open state and disrupted inactivation at negative voltages. Consistent with this idea, closed-state inactivation of the VI channels occurred at a significantly slower rate . Interestingly, the V406I channels, which also exhibited slow inactivation at positive potentials, did not show altered closed-state inactivation. As suggested above, V404I in the double mutant is necessary to slow closed-state inactivation at negative voltages. V406I could slow inactivation indirectly by favoring the open state. In such a case, the relative occupancy of an inactivation-permissive closed state that precedes channel opening decreases and the apparent rate of macroscopic inactivation decreases too (see discussion ). To extract more direct information about the stability of the open state, we examined channel deactivation at hyperpolarized membrane potentials. Deactivation rates and 4-AP affinities are correlated in Kv channels . This is consistent with observations showing that channel opening influences 4-AP binding in Kv4 open channels . Accordingly, VI channels, which feature reduced blockade by 4-AP (Table I ), also exhibited drastically slower tail currents . Wild-type tail currents were well described assuming an exponential relaxation, but a good description of the mutant tail currents required the sum of two exponential terms. In both cases, however, the resulting time constants showed little voltage dependence between −140 and −100 mV . The slow time constant (∼50 ms) dominated the time course of the tail current (∼70%). Relative to the deactivation time constant of wild type, the fast and slow time constants of deactivation of the mutant tail currents at −140 mV were ∼4- and ∼40-fold slower, respectively . Earlier studies have shown that mutations in the S6 region alter the single channel conductance of certain Kv channels . Because the VI mutations are located near the end of the S6 segment, we examined the single channel properties of the wild-type channel and the VI mutant channel. Fig. 5 , A and C, shows sets of six consecutive single channel traces evoked by 900-ms step depolarizations to +50 mV from a holding potential of −100 mV and the corresponding ensemble averages (cell-attached patches). The wild-type channels exhibited rapid flickering between the closed and open levels before inactivating. Additional noise was apparent when these channels seemed to fluctuate between the fully open level and subconductance levels . This complex single channel behavior produced an ensemble average trace that closely matches rapidly inactivating macroscopic currents recorded from macropatches or whole oocytes. Single VI channels produced a significantly more stable open level and do not seem to inactivate significantly because channels frequently reopen during the depolarizing pulse . Occasionally, a subconductance level was clearly apparent , and although the last trace suggests a retarded latency to the first opening this change was not consistently observed (see above for the estimation of activation delay). The average open time of well-resolved full openings was computed to estimate the relative change in the apparent open time between wild-type and VI channels. The resulting values were 8.2 ± 3.1 ms ( n = 5 patches) and 21.9 ± 3.7 ms ( n = 7 patches) for wild-type and mutant channels, respectively (because only well-resolved openings were examined, the apparent open times are overestimated). The increase in the apparent open time was qualitatively consistent with the slow deactivation of the VI channels at hyperpolarized membrane potentials, and the ensemble average trace closely matches slowly inactivating macroscopic currents recorded from macropatches or whole oocytes. These results indicated that the recorded single channel activity represents gating of the channels under study. Therefore, the single channel records were analyzed further to test whether VI affected the single channel conductance. Two different experimental protocols were used to estimate the single channel conductance. In one, the membrane potential was ramped between −100 and +70 mV. Single channel currents evoked during this voltage ramp are shown in Fig. 5 , B and D from patches expressing wild-type and VI channels, respectively. In the other, the mean amplitude of discrete single channel openings was estimated from amplitude histograms at different membrane potentials . Clearly, both protocols gave similar results. The estimated slope conductances were 5.1 ± 0.7 pS ( n = 3 patches) and 5.8 ± 0.7 pS ( n = 4 patches), for wild-type and mutant channels, respectively. Thus, VI did not significantly affect the unitary conductance of Kv4.1 K + channels. The reversal potentials of wild-type and mutant tail currents in normal external solution (2 mM K + ) were also similar (approximately −93 mV; Table I ). Furthermore, the relation between the reversal potential and the concentration of external K + (substituting Na + for K + ) was examined to verify that the P Na / P K was not affected by the mutation. The slopes of these relations were nearly identical (58.7 and 57.6 mV/10-fold change in the concentration of external K + for the wild-type and the VI currents, respectively; data not shown). Therefore, V404 and V406 in Kv4.1 K + channels do not appear to be critical in determining ion permeation and K + selectivity. The S4–S5 loop is thought to be an important component of the gating machinery of Kv channels . In addition, the S4–S5 loop appears to contribute to the receptor of the inactivation particle and ion permeation . Presumably, the S4–S5 loop could play similar roles in Kv4 K + channels, but that has not been directly investigated. In particular, it is intriguing that position 322 in Kv4.1 (or the equivalent position in all Shal K + channels) is occupied by cysteine, whereas in other Kv channel subunits the equivalent position is occupied by serine . Mutation of this serine to cysteine in Shaker B K + channels (S392C) destabilizes the inactivated state , probably by affecting the interactions of the S4–S5 loop with the inactivation particle. Thus, because macroscopic inactivation of Shaker B K + channels is much faster than that of Kv4 K + channels, we had hypothesized that the reverse substitution in Kv4.1 (C322S) could favor inactivation. By contrast, C322S channels exhibit slower macroscopic inactivation and deactivation kinetics . The degree of inactivation [I (450 ms) / I peak ] at +50 mV is 0.69 ± 0.03 ( n = 6). Compared with wild-type currents, this figure is approximately eightfold larger. When the current was evoked by a long depolarization (+50 mV, 10 s), there was little or no current remaining by the end of the pulse . The 50% rise time at +50 mV was also slowed by an approximately twofold (1.7 ± 0.3 and 3.4 ± 0.8 ms for wild type ( n = 5) and mutant ( n = 5), respectively; Fig. 6 B, inset) and the activation delay was modestly increased (0.97 ± 0.3 and 1.4 ± 0.3 ms for wild type ( n = 6) and mutant ( n = 5), respectively). Tail currents at −140 mV were best described assuming the sum of two exponential terms , with the slow component (τ = 13.9 ± 4 ms, n = 5) dominating the time course of the tail current (∼60%). These results are not consistent with the idea that C322 in Kv4.1 and S392 in Shaker B play equivalent functions. Interestingly, however, the kinetics of macroscopic activation and inactivation, recovery from inactivation and deactivation of C322S, VI, and V406I are similar (Table I ). Like the V406I mutant, the prepulse inactivation curve and the peak G/V relation of C322S were only modestly affected (Table I ). Also, closed-state inactivation remained unchanged . Notably, however, the blockade of C322S channels by 4-AP was also reduced (Table I ). The single channel conductance is one of the most significant differences between the VI and C322S mutant channels. The latter mutant roughly doubled the single channel conductance (Table I ), a result that is consistent with a contribution of the S4–S5 loop to the ion permeation pathway . The ability C322S to mimic the inactivation gating properties of VI and V406I suggests that two regions that are thought to form the inner vestibule of Kv channels (the S4–S5 loop and the distal section of S6) may in a unique way contribute to inactivation gating in Kv4 K + channels. This idea is particularly significant in light of the distinct effects of similar mutations in Shaker K + channels (see below). One of the most striking effects of the mutations studied here was the dramatic slowing of deactivation, which in all cases is associated with slow current inactivation . This relationship is the key to the mechanism of inactivation of Kv4 channels (see discussion ). The cytoplasmic NH 2 - and COOH-terminal domains are necessary to maintain the fast inactivation process . In the absence of most of the terminal domains, slower inactivation processes remained relatively unchanged . Thus, there is little or no interaction between the fast and slow processes of inactivation. To examine whether the slow inactivation processes are affected by the S6 and S4–S5 mutations in the absence of the termini, we introduced the VI and C322S mutations into a previously studied double deletion mutant . The main effect of the deletion alone is to eliminate the fast phase of macroscopic inactivation observed at positive membrane potentials . ΔN71/ΔC158/VI dramatically slowed the rising and decaying phases of the current . ΔN71/ΔC158/C322S also slowed macroscopic inactivation and the rising phase but to a lesser extent than ΔN71/ΔC158/VI . These observations are qualitatively in agreement with observations made in the presence of the termini , including the fact that ΔN71/ΔC158/ VI and ΔN71/ΔC158/C322S continued to inactivate over a period of 9 s (not shown). These results showed that independently of the termini, V404, V406, and C322 help to control inactivation in Kv4 K + channels. Thus, it is unlikely that the main function of these residues is to contribute to a docking site for a putative cytoplasmic inactivation gate in these channels. Further support for a distinct form of inactivation in Kv4 channels can be found by examining Kv channels that undergo inactivation mainly coupled to channel opening . In such a case, how does a change in deactivation kinetics affect inactivation gating? We hypothesized that, by contrast to VI in Kv4.1, the equivalent mutation in Kv1.4 (VI) may not slow the development of macroscopic inactivation, but significantly slow current deactivation and recovery from inactivation. This result is expected because K + channels of this kind pass through the open state when they recover from inactivation , and the distal section of S6 influences gating . Both the wild-type and mutant Kv1.4 channels expressed nearly identical outward currents that rapidly activate and inactivate . The decay of these currents is well described assuming a biexponential relaxation (Table II ) with time constants that are voltage independent at positive membrane potentials (not shown). As predicted, the VI mutation slows the kinetics of deactivation and recovery from inactivation but does not significantly alter the development of macroscopic inactivation (Table II ). The time constants of deactivation (at −140 mV) and recovery from inactivation (at −100 mV) increased 10- and 5-fold, respectively (Table II ). This result agrees with the hypothesis because channel closing is rate limiting when the channels recover from inactivation passing through the open state (a direct consequence of inactivation mainly coupled to channel opening). The VI mutation also destabilizes voltage-dependent activation of Kv1.4 K + channels because the peak G/V relation and the prepulse inactivation curve exhibit a depolarizing shift of ∼10 mV (Table II ). Note that VI in Kv4.1 channels caused a hyperpolarizing shift of the peak G/V curve and a depolarizing shift of the prepulse inactivation curve . In addition to the effects on channel gating, the VI mutation drastically reduced the sensitivity of the Kv1.4 channels to 4-AP. This property was evaluated by recording two successive current–voltage relations in the absence and presence of 20 mM 4-AP (separated by ∼7 min). Although this protocol is not sufficient to develop steady state use-dependent blockade, in wild-type channels we observed a characteristically faster decay of the current in the presence of 4-AP (the fast time constant of inactivation decreased by two- to threefold) and 66% inhibition of the peak current (Table II ). This inhibition is influenced by N-type inactivation and has been previously characterized in detail as an open channel blockade . The VI mutation eliminated the blockade of the current by 4-AP (Table II ). Overall, these results demonstrate that valines in the distal section of S6 in Kv4.1 and Kv1.4 influence channel gating and 4-AP binding, but exert radically different actions on channel inactivation. Clearly, two classes of A-type K + channels inactivate by distinct mechanisms. Kv4 K + channels play critical roles in both the central nervous system and the heart. In brain, these channels constitute the somatodendritic subthreshold A-type K + current that prevents back propagation of action potentials and helps to regulate the frequency of slow repetitive spike firing . In ventricular myocytes, Kv4 channels underlie the Ca-independent A-type outward current known as I to . This current helps to shape the early phase of the cardiac action potential. Inactivation gating is crucial for these functions. To investigate the molecular mechanism underlying inactivation gating of Kv4 K + channels, we examined the biophysical effects of mutations in the distal section of the S6 region (VI and V406I) and the S4– S5 loop (C322S) of Kv4.1. Both regions are putative components of the inner vestibule of the pore . Valines 404 and 406 are conserved in most Kv channels and we hypothesized that they may contribute to the mutually exclusive interaction between 4-AP and inactivation gating in Kv4 K + channels. C 322 is a Kv4-specific residue that in conjunction with V404 and V406 may play a key function in the inactivation mechanism. The VI and C322S mutations similarly slowed inactivation, deactivation, and inhibited blockade by 4-AP in a manner that is independent of the cytoplasmic NH 2 - and COOH-terminal domains. Although the effects appeared to be complex, the key to the mechanism of inactivation in Kv4.1 K + channels was the parallel relation between macroscopic inactivation and deactivation (see below). The specificity and novel nature of these findings is supported by the starkly different effects of homologous mutations in the S6 region of a Kv channel that undergoes N- and C-type inactivation (Kv1.4). Our previous study and the current data suggest the presence of at least two relatively independent pathways of inactivation at positive membrane potentials in Kv4.1 K + channels. One is relatively fast (τ ∼ 15 ms), contributes to a small percentage of the total decay (∼17%), and is affected by both NH 2 - and COOH-terminal deletions. The other is slow and exhibits two measurable relaxations: intermediate (τ ∼ 75 ms) and slow (τ ∼ 250 ms); contributing to ∼35 and ∼45% of the total decay, respectively. The S6 and S4–S5 loop mutations characterized in this study drastically affect the more prominent intermediate and slow processes. Also, slower macroscopic inactivation induced by the mutations was associated with slower current deactivation and a slightly accelerated rate of recovery from inactivation. Thus, we concluded that to enter the dominant inactivated states at depolarized voltages channel closing and inactivation are coupled (i.e., to inactivate, the channel must close). This coupling is disrupted by 4-AP because this agent interacts with the closed channels and slows macroscopic inactivation. Therefore, to explain our observations with Kv4.1 channels, a kinetic model must account for the apparent coupling between inactivation, deactivation, and channel blockade by 4-AP, which constitutes the main experimental constraint on the kinetic analysis. Qualitatively, the simplified Schemes Ia and Ib can explain the main results. In Schemes Ia and Ib, [C] represents the rapid equilibrium of voltage-dependent state transitions that precede channel opening, and [I] represents an aggregate of inactivated states (transitions outside of the main activation pathway are assumed to be voltage independent). Channels can rapidly reach an inactivated state from the open state (the rapid pathway). However, such a state is unstable (λ > κ). This is consistent with the presence of a small fast component in the decay of the current (see above). Thus, if the equilibrium is shifted toward the closed state (e.g., k −1 > k 1 ), the channels may inactivate from a closed state (preferably from that state that precedes channel opening). This pathway may correspond to the slower processes of inactivation. If the C ↔ O and O ↔ I equilibria are relatively rapid in Scheme Ia, the slow time constant of inactivation from the preopen closed state can be approximated as τ i ≈ ( P c γ + δ) −1 , where P c is the equilibrium probability of occupying the inactivation permissive preopen closed state [ k −1 P ro ( k −1 P ro + k +1 ) −1 ], and P ro is the reopen equilibrium probability [λ(λ + κ) −1 ]. Thus, by changing one rate constant ( k −1 ) in Scheme Ia, it can be seen that channel closing influences inactivation. Because 4-AP mainly blocks closed channels, Scheme Ib shows that inactivation and 4-AP binding are mutually exclusive, as demonstrated by Campbell et al. and Tseng et al. . As for the effects of the mutations, inactivation from the preopen closed state becomes unfavorable when the C ↔ O equilibrium in Scheme Ia is shifted toward the open state (e.g., k −1 is reduced). Therefore, the macroscopic current decays at a slower rate and 4-AP binding is reduced. This is a likely mechanism because the mutations slowed the closing rate . Other important but less significant changes are discussed later assuming an expanded version of Scheme Ia. A strictly sequential scheme, which assumes inactivation coupled to channel opening (Scheme II), cannot explain the results with Kv4.1 channels. If the C ↔ O equilibrium is relatively rapid, the time constant of inactivation in Scheme SII is approximated as τ i ≈ ( P o κ + λ) −1 . Thus, a shift in the opening equilibrium toward the open state (by simply slowing the closing rate) accelerates macroscopic inactivation (i.e., τ i decreases). This prediction is opposite of the observations with Kv4.1 channels (i.e., deactivation parallels inactivation). Also, Scheme SII with a slower closing rate predicts slower recovery from inactivation because channels revisit the open state when they recover from inactivation. This change is also opposite of the effect of the mutations in Kv4.1, which in fact slightly accelerated the recovery from inactivation. It could be argued that a relatively large simultaneous reduction of both k −1 and κ may explain the apparent coupling between channel closing and inactivation. However, such changes in Scheme SII also predict a significant increase in the level of the steady state current, which was not observed with the Kv4.1 mutants (and these mutants exhibited only slightly accelerated recovery from inactivation). Scheme SII appears more appropriate to describe the results with Kv1.4 channels (see below). To demonstrate more quantitatively the mechanism described above, we simulated the currents assuming a nine-state time-homogeneous Markov model (Scheme III) as an expanded version of Scheme Ia. This expansion is necessary to account for voltage-dependent gating and sigmoidal activation kinetics. Scheme SIII can model the main kinetic features of the macroscopic currents at positive voltages (over a range of four orders of magnitude in time) and the tail current at hyperpolarized voltages . Similar schemes have been previously proposed to describe inactivation gating of A-type K + channels . However, in contrast to these models, Scheme SIII assumes that channel closing is faster than opening ( k −1 > k 1 ), and that the inactivated state that originates from the open state is unstable ( k −3 ≥ k 3 ). These two assumptions are compatible with single channel recordings showing rapid flickering behavior . It also assumes, as explained above, that the channels mainly inactivate from the preopen closed state and that this pathway includes at least two inactivated states. Scheme SIII accounts for the complexity of the macroscopic current at positive voltages. Table III summarizes the sets of parameters that were used to model the wild-type and mutant currents . As explained above, the main experimental observation that constrains the kinetic analysis of Kv4.1 inactivation is the apparent parallel relation between the development of current inactivation and deactivation. Three changes of the model's parameters were sufficient to closely reproduce the effects of the mutations. (a) A 19-fold reduction of the closing rate k −1 . This change alone significantly slows the tail current and the rising and decaying phases of the macroscopic outward current . (b) To account for the slow closed-state inactivation observed with the VI mutant, the transition rates governing C 4 → I 1 ( k 2 ) and I 1 → I 3 ( k 4 ) were reduced by two- and fivefold, respectively. (c) The inactivation rate k 3 was reduced by ∼30% and the reopening rate k −3 was approximately doubled because the fast phase of inactivation was also slowed and reduced by the mutations. These changes suggest that the S6 mutations studied here may also modestly affect the process of inactivation that involves the concerted action of the cytoplasmic NH 2 - and COOH-terminal domains . It should be noted, however, that the most dramatic kinetic changes induced by the VI mutation appear to result from slowing channel closing and closed-state inactivation. The slower inactivation of C322S, on the other hand, was mainly the result of slowing channel closing ( k −1 ) and, to some extent, of slowing k 3 and accelerating k −3 . Scheme SIII also accounted for the kinetics of the tail currents from mutant channels, which exhibited two time constants. The fast and slow relaxations are probably associated with the rapid equilibrium O ↔ I 2 (at the peak of the current ∼15% of the mutant channels have entered I 2 ) and channel closing, respectively . The voltage-dependent activation rate constant α did not seem affected because the activation delay was not significantly different between wild-type and mutant channels (see results ). When β is very small (as expected at depolarized voltages), the activation delay approximates the mean latency to arrive at the open state . The VI mutation in Kv1.4 channels also slowed current deactivation . However, by contrast to VI in Kv4.1, it had little impact on the time course of macroscopic inactivation but also significantly slowed the recovery from inactivation . As discussed above, these changes are more consistent with inactivation coupled to channel opening (Scheme II), which is a favored pathway of inactivation of Kv1.4 channels and other A-type K + channels in the Shaker family . Because macroscopic inactivation did not appear accelerated (as predicted earlier for Scheme SII when channel closing is slower), it is possible that the VI mutation in Kv1.4 might have also modestly reduced the rate of inactivation. Modeling of inactivation coupled to channel opening showed that accelerated macroscopic inactivation caused by a 10-fold slower closing rate can be compensated by a 30% slower rate of inactivation. Altogether, a kinetic model that mainly assumes inactivation coupled to channel opening can explain the results with Kv1.4 channels but fails to account for the apparent coupling between channel closing and the development of inactivation observed with Kv4.1 channels. The initial analysis adopted Scheme Ia to explain inactivation coupled to channel closing and hypothesized that the main effect of the mutations is to slow the closing rate. Thus, to test this hypothesis further, the main goal of the simulations was to determine whether Scheme SIII (the expanded version of Scheme Ia) can model wild-type and mutant currents in the time domain when k −1 > k 1 and when k −1 is reduced. To evaluate the simulations, we focused our attention on two sets of experiments that examine: (a) currents evoked by short and long depolarizing steps to positive membrane potentials (up to +70 mV), which covered about four orders of magnitude in the time domain (1 ms to 10 s); and (b) tail currents at negative membrane potentials (−140 to −100 mV). The simulations succeeded in simulating the results of these experiments and revealed that slower closed-state inactivation may also significantly contribute to slower inactivation of macroscopic mutant currents. However, we did not examine in detail the voltage dependence of activation and inactivation. Therefore, there is uncertainty about the complexity of the activation pathway, which may include multiple closed and inactivated states . Nevertheless, at a qualitative level, the same set of parameters (Table III ) predicted the observed shifts in the peak G/V relation and the prepulse inactivation curve, and little change in the recovery from inactivation. Also, because of the complexity of the single channel records of wild-type currents, we have not yet obtained more quantitative constraints of the model parameters. To account for the presence of subconductance levels, we have assumed that: (a) the open state in Scheme SIII represents an aggregate of states with different unitary conductances, and (b) partly and fully open channels can undergo rapid inactivation. In Kv4.1 channels, the fast component of macroscopic inactivation is eliminated by deletion of the first 31 amino acids at the NH 2 terminus and certain COOH-terminal deletions . However, additional experimental criteria that are crucial in defining N-type inactivation in Shaker K + channels are not satisfied by Kv4.1 K + channels : (a) internal tetraethylammonium does not compete with a putative inactivation particle; (b) basic residues within the first 40 amino acids at the NH 2 -terminal domain are not critical in determining the rate of inactivation; (c) elevated external K + slows recovery from inactivation (in disagreement with the presence of an internal inactivation particle that acts as an open channel blocker); and (d) the S4–S5 loop does not appear to contribute to the docking site of a putative inactivation particle because C322S does not significantly compromise the stability of the inactivated state (for an extended argument, see results ), and other mutations of a highly conserved glutamate in the S4–S5 loop (E325Q and E325D, which disrupt inactivation of Shaker channels, in fact accelerated macroscopic inactivation in Kv4.1 . The removal of fast inactivation by NH 2 - and COOH-terminal deletions in Kv4.1 channels leaves slower inactivation processes that appear to function independently from the fast process . Additional results rendered the slow Shaker C-type mechanism in Kv4 K + channels also unlikely : (a) high external tetraethylammonium (96 mM) moderately inhibits the current but does not interfere with inactivation; (b) elevated external K + accelerates recovery from C inactivation , but slows recovery from inactivation in Kv4.1; and (c) elevated external K + slows the rate of C-type inactivation , but accelerates macroscopic Kv4.1 inactivation. C-type inactivation involves residues in the S5–S6 linker and the S6 transmembrane segment . In particular, mutation of a threonine to valine at position 449 in Shaker B (T449V) nearly eliminates C-type inactivation . In Kv4 proteins, a valine already exists at the equivalent position. The oxidizing agent chloramine-T accelerates C-type inactivation and induces irreversible current rundown in the NH 2 -terminal deleted Shaker B channels . M448 is implicated as a target for chloramine-T. Although in Kv4.1 channels a methionine occupies the equivalent position, exposure to 1 mM chloramine-T caused no effect on these channels (Jerng, H.H., unpublished observations). Altogether, the data suggest the presence of new components of inactivation gating in Kv4 channels. The external vestibule of Kv channels has been extensively studied and there is a consensus about its structure and function . The inner vestibule of these channels is, by contrast, more complex and less well understood. Nevertheless, several studies have begun to define the structural components of the inner vestibule of Kv channels (see introduction ). In particular, it has been demonstrated that components of the inner vestibule (S4–S5 loop and the distal section of S6) undergo conformational changes that can be related to channel gating . Here, we have characterized a form of inactivation gating in Kv4.1 K + channels that depends on the coupling between channel closing and inactivation at depolarized membrane potentials (channels must close before they inactivate), and involves components of the inner vestibule of the pore. The results demonstrated that a Kv4-specific cysteine in the S4–S5 loop (C322) and two valines located in the distal section of S6 (V404 and V406) are important in controlling the inactivation mechanism that is coupled to channel closing. A corollary of this result implies that other residues (or processes) that control channel closing may also influence inactivation, but this remains to be investigated. Because Kv4.1 channels do not exhibit the hallmarks of N- and C-type inactivation (see previous section) and the new observations described here, a novel mechanism of inactivation might be present in these channels. However, the current evidence cannot completely rule out that channel closing at the inner vestibule influences C-type inactivation from partly activated closed states , which could involve a P-type component at the external mouth of the pore . We suggest the term “V-type” inactivation to indicate that putative components of the inner vestibule affect inactivation gating of Kv4 channels in an unexpected way. In current structural models of the cytoplasmic side of Kv channels, the S4–S5 loop and the distal section of S6 contribute to the inner mouth of the pore . Thus, in Kv4 channels the inner mouth of the pore (acting as a single gate) may undergo shutter-like conformational changes that sequentially close and inactivate the channel. Closed-state inactivation is also a major gating pathway in slow-inactivating Kv2 channels and the L382I Shaker B mutant . However, it is not yet clear whether the V-type component is also present in these channels. | Study | biomedical | en | 0.999998 |
10228181 | Depolarization-activated K + currents play key roles in determining the amplitudes and durations of action potentials in cardiac cells, and several distinct types of voltage-gated K + channels that subserve these functions have been identified . This diversity has a physiological significance in the heart in that the various K + currents underlie distinct phases of action potential repolarization , and cell type–specific differences in K + channel expression contribute to regional variations in action potential waveforms . Voltage-gated K + channels are primary targets for the actions of a variety of endogenous neurotransmitters and neurohormones, as well as exogenous drugs that modulate cardiac functioning . In addition, changes in the densities and/or the properties of K + currents occur in conjunction with myocardial damage or disease and these changes can have profound physiological consequences, including leading to the generation of life threatening arrhythmias . For all of these reasons, there is considerable interest in defining the molecular correlates of functional cardiac K + channels , and in understanding the mechanisms controlling the regulation, modulation, and functional expression of these channels. A number of voltage-gated K + channel pore-forming (α) and accessory (β) subunits have now been cloned from heart cDNA libraries, and a variety of experimental approaches are being exploited to probe the molecular basis of functional K + channel diversity in mammalian cardiac cells . Of these, transgenic and knockout strategies seem particularly promising because the functional consequences of manipulating K + channel expression in vivo can also be explored directly. Indeed, mice with targeted Kv α subunit deletions, including Kv3.1, Kv1.1, and Kv1.4, have been described recently . None of these animals appears to display a cardiac phenotype, however, leading to suggestions that Kv1.1, Kv3.1, and Kv1.4 likely do not play roles in the generation of functional cardiac K + channels, at least in the mouse . London et al. , however, recently reported the generation of transgenic mice expressing a truncated Kv1.1 ( Kv1 . 1N206Tag ), driven by the α-myosin heavy chain promoter to direct cardiac-specific expression of the transgene . In vitro, Kv1 . 1N206Tag functions as a dominant negative, reducing or eliminating heterologously expressed Kv1.4 and Kv1.5 currents . In ventricular myocytes isolated from Kv1 . 1N206Tag -expressing mice, a 4-aminopyridine–sensitive, slowly decaying outward K + current, I K,slow , was found to be selectively attenuated . In addition, action potentials and QT intervals are prolonged in the Kv1 . 1N206Tag -expressing transgenics, and electrocardiographic recordings revealed increased frequency of premature ventricular beats and spontaneous ventricular tachycardia in these animals . A dominant negative strategy has also been exploited by Barry et al. in studies focussed on identifying the molecular correlate of the cardiac transient outward K + current, I to . Using an approach previously used to make Kv1 α subunits that gate on membrane depolarization but do not conduct , mutations were introduced into the coding sequence of the pore region of Kv4.2 to convert the tryptophan (W) in position 362 to phenylalanine (F) to produce Kv4.2W362F, and, in in vitro experiments, Kv4.2W362F was shown to function as a dominant negative against Kv4.2 and Kv4.3 . In ventricular myocytes isolated from Kv4.2W362F-expressing animals, I to (I to,f ) is eliminated, and action potential durations are increased significantly . Although QT intervals are also markedly prolonged in Kv4.2W362F-expressing transgenics, these animals do not develop spontaneous arrhythmias . However, a “novel” rapidly activating, slowly inactivating K + current, not clearly evident in wild-type cells, was identified in ventricular myocytes isolated from Kv4.2W362F-expressing animals, an observation interpreted as suggesting that electrical remodeling occurs in the myocardium when the expression of endogenous K + channels is altered . In spite of the growing interest in using transgenic and knockout mice, the electrophysiological properties of adult mouse cardiac myocytes have not been characterized in detail to date. This fact and the finding of the novel K + current in Kv4.2W362F-expressing ventricular myocytes prompted us to undertake studies focussed on examining the time- and voltage-dependent properties and the pharmacological sensitivities of the voltage-gated K + currents in adult mouse ventricular myocytes. The results of these experiments reveal the presence of four kinetically and pharmacologically distinct voltage-gated K + currents in these cells: (a) a rapidly activating and inactivating, “fast transient” K + current, I to,f ; (b) a rapidly activating, slowly inactivating, “slow transient” current, I to,s ; (c) a rapidly activating, very slowly inactivating current, I K,slow ; and (d) a slowly activating, noninactivating K + current, I ss . Interestingly, the results presented here reveal that I K,slow and I ss are expressed in all adult mouse ventricular cells, whereas I to,f and I to,s are differentially distributed. Ventricular myocytes were isolated from adult (after postnatal day 45) C57BL6 mice using a procedure previously developed and used to isolate rat cardiomyocytes . In brief, hearts were excised from anesthetized (5% halothane/95% O 2 ) adult animals, mounted on a Langendorf apparatus, and perfused retrogradely through the aorta with 40 ml of a Ca 2+ -free HEPES-buffered Earles balanced salt solution ( Gibco Laboratories ) supplemented with 6 mM glucose, amino acids, and vitamins (Buffer A). Hearts were then perfused with 50 ml of Buffer A containing 0.8 mg/ml collagenase B ( Boehringer Mannheim Biochemicals ) and 10 μM CaCl 2 , and the temperature of the tissue and the perfusate were maintained at 34–35°C. The enzyme solution was filtered (at 5 μm) and recirculated through the heart for ∼15–20 min. In initial experiments, the lower two thirds of the left and right ventricles were removed after the perfusion, and, after mincing in enzyme-containing Buffer A, were transferred to fresh (enzyme-free) Buffer A supplemented with 1.25 mg/ml taurine, 5 mg/ml bovine serum albumin ( Sigma Chemical Co. ), and 150 μM CaCl 2 (Buffer B). In experiments focussed on determining whether there are regional differences in K + current expression, the heart was cut open after perfusion, and the ventricular septum and the top ∼0.3 mm of tissue at the apex of the left ventricle were removed. The tissue pieces from the apex and septum were placed separately in fresh Buffer B. After mechanical dispersion (by gentle trituration), cell suspensions were filtered to remove large undissociated tissue fragments, and cells were collected by sedimentation. Isolated myocytes were resuspended in fresh Buffer B, plated on laminin-coated coverslips, and placed in a 95% air/5% CO 2 incubator at 37°C. Approximately 30 min after plating, serum-free medium-199 (M-199; Irvine Scientific), supplemented with antibiotics (penicillin/streptomycin), was added. Ca 2+ -tolerant ventricular myocytes adhered to the laminin, and damaged cells were removed by replacing the medium with fresh M-199 ≈ 1 h after plating. Cells were examined electrophysiologically within 48 h of isolation. The conventional whole-cell gigaohm seal recording technique was employed to record Ca 2+ -independent, depolarization-activated K + currents from isolated adult mouse ventricular myocytes. Electrophysiological recordings were only obtained from Ca 2+ -tolerant, rod-shaped ventricular cells, and all experiments were conducted at room temperature (22–24°C). The bath solution contained (mM): 136 NaCl, 4 KCl, 1 CaCl 2 , 2 MgCl 2 , 5 CoCl 2 , 10 HEPES, 0.02 tetrodotoxin, and 10 glucose, pH 7.35, and 295–305 mOsm. Recording pipettes contained (mM): 135 KCl, 1 MgCl 2 , 10 EGTA, 10 HEPES, 5 glucose, pH 7.2, and 300–310 mOsm. α-dendrotoxin (α-DTX; Alamone Labs), 1 Heteropoda toxin-3 (HpTx-3; NPS Pharmaceuticals), and 4-aminopyridine (4-AP; Sigma Chemical Co. ) stock solutions were prepared in distilled water, and diluted to the appropriate concentration in bath solution immediately before use. Tetraethylammonium (TEA; Sigma Chemical Co. )-containing bath solutions were prepared by equimolar substitution of TEACl for NaCl in the standard bath solution. α-DTX, HpTx-3, 4-AP, or TEA was applied to isolated myocytes during recordings using narrow-bore capillary tubes (300 μm i.d.) placed within ≈ 200 μm of the cell. Experiments were conducted using an Axopatch-1D (β = 1) ( Axon Instruments ) or a Dagan 3900A (Dagan Corp.) amplifier. Recording pipettes, fabricated from soda lime glass, had tip diameters of 1–2 μm and resistances of 2–3 MΩ when filled with recording solution. Tip potentials were zeroed before membrane pipette seals were formed; seal resistances were ≥5 GΩ. After establishing the whole-cell configuration, ±10-mV steps were applied to allow measurement of whole cell membrane capacitances and input resistances. Series resistances were estimated by dividing the time constant of the decay of the capacitative transient by the membrane capacitance. Whole-cell membrane capacitances and series resistance were routinely compensated (≥85%) electronically; voltage errors resulting from the uncompensated series resistance were ≤8 mV and were not corrected. Only data obtained from cells with input resistance ≥0.7 GΩ were analyzed. Voltage-gated outward K + currents were routinely evoked during 500-ms or 4.5-s depolarizing voltage steps to potentials between −40 and +60 mV from a holding potential of −70 mV; voltage steps were presented in 10-mV increments at 15-s intervals. Experiments were controlled and data were collected using a Gateway 300 MHz microcomputer equipped with a Digidata 1200 ( Axon Instruments ) analogue/digital interface and pClamp 6.1 ( Axon Instruments ). Data were acquired at variable sampling frequencies (ranging from 0.1 to 50 kHz), and current signals were filtered on-line at 5 kHz before digitization and storage. Analyses of digitized data were completed using pClamp 6.1. Whole-cell membrane capacitances were determined by integrating the capacitative transients evoked during ±10-mV voltage steps from a holding potential of −70 mV (before series resistance and capacity compensation). Peak currents at each test potential were measured as the difference between the maximal outward current amplitudes and the zero current level. The waveforms of the 4-AP–, TEA-, and α-DTX–sensitive currents were determined by subtraction of the currents recorded in the presence of 4-AP, TEA, or α-DTX from the control currents (in the same cell). Activation time constants were determined from single exponential fits to the rising phases of the outward K + currents evoked during depolarizing voltage steps to test potentials between 0 and +60 mV from a holding potential of −70 mV; the fits were constrained to data points acquired 300 μs after the onset of the voltage step (thereby ignoring the delay) to the peak of the outward current. The decay phases of the currents evoked during long (4.5 s) depolarizing voltage steps to test potentials between +10 and +60 mV from a holding potential of −70 mV were fitted by the sum of two or three exponentials using one of the following expressions: y ( t ) = A 1 * exp(− t /τ 1 ) + A 2 * exp(− t / τ 2 ) + A ss or y ( t ) = A 1 * exp(− t /τ 1 ) + A 2 * exp(− t /τ 2 ) + A 3 * exp(− t /τ 3 ) + A ss , where t is time, τ 1 , τ 2 , and τ 3 are the time constants of decay of the inactivating K + currents, A 1 , A 2 , and A 3 are the amplitudes of the inactivating current components, and A ss is the amplitude of the steady state, noninactivating component of the total outward K + current. For all fits, time zero was set at the peak of the outward current. For all analyses, correlation coefficients ( R ) were determined to assess the quality of fits, R values for the fits reported here were ≥0.98. All averaged and normalized data are presented as means ± SEM. The statistical significance of observed differences between groups of cells or between different parameters describing the properties of the currents were evaluated using a one way analysis of variance or a two-tailed Student's t test; P values are presented in the text, and statistical significance was set at P < 0.05. In the initial experiments here, whole-cell depolarization-activated outward K + currents in myocytes isolated from the (lower two thirds of the left and right) ventricles of adult C57BL6 mice were recorded and analyzed (see materials and methods ). With voltage-gated Ca 2+ and Na + currents blocked, outward currents were routinely recorded during 500-ms and 4.5-s depolarizing voltage steps to potentials between −40 and +60 mV from a −70-mV holding potential ( n = 72). The rates of rise and the amplitudes of the currents increase with increasing depolarization; the largest and most rapidly activating current in Fig. 1 was evoked at +60 mV. No outward K + currents were recorded during depolarizing voltage steps when the K + in the pipettes was replaced by Cs + ( n = 6). The currents characterized here, therefore, reflect only the activation of Ca 2+ -independent, depolarization-activated K + channels. Although the outward K + currents in all cells activated rapidly, the absolute current amplitudes (densities) and the decay phases of the currents were somewhat variable among cells . In the majority of cells (65 of 72, ≈90%), outward current waveforms similar to those in Fig. 1 , A and B, were recorded. There is a rapid component of current decay in these cells, consistent with the presence of a transient outward K + current, previously described in these cells , as well as in cardiac myocytes in other species . We refer to this current as I to, fast , or I to,f , to distinguish it from another, more slowly inactivating, transient outward K + current, I to,slow , or I to,s , seen in some cells (see below). As noted in the introduction , it was recently demonstrated that I to,f is eliminated in adult mouse ventricular myocytes isolated from animals expressing a mutant Kv4.2 α subunit (Kv4.2W362F) that functions as a dominant negative , an observation consistent with previous suggestions that members of the Kv4 α subunit subfamily underlie cardiac I to,f . In the other (7 of 72, ≈10%) cells studied in these initial experiments, I to,f was not evident . Peak outward K + current densities in this subset of cells were significantly ( P < 0.001) lower than in the cells with I to,f ; the mean ± SEM peak outward K + current densities at +40 mV in cells with and without I to,f , for example, were 47.0 ± 2.5 ( n = 65) and 31.5 ± 4.1 ( n = 7) pA/pF, respectively (Table I ). In other respects, however, the properties of the cells with and without I to,f were indistinguishable; for example, the mean ± SEM whole cell membrane capacitances and input resistances were 142 ± 4 pF and 1.00 ± 0.09 GΩ for the cells with I to,f ( n = 65) and 151 ± 14 pF and 0.95 ± 0.15 GΩ for the cells lacking I to,f ( n = 7). Subsequent experiments were focussed, therefore, on characterizing the depolarization-activated K + current components in these cells in further detail. Analysis of the decay phases of the outward K + currents evoked during long depolarizations in the majority of cells revealed that current decay is well described by the sum of two exponentials, with decay time constants (τ decay ) that differ by an order of magnitude, and a noninactivating (i.e., steady state) current. The mean ± SEM τ decay ( n = 65) for the fast and slow components derived from these fits were 85 ± 2 and 1,162 ± 29 ms (Table I ); neither time constant displays any appreciable voltage dependence . Two components of outward K + current decay (with τ decay values of 80 ms and 1 s) in adult mouse ventricular myocytes were previously described, although both were attributed to inactivation of I to . In another recent study, however, the two components of inactivation were considered distinct current components: the rapidly inactivating current was referred to as I to and the slowly inactivating current was termed I K,slow . Consistent with this distinction, I K,slow was selectively attenuated in ventricular myocytes isolated from transgenic mice expressing a truncated Kv1.1 construct, Kv1 . 1N206Tag that functions as a dominant negative . These observations were interpreted as suggesting that Kv1 α subunits, likely Kv1.5, underlie mouse ventricular I K,slow . The slowly decaying current component will also be referred to here as I K,slow and as noted above the rapidly inactivating current is referred to as I to,f to distinguish it from I to,s . The densities of I to,f and I K,slow vary considerably among cells. For I to,f , for example, the peak current density at +40 mV ranged from 7.9 to 62.4 pA/pF, with a mean ± SEM of 26.2 ± 1.6 pA/pF ( n = 65; Table I ). I K,slow density at +40 mV ranged from 2.8 to 39.9 pA/pF with a mean ± SEM of 14.9 ± 0.9 pA/pF ( n = 65; Table I ). The noninactivating K + current component that remains at the end of 4.5-s voltage steps is referred to here as I ss (steady state). It is important to note that I ss is not the same as the “sustained” K + current recently described by Fiset et al. that was measured at the end of 500-ms voltage steps, and almost certainly reflects the sum of two K + currents, I K,slow and I ss (see discussion ). As with I to,f and I K,slow , I ss densities varied measurably among cells: at +40 mV, for example, I ss density ranged from 1.7 to 10.5 pA/pF, with a mean ± SEM of 5.5 ± 0.3 pA/pF ( n = 65; Table I ). Analysis of the decay phases of the outward currents evoked during 4.5-s depolarizations in cells lacking I to,f also revealed two inactivating components with mean ± SEM ( n = 7) decay time constants (τ decay ) of 196 ± 7 and 1,368 ± 101 ms (Table I ); neither time constant displays any appreciable voltage dependence . The τ decay (196 ± 7 ms) for the faster component of current decay in these cells is significantly ( P < 0.001) larger than the τ decay (85 ± 2 ms) for I to,f . In addition, when all of the fast τ decay values are compared , the cells with a mean ± SEM τ decay of 196 ± 7 ms fall well outside of the otherwise normal distribution of decay time constants. Taken together, these observations suggest a distinct subpopulation of cells (see below), and this current is referred to here as I to,s for transient outward, slow (see below). I to,s density in these cells ranged (at +40 mV) from 4.8 to 16.8 pA/pF, with a mean ± SEM ( n = 7) density of 11.4 ± 1.9 pA/pF (Table I ). In contrast to the differences in the rates of inactivation of the rapid components (i.e., I to,f and I to,s ) of current decay, the mean ± SEM τ decay (1,368 ± 101 ms) for the slowly inactivating current in the cells lacking I to,f is not significantly different from the mean ± SEM τ decay for I K,slow (1,162 ± 29 ms) in cells with I to,f (Table I ), consistent with the presence of I K,slow in both populations of cells. In addition, the mean ± SEM ( n = 7) I K,slow density of 15.6 ± 2.1 pA/pF in cells lacking I to,f (Table I ) is not significantly different from the mean ± SEM I K,slow density (of 14.9 ± 0.9 pA/pF) in cells with I to,f (Table I ). In cells lacking I to,f , however, I K,slow contributes, on average, 50% to the peak outward currents; i.e., substantially more than the average contribution (32%) of this current to the peak in cells with I to,f (Table I ). I ss is also evident in adult mouse ventricular myocytes expressing I to,s ; I ss density at +40 mV in these cells ranged from 2.5 to 6.2 pA/pF, with a mean ± SEM ( n = 7) of 4.5 ± 0.5 pA/pF, a value that is not significantly different from the mean ± SEM I ss density of 5.5 ± 0.3 pA/pF ( n = 65) in cells with I to,f (Table I ). The finding of the slowly inactivating, transient outward K + current, I to,s , in a subset of adult mouse ventricular myocytes prompted us to consider the possibility that this current might also be present in cells with I to,f , but simply not be readily detected because the density is too low (particularly relative to I to,f ) to be resolved. To test this hypothesis, the decay phases of the currents were fitted assuming three exponential components (see materials and methods ) with fixed inactivation time constants of τ 1 = 80 ms (for I to,f ), τ 2 = 1,200 ms (for I K,slow ), and τ 3 = 200 ms (for I to,s ). These analyses revealed that the decay phases of the total outward K + currents in some (40 of 65) cells could indeed be fitted by the sum of three exponentials; in the other 25 cells, the fits did not converge or the amplitude of the component with the τ decay = 200 ms was negative. Importantly, even when current records could be fitted to the sum of three exponentials, the quality of the fits was not improved significantly by the inclusion of the third component. Based on these analyses, therefore, it was not possible to determine whether some mouse ventricular cells do indeed express both I to,f and I to,s . The finding of cells expressing I to,s and lacking I to,f prompted us to explore the possibility that there might be regional differences in the expression/densities of these functionally distinct K + conductance pathways in mouse ventricle. To test this hypothesis directly, tissue pieces were dissected from the ventricular septum and from the apex of the left ventricle and dispersed (see materials and methods ). Electrophysiological recordings revealed that the waveforms of the outward K + currents in left ventricular myocytes isolated from these two regions are indeed distinct . Peak outward K + current densities in cells from the apex (mean ± SEM = 57.2 ± 3.5 pA/pF, n = 35) are significantly ( P < 0.001) greater than in cells isolated from the septum (mean ± SEM = 28.5 ± 1.5 pA/pF, n = 28; Table II ). In addition, for all cells isolated from the apex, analysis of the waveforms of the outward currents evoked during long (4.5 s) depolarizations revealed the presence of I to,f , I K,slow , and I ss (Table II ). The mean ± SEM τ decay for I to,f in these cells was 59 ± 2 ms ( n = 35; see discussion ); I to,f density (at +40 mV) in these cells ranged from 13.8 to 79.5 pA/pF, with a mean ± SEM ( n = 35) density of 34.6 ± 2.6 pA/pF and, on average, I to,f contributes 60% to the peak outward K + currents in apex cells (Table II ). Mean ± SEM ( n = 35) I K,slow and I ss densities at +40 mV in these cells were 17.2 ± 1.1 and 5.5 ± 0.4 pA/pF (Table II ). The waveforms of the outward K + currents in cells isolated from the septum appear different and are more variable than those seen in cells from the apex. Two distinct outward K + current waveforms were recorded in cells isolated from the septum . Some cells, for example, clearly lack I to,f , and analyses of the decay phases of the currents in these cells revealed two components with τ decay of 194 ± 11 and 1,143 ± 64 ms (Table II ). These values are indistinguishable for those determined above for I to,s and I K,slow (Table I ). I to,s density (at +40 mV) ranged from 2.8 to 8.5 pA/pF, with a mean ± SEM ( n = 6) density of 5.4 ± 0.9 pA/pF (Table II ) and, on average, I to,s contributes 26% to the peak outward K + currents in these cells (Table II ). The mean ± SEM I K,slow density (10.4 ± 1.5 pA/pF) in septum cells lacking I to,f (Table II ) is similar to the density of this current component in cells with I to,f (Tables I and II ). In cells lacking I to,f , however, I K,slow contributes, on average, ≈50% to the peak outward currents; i.e., substantially more than the average contribution (of 30–40%) seen in cells with I to,f (Tables I and II ). In the other subset of cells from the septum , three exponentials with mean ± SEM ( n = 22) time constants of 53 ± 2, 258 ± 15, and 1,180 ± 45 ms were required to fit the decay phases of the currents; the decay time constants for these three components suggest the coexpression of I to,f , I to,s , and I K,slow (as well as I ss ; Table II ). The mean ± SEM I to,f density in these cells (6.8 ± 0.5 pA/pF at +40 mV, n = 22) in these cells is significantly lower ( P < 0.001) than the mean ± SEM I to,f density (34.6 ± 2.6 pA/pF at +40 mV, n = 35) in cells from the left ventricular apex, and I to,f only contributes ∼20% to the peak outward currents in these cells, compared with 60% in apex cells (Table II ). In addition, the densities of I K,slow and I ss in cells isolated from the septum are slightly lower than in apex cells. I ss density at +40 mV in these cells ranged from 2.5 to 6.2 pA/pF, with a mean ± SEM ( n = 7) of 4.5 ± 0.5 pA/pF. Also, in septum cells, I ss contributes significantly more to the total outward current than does I ss in apex cells (Table II ). Subsequent experiments were focussed on examining the pharmacological sensitivities of the outward K + currents in adult mouse ventricular myocytes and on determining if the kinetically distinct K + current components could also be distinguished pharmacologically. In initial experiments, the effects of varying concentrations (10 μM to 5 mM) of 4-AP on whole-cell K + currents were examined. Control K + currents, evoked during 500-ms and 4.5-s voltage steps, were recorded before superfusion of 4-AP–containing bath solutions and, when the effect of 4-AP had reached a steady state, outward currents were again recorded. To obtain the current(s) blocked by 4-AP, records obtained in the presence of each concentration of 4-AP were digitally subtracted from the controls. Examples of typical experiments and the currents blocked by varying concentrations of 4-AP are presented in Fig. 4 . The currents blocked by 10 μM 4-AP activate rapidly and inactivate slowly . Analysis of the decay phases of the 10 μM 4-AP–sensitive currents suggests that I K,slow is selectively attenuated (by ≈35%), consistent with previous reports of the 4-AP sensitivity of this conductance pathway . In the presence of 50 μM 4-AP, I K,slow is further reduced to <50% of control ( n = 7), whereas I to,f is slightly (≈16%) reduced, and I ss is unaffected (Table III ). Exposure of adult mouse ventricular myocytes to concentrations of 4-AP > 100 μM also blocks I to,f (Table III ). After application of 0.5 mM 4-AP, for example, peak outward currents are attenuated markedly , and comparison of the control currents and the currents blocked by 0.5 mM 4-AP revealed that I K,slow is blocked by ≈80% and I to,f is reduced by ≈50% at 0.5 mM 4-AP; I ss , in contrast, is unaffected by 0.5 mM 4-AP (Table III ). When the 4-AP concentration is increased to 5 mM, outward currents are markedly reduced , and analysis of the control and the 5 mM 4-AP–sensitive currents revealed that I to,f (as well as I K,slow ) is blocked completely at this (5 mM) 4-AP concentration. The currents remaining in 5 mM 4-AP are interpreted as reflecting only I ss , although I ss is attenuated by ≈35% at high concentrations of 4-AP (Table III ). Experiments conducted on mouse ventricular myocytes isolated from the septum reveal that I to,s is also blocked by 4-AP . Application of 10 μM 4-AP to septum cells selectively attenuates I K,slow , and higher concentrations of 4-AP are required to also affect I to,s . In the presence of 0.5 mM 4-AP, I to,s is attenuated by ≈70% and I K,slow is blocked completely . To test the validity of the separation of the currents based on differential sensitivities to 4-AP , the effects of varying concentrations of other K + channel blockers, TEA, α-DTX, and HpTx-3 were also examined. As in the experiments with 4-AP, outward K + currents were recorded before and after bath applications of these blockers, and the drug-sensitive currents were obtained by off-line digital subtraction of these records. Exposure to 25 mM TEA resulted in marked attenuation of the peak currents and the currents remaining at the end of long depolarizing voltage steps. Analysis of the 25 mM TEA-sensitive currents reveals that these currents activate rapidly and inactivate slowly to a steady state level. The decay phases of the 25 mM TEA-sensitive currents were well described by single exponential with a mean ± SEM τ decay of 1,234 ± 197 ms ( n = 4), a value that is similar to the τ decay of I K,slow (Tables I and II ). In addition, comparison of the current waveforms in Fig. 6 B reveals that both I ss and I K,slow are reduced by ≈60% at this TEA concentration, whereas I to,f is unaffected (Table III ). In similar experiments completed on cells isolated from the septum, I to,s is also found to be unaffected by 25 mM TEA (Table III ). When the TEA concentration was increased to 135 mM, the currents remaining are rapidly inactivating . Both I ss and I K,slow are blocked completely and I to,f is blocked by ≈40% by isotonic TEA (Table III ). In experiments completed with α-DTX at concentrations up to 100 nM ( n = 4), no measurable effects on the outward K + currents in adult mouse ventricular myocytes were observed (not shown). In contrast, local applications of 100–300 nM HpTx-3 resulted in the selective attenuation of I to,f . In cells isolated from the apex of the left ventricle, for example, 100 nM HpTx-3 reduced I to,f by 30–40% . In cells isolated from the septum that express I to,f , this current is selectively attenuated by HpTx-3 , whereas I to,s in cells with and without I to,f is unaffected by HpTx-3 at concentrations up to 300 nM. Having identified four distinct depolarization-activated K + currents, I to,f , I to,s , I K,slow , and I ss , in adult mouse ventricular myocytes, subsequent experiments were focussed on characterizing the time- and voltage-dependent properties of these currents. For most of these analyses, the amplitudes of the currents were determined from exponential fits to the decay phases of the currents evoked during long depolarizing voltage steps. In some cases, the individual current components, separated based on differential sensitivities to 4-AP and TEA, were also analyzed. In the pharmacological analyses, I K,slow was defined as the 10-μM 4-AP–sensitive current ; the current remaining in the presence of 5 mM 4-AP was analyzed as I ss , and the current remaining in 135 mM TEA was analyzed as I to,f . Most of the initial experiments were completed on randomly dispersed adult mouse ventricular myocytes; detailed analysis of I to,s was completed on cells isolated from the septum. In some cases, the properties of I to,f , I K,slow , and I ss in apex and septum cells were determined and compared. To examine the voltage dependences of activation, the amplitudes of the individual current components at each test potential (in each cell) were measured and normalized to the current amplitude determined (in the same cell) at +30 mV; mean normalized currents are plotted as a function of test potential in Fig. 8 A. Although the voltage dependences of activation of I to,f , I to,s , I K,slow , and I ss are similar in that the currents begin to activate at less than −30 mV, the foot of the current–voltage plot for I to,f is less steep than for I to,s , I K,slow , or I ss . Time constants of activation for I to,f , I K,slow , and I ss were determined from single exponential fits to the rising phases of the currents separated using 4-AP and TEA. For these analyses, I K,slow was defined as the 10 μM 4-AP–sensitive current , and I to,f and I ss were defined as the currents remaining in the presence of 135 mM TEA or 5 mM 4-AP , respectively. For all current components, the rising phases of the currents at each test potential were well described by single exponentials. Mean (± SEM) activation time constants for I to,f , I K,slow , and I ss are plotted as a function of test potential in Fig. 8 B. As is evident, I to,f and I K,slow activate rapidly, and with similar activation time constants. I ss , in contrast, activates much more slowly than either I to,f or I K,slow at all test potentials . In addition, analyses of the rising phases of the currents in records such as those in Figs. 4 C and 6 C revealed that the time constants of activation of the 25-mM TEA-sensitive currents and 10-μM 4-AP–sensitive currents are indistinguishable: mean ± SEM activation time constants at +40 mV for the 25-mM TEA-sensitive currents and the 10-μM 4-AP–sensitive currents , for example, are 1.9 ± 0.5 ms ( n = 4) and 2.1 ± 0.2 ms ( n = 4), respectively. I K,slow , therefore, is blocked selectively by both 10 μM 4-AP and 25 mM TEA. The activation time constants (tau, τ) are voltage dependent, decreasing with increasing depolarization, and the variations with voltage are well described by single exponential functions of the form: tau(V) = a + b [exp(−V m / c )], where V m is the test potential and c is a constant that defines the steepness of the voltage dependence. The best fits to the data yielded: c = 27.1 ( a , 0.54; b, 11.2) for I to,f ; c = 18.1 ( a , 1.1; b , 10.1) for I K,slow ; and c = 13.0 ( a, 12.5; b , 3.9) for I ss . Comparison of the c values derived from these fits indicates that I to,f and I K,slow activation rates vary similarly as a function of voltage, whereas the voltage dependence of I ss activation is much steeper . The voltage dependences of steady state inactivation of I to,f , I to,s , and I K,slow were examined during voltage steps to +50 mV presented after 5-s conditioning prepulses to potentials between −100 and −10 mV; the protocol is shown below the current records in Fig. 9 . Experiments were completed on randomly selected myocytes, as well as on cells isolated from the left ventricular apex and septum (B). In the first two cases, the decay phases of the currents evoked at +50 mV from each prepulse potential were fitted to the sum of two exponentials to provide I to,f and I K,slow . For cells isolated from the septum, the amplitudes of I to,s , I to,f (when present) and I K,slow were determined from (double or triple) exponential fits to the decay phases of the currents evoked at +50 mV from each prepulse potential. The amplitudes of I to,f , I to,s , and I K,slow evoked from each conditioning potential were then normalized to their respective maximal current amplitudes (in the same cell) evoked from −100 mV. Mean (± SEM) normalized I to,f , I to,s , and I K,slow amplitudes are plotted as a function of conditioning potential in Fig. 9 C; the continuous lines represent the best Boltzmann fits to the averaged data. The steady state inactivation data for I to are well described by single Boltzmann with a V 1/2 of −24 mV . For I to,s and I K,slow , in contrast, the variations in current amplitudes with conditioning voltage were not well described by a single Boltzmann, and two Boltzmanns were required to fit the data . For I K,slow , the V 1/2 values derived from these fits were −73 mV ( k = 6.7 mV) and −19 mV ( k = 6.0 mV), and for I to,s , the V 1/2 values were −64 mV ( k = 9.0 mV) and −19 mV ( k = 6.3 mV). The voltage dependences of steady state inactivation of I to,s and I K,slow are very similar , both in terms of the steepness ( k values) of the curves and the relative amplitudes (≈35%, V 1/2 = −73 mV, and ≈65%, V 1/2 = −19 mV) of the two components. The finding of two components of steady state inactivation suggest the presence of two populations of channels contributing to I K,slow (and I to,s ) or, alternatively, the complex gating of a single population of I K,slow (I to,s ) channels (see discussion ). To examine the time dependences of recovery from steady state inactivation of I to,f , I to,s , and I K,slow , cells were first depolarized to +50 mV for 9.5 s to inactivate the currents (longer depolarizations did not lead to further inactivation), subsequently hyperpolarized to −70 mV for varying times ranging from 10 to 9,064 ms, and finally stepped to +50 mV (to activate the currents and assess the extent of recovery); the protocol is illustrated in Fig. 10 . Experiments were completed on randomly selected myocytes, as well as on cells isolated from the left ventricular apex and septum (B). Typical records obtained from cells isolated from the apex and septum are illustrated in Fig. 10 , A and B. For the randomly selected and the apex cells, the decay phases of the currents evoked at +50 mV after each recovery period were fitted to the sum of two exponentials to provide I to,f and I K,slow . For cells isolated from the septum, the amplitudes of I to,s , I to,f (when present), and I K,slow were determined from (double or triple) exponential fits to the decay phases of the currents evoked at +50 mV after each recovery period; note that for the cell illustrated in Fig. 10 B, I to,f (as well as I to,s ) is present. The amplitudes of I to,f , I to,s , and I K,slow after each recovery period were then normalized to their respective maximal current amplitudes (in the same cell) evoked after the 9.5-s recovery time. Mean (± SEM) normalized I to,f , I to,s , and I K,slow amplitudes are plotted as a function of recovery time in Fig. 10 C; the continuous lines represent the best single exponential fits to the averaged data. The mean normalized recovery data for I to,f (in left ventricular apex and septum cells) are well described by a single exponential characterized by a time constant of 27 ms ; note that the recovery data for cells isolated from the septum and apex are indistinguishable. The mean normalized recovery data for I to,s and I K,slow also follow a monoexponential time course , and the recovery data are well described by single exponentials with time constants of 1,298 (I to,s ) and 1,079 (I K,slow ) ms. Thus, in addition to differences in inactivation rates, I to,f and I to,s also recover from steady state inactivation at markedly different rates . The results presented here demonstrate the presence of four kinetically and pharmacologically distinct Ca 2+ -independent, voltage-gated K + currents in isolated adult mouse ventricular myocytes: I to,f , I to,s , I K,slow , and I ss . Although I K,slow and I ss were found in all ( n = 132) adult mouse ventricular myocytes studied, I to,f and I to,s were not. In randomly selected mouse ventricular cells, I to,f was evident in the majority (65 of 72, ≈90%) of the cells, whereas I to,s was identified in only 7 of 72 (≈10%) cells; i.e., the 7 cells lacking I to,f . Importantly, the densities and the properties of I K,slow and I ss in I to,s -expressing cells were indistinguishable from the corresponding currents in cells with I to,f . Subsequent experiments revealed regional differences in I to,f and I to,s expression in mouse left ventricles: I to,f was identified in all cells ( n = 35) isolated from the apex and I to,s was not detected in these cells ( n = 35); in the septum, by contrast, all cells expressed I to,s ( n = 28), and in the majority (22 of 28, 80%) of cells, I to,f was also present. The density of I to,f (mean ± SEM at +40 mV = 6.8 ± 0.5 pA/pF, n = 22) in septum cells, however, is significantly ( P < 0.001) lower than I to,f density in cells from the apex (mean ± SEM at +40 mV = 34.6 ± 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, I to,f , I to,s , and I K,slow were also distinguished here by marked differences in the rates of recovery (from inactivation), as well as differential sensitivities to 4-AP, TEA, and HpTx-3; none of the currents in mouse ventricular myocytes was found to be sensitive to the dendrotoxins. The absolute amplitudes of the individual current components varied among cells and, in all randomly selected cells with I to,f ( n = 65) and in cells from the apex ( n = 35), the density of I ss was substantially less than the density of either I to,f or I K,slow (Tables I and II ). On average, the ratio of current densities in these (randomly selected and apex) cells was 5–6:3:1 for I to,f , I K,slow , and I ss , respectively. In the I to,s -expressing cells from the septum, peak outward K + current densities are significantly ( P < 0.001) lower (in cells with and without I to,f ) than the peak outward current density in cells from the apex (Table II ), although no differences in cell sizes or input resistances were evident when these two groups of cells were compared. The mean ± SEM I K,slow density is also significantly ( P < 0.001) lower in septum than in apex cells (Table II ). There were no significant differences, however, in mean ± SEM I ss densities among the various populations of adult mouse ventricular (i.e., randomly selected) cells from the apex or cells from the septum (both the cells with and without I to,f ; Tables I and II ). On average, the ratio of current densities in I to,s -expressing cells was 1:2:1 for I to,s , I K,slow , and I ss , respectively, in cells lacking I to,f and 1:1:2:0.7 for I to,f , I to,s , I K,slow , and I ss , respectively, for the cells with I to,f (Table II ). Functionally, I to,f , I to,s , and I K,slow underlie the peak outward currents in all mouse ventricular cells ; since activation is slow, however, I ss does not contribute appreciably to the peak. Rather, I ss , together with I K,slow , determines current amplitude at times late after the onset of depolarization(s), whereas I to,f and I to,s , which inactivate rapidly (τ decay ≈ 60 and 200 ms, respectively) also do not contribute to the late currents. The fact that peak current densities are significantly lower in I to,s - than I to,f -expressing cells and that I K,slow densities are similar suggests that action potentials likely are broader in I to,s -expressing adult mouse ventricular myocytes than in cells lacking I to,s and expressing I to,f . The finding that I to,f and I to,s are differentially distributed also suggests that there will be differences in action potential waveforms in cells isolated from different regions of the ventricles. In addition, the marked differences in the rates of inactivation and recovery from inactivation for I to,f and I to,s suggest regional differences in rate-dependent variations in action potential waveforms. Further experiments will be necessary to test these hypotheses directly. Several previous studies have examined outward K + currents in adult mouse ventricular cells, and have been focussed primarily on I to,f . The time- and voltage-dependent properties of I to,f described in these studies are quite similar to those reported here, in that current activation and inactivation are rapid. The results presented here also show that mouse ventricular I to,f recovers from steady state inactivation very rapidly (τ = 27 ms), and the currents resemble those of I to,f in a variety of other species . The slowly inactivating outward K + current, I K,slow , has also been identified previously in adult mouse ventricular myocytes . The mean I K,slow inactivation time constant reported in these studies was 595 ± 29.3 ms ( n = 7), a value that is considerably smaller than the time constant (mean ≈1,200 ms, n = 132) determined here for I K,slow . The reason for the discrepancy between the absolute values of these time constants is unclear. Similar to previous findings , however, I K,slow is highly sensitive to 4-AP. A current sensitive to micromolar concentrations of 4-AP and referred to as I sus (for sustained), has also been described by Fiset et al. . Because I sus was measured at the end of 500-ms voltage steps, however, it almost certainly reflects the sum of I ss and I K,slow . In contrast to I to,f and I K,slow , neither I to,s nor I ss has been characterized previously in adult mouse ventricular myocytes. Interestingly, however, I to,s is similar to the novel current recently described in ventricular myocytes isolated from adult Kv4.2W362F-expressing transgenic mice, in which I to,f is eliminated . The time constants of inactivation of the novel current and I to,s are similar, and preliminary experiments suggest that the pharmacological properties of the two currents are also similar (H. Xu and J.M. Nerbonne, unpublished observations). Thus, it seems reasonable to suggest that I to,s is the novel current seen in the Kv4.2W362F transgenics and, further, that the functional expression of this conductance pathway is influenced, and perhaps regulated, by I to,f expression. Further experiments will be necessary to test this hypothesis directly. Several recent studies focused on identifying the molecular correlates of cardiac transient outward K + currents have provided considerable evidence supporting the hypothesis that α subunits of the Kv4 subfamily underlie I to,f . In mouse heart, for example, it has been demonstrated that I to,f is selectively eliminated in both ventricular and atrial myocytes isolated from transgenic animals expressing the dominant negative construct, Kv4.2W362F . The finding that the novel transient outward current (i.e., I to,s ) is expressed in all transgenic cells lacking I to,f , in contrast, suggests that Kv4 α subunits do not contribute to I to,s . The slow kinetics of inactivation and recovery from inactivation of I to,s , however, are reminiscent of the properties of another Kv α subunit, Kv1.4 , that generates slowly decaying transient K + currents when expressed in heterologous systems . In addition, it was reported recently that regional differences in the properties of the transient outward K + currents in ferret left ventricular epicardial and endocardial myocytes are correlated with differences in the expression of Kv4.2/Kv4.3 (epicardium, fast I to ) and Kv1.4 (endocardium, slow I to ) . Although these observations make it tempting to speculate that Kv1.4 underlies mouse ventricular I to,s , it has recently been reported that targeted deletion of the Kv1.4 gene does not affect the membrane properties of adult mouse ventricular myocytes . In these experiments, however, mouse ventricular myocytes were randomly dispersed and a rather small number of cells was studied. It is certainly possible, therefore, that the small subset of cells expected to be affected by elimination of Kv1.4 (if indeed this subunit underlies I to,s ) might not have been identified. Additional experiments aimed at examining regional differences in the K + currents expressed in the Kv1.4 knockout mice and the molecular correlate(s) of I to,s will certainly be of interest. In addition, two components of steady state inactivation of I to,s were identified in the experiments here. These could reflect two (molecularly) distinct populations of I to,s channels or, alternatively, the complex gating of a single population of I to,s channels. Further experiments focused on resolving this question will be of interest. A dominant negative strategy was also exploited recently in the generation of transgenic mice expressing a truncated Kv1.1, Kv1 . 1N206Tag . Electrophysiological experiments revealed that the 4-AP–sensitive, slowly decaying outward K + current I K,slow was selectively attenuated in ventricular myocytes isolated from Kv1 . 1N206Tag -expressing mice . These observations, together with the sensitivity of I K,slow to 4-AP, suggest that a member of the Kv1 subfamily, likely Kv1.5, underlies I K,slow . Interestingly, the experiments here have revealed the presence of two components of steady state inactivation of I K,slow . The finding of two components of steady state inactivation could reflect the fact that there are two (molecularly) distinct populations of K + channels contributing to I K,slow or, alternatively, the complex gating of a single population of I K,slow channels. It will be of interest to pursue further studies on ventricular myocytes isolated from Kv1 . 1N206Tag -expressing animals to determine whether one (or both) component of steady state inactivation is reduced by Kv1 . 1N206Tag expression. Further experiments will also be necessary to define the molecular correlate of the slowly activating and noninactivating component of the mouse ventricular K + currents, I ss . | Review | biomedical | en | 0.999999 |
10228182 | Among the different types of neuronal voltage-gated calcium channels, L-type channels play a specific role in regulating activity-dependent gene expression , neuronal survival and differentiation , and some forms of synaptic plasticity . Multiple functionally and structurally different neuronal L-type channels have been described . In addition to classical cardiac-type channels, anomalous L-type channels, which reopen upon repolarization to resting potentials after a depolarization, have been reported in cerebellar, hippocampal, and sensory neurons . The probability of reopening of these L-type channels is voltage dependent, increasing with increasing voltage of the previous depolarization. Different authors have proposed and supported different mechanisms accounting for voltage-dependent reopenings of L-type channels. Forti and Pietrobon have proposed that long reopenings of anomalous L-type channels are a manifestation of a voltage- dependent equilibrium between gating modes, whereby increasing voltage drives the channel from a short- to a long-opening mode , and also increases the occupancy of a closed state outside the activation pathway, which is connected to the open state through a voltage-dependent transition within each mode. This additional closed state accounts for both the delay with which long openings occur upon repolarization of the membrane and the anomalous voltage dependence of the mean open/closed times and of the open probability, found by the same authors . Alternatively, reopenings have been interpreted as a manifestation of recovery from voltage- and/or current-dependent inactivation , resulting from voltage-dependent block of the channel pore by a positively charged cytoplasmic particle . Kavalali and Plummer have proposed that reopenings reflect a particular form of voltage-dependent potentiation (LVP) in which the conditioning depolarization essentially reduces the voltage necessary to activate the channel. The different interpretations might reflect real differences in the L-type channels under study or simply derive from the emphasis of different aspects of the functional properties of essentially similar anomalous L-type channels. The data presented in this paper favor the second hypothesis. As in most other neurons, the high-voltage activated whole-cell calcium current of embryonic and neonatal motoneurons can be dissected into four (L-, N-, P-, and R-type) pharmacological components . It is not known whether motoneurons express anomalous L-type channels. In the only single channel characterization of calcium channels in motoneurons , a classical L-type channel has been described. Here we show that embryonic rat spinal motoneurons express L-type channels that reopen at negative repolarization voltages and display anomalous gating properties similar to those of anomalous L-type channels of cerebellar granule cells. We have investigated the mechanism giving rise to the anomalous gating in motoneurons. Our data are consistent with the model proposed by Forti and Pietrobon . They are also consistent with reversible block of the open pore by a positively charged cytoplasmic particle, but exclude block by diffusible ions, and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels. Spinal motoneurons from embryonic day 15 (E15) Wistar rat embryos were grown in primary culture after purification by a two-step metrizamide-panning method according to the procedure of Camu et al. . In brief, ventral spinal cords were dissociated after trypsin digestion, and centrifuged over 6.5% metrizamide (Serva) cushions to eliminate the four-plate cells, which are dense enough to sediment through the cushion. The large cells were further enriched by immunopanning on Petri dishes coated with the IgG-192 antibody specific for the p75 neurotrophin receptor, which is specifically expressed by motoneurons at this stage. The hybridoma was generously provided by Dr. C.E. Henderson (University Mediterrane, Marseille, France). Routinely, 90% of purified neurons express p75 immunoreactivity . The cells were plated on polyornithine- ( Sigma Chemical Co. ) and laminin- ( GIBCO BRL ) coated glass coverslips, and cultured in Dulbecco's modified Eagle medium ( GIBCO BRL ) supplemented with 17 mM glucose, 0.87 μM insulin, 0.99 mM putrescine, 0.93 μM sodium selenite, 1.32 μM transferrin, 0.19 μM progesterone, 0.51 μM triiodothyronine, 0.45 μM tiroxine, 1.32 μM BSA (all purchased from Sigma Chemical Co. ), 2 mM glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all purchased from GIBCO BRL ). 12 h after plating, the culture medium was supplemented with 25% muscle-conditioned medium, obtained from myotube cultures of newborn rat. Experiments were performed on motoneurons kept in culture from 1 to 5 d. Single channel patch-clamp recordings followed standard techniques . Currents were recorded with a DAGAN 3900 patch-clamp amplifier, low-pass filtered at 1 kHz (−3 dB; eight-pole Bessel filter), sampled at 5 kHz and stored for later analysis on a PDP-11/73 computer. Experiments were performed at room temperature (21–25°C). Linear leak and capacitative currents were digitally subtracted from all records used for analysis. Current amplitude histograms were obtained from the data directly, with bin width equal to our maximal resolution (323.6 points/pA). For display, each histogram was normalized to the value of the zero current peak. Open probability, P o , was computed by measuring the average current in a given single channel current record and dividing it by the unitary single channel current. Open-channel current amplitudes were measured by manually fitting cursors to well-resolved channel openings. A channel opening or closure was detected when more than one sampling point crossed a discriminator line at 50% of the elementary current. Histograms of open and closed times were fitted with sums of decaying exponentials. The best fit was determined by maximum likelihood maximization and the best minimum number of exponential components was determined by the maximum likelihood ratio test . Log binning and fitting of the binned distributions were done as described by McManus et al. and Sigworth and Sine . Openings occurring with a delay of more than one sampling point after repolarization of the membrane at −80 or −60 mV were considered as reopenings. In the measurement of the fraction of traces with reopenings, reopenings were detected using a discriminator line at 33% of the elementary current, to decrease the number of missed short reopenings. To calculate the fraction of traces with long and short reopenings, we used a discriminating open time value, calculated from the double exponential open time histogram as the open time that equalized the number of openings of the fast exponential component falsely assigned as long openings and the number of openings of the slow exponential component falsely assigned as short openings . Reopenings of duration longer than this value were considered as long reopenings and those of shorter duration as short reopenings. The pipette solution contained (mM) 90 BaCl 2 , 10 TEACl, 15 CsCl, 10 HEPES, pH 7.4 with TEAOH. The bath solution was (mM) 140 K-gluconate, 5 EGTA, 35 l -glucose, 10 HEPES, pH 7.4 with KOH. The high-potassium bath solution was used to zero the membrane potential outside the patch. The dihydropyridine agonist (+)-(S)-202-791 (gift from Dr. Hof, Sandoz Co., Basel, Switzerland) was added (1 μM) to the bath solution in most recordings. Liquid junction potential at the pipette tip was +12 mV (pipette positive), and this value should be subtracted to all voltages to obtain the correct values of membrane potentials in cell attached recordings . Figs. 1 and 2 show that embryonic rat spinal motoneurons express L-type channels which reopen after repolarization of the membrane and display anomalous gating properties similar to those of anomalous L-type channels of cerebellar granule cells . The single channel current recordings in Figures 1 and 2 were obtained from cell-attached membrane patches of rat spinal motoneurons in primary culture, which contained only one channel. The membrane was held at −80 mV and depolarized to four different voltages for 724 ms, every 4 seconds, in the presence of the dihydropyridine (DHP) 1 agonist (+)-(S)- 202-791 in the bath in Figure 1 and in its absence in Figure 2 . The representative current traces and the normalized current amplitude histograms from all traces with activity display the main unusual voltage- dependent properties of anomalous L-type channels. The first unusual property is represented by the reopenings occurring with some delay after repolarization of the membrane at −80 mV, a voltage well below the threshold for channel activation. Some of these reopenings are quite long, much longer than the openings of the same channel during the preceding depolarization . The second unusual property is represented by the voltage dependence of the open probability and of the open and closed times. The open probability ( P o ) does not increase with voltage in the usual sigmoidal manner, but reaches a maximum at +20 mV, and then decreases with increasing voltage, remaining low in the entire voltage range . In three single-channel patches, the maximal P o at +20 mV in the presence of DHP agonist was 0.12 ± 0.02. In each patch, average open probabilities were obtained from the traces with activity, without including nulls, which were a minority at each voltage (0–7% at +10 mV and 10–20% at +40 mV). In the absence of DHP agonist, the activity of anomalous L-type channels is characterized by brief, mostly unresolved and infrequent openings and by an extremely low open probability at all voltages: the maximal value of P o was 0.024 in the single channel patch of Fig. 2 . Kinetic analysis of the open and closed time histograms reveals that the low open probability and its anomalous voltage dependence are due to the anomalous voltage dependence of both open and closed time constants. As shown in Fig. 3 , the time constants of the two exponential components best fitting open time histograms both decrease with increasing voltage, and the two larger time constants of the three exponential components best fitting closed time histograms decrease with voltage up to +20 mV, and then start to increase . Interestingly, the contribution of the slow exponential component in the open time histograms increases with increasing voltage, with a symmetrical decrease of the fast component. Thanks to these anomalous gating properties, anomalous L-type channels in the presence of DHP agonist could be easily distinguished from the other L- and non–L-type channels of rat spinal motoneurons. We have found that rat spinal motoneurons coexpress, together with the anomalous L-type channels characterized in this study, two additional DHP-sensitive channels, one similar to cardiac L-type channels and the other inactivating quite rapidly . They can be distinguished from L-type channels with anomalous gating on the basis of their larger unitary current and conductance (24 vs. 20 pS), their larger mean open time and open probability at V > +20 mV (not decreasing with increasing voltage), and the complete absence of reopenings. Moreover, rat spinal motoneurons express several different DHP-insensitive calcium channels, including two channels sharing the same conductance of 20 pS but differing in inactivation and pharmacological properties . In this study, we have investigated the mechanism giving rise to the unusual voltage-dependent properties of anomalous L-type channels of rat spinal motoneurons. To discriminate between different mechanisms, it was essential to be able to study the activity of a single anomalous channel during both the depolarization and repolarization periods. Since anomalous L-type channels represent only a small fraction of the different types of calcium channels with similar conductance expressed in motoneurons and patches with only one anomalous L-type channel were very rare, and since, in addition, the open probability of single anomalous L-type channels in the absence of DHP agonist is extremely low , a property that they share with the more abundant inactivating L-type channel of 24 pS, it was necessary to prolong the openings of L-type channels with a DHP agonist to be sure that only one anomalous L-type channel was present in the patch. The comparison between Figs. 1 and 2 shows that the peculiar voltage-dependent properties of anomalous L-type channels above described are essentially similar with or without agonist, as previously shown in cerebellar granule cells . It has been proposed that reopenings of anomalous L-type channels reflect recovery from voltage- and/or current-dependent inactivation . If this interpretation is correct, then, in an experiment in which the membrane is depolarized at increasingly positive voltages, one should find a correlation between the extent of inactivation of single anomalous L-type channels during the depolarization and the fraction of traces with reopenings upon repolarization. Fig. 4 shows that such a correlation is absent. The ensemble average currents from a patch containing a single anomalous L-type channel in Fig. 4 A shows a lack of inactivation during long depolarizations at positive voltages (+10 to +40 mV) elicited from quite negative holding potentials (−80 mV). In the same voltage range, the fraction of traces with reopenings at −80 mV of the same channel increased as shown in Fig. 4 B (from 0 to 62%). Similar results were obtained for single anomalous L-type channels in cerebellar granule cells (Forti and Pietrobon, unpublished observations). Thus, the previously reported absence of inactivation of cerebellar anomalous L-type channels during depolarizations effective in inducing reopenings cannot be ascribed to the relatively depolarized holding potentials, as recently suggested , but appears as a general distinctive property of anomalous L-type channels. As already pointed out, after long depolarizations, both short and long reopenings at −80 mV could be observed. The existence of two clearly different open states, one short and the other long lasting, is even more evident if one analyzes the reopenings at −60 mV after a 400-ms long depolarization to +40 mV . The recordings in Fig. 5 were obtained from a cell-attached patch containing only one anomalous L-type channel. The open time histogram of reopenings at −60 mV required two exponential components with time constants of 1.8 and 29 ms for best fit according to the maximum likelihood criterion. Strikingly, both open time constants were larger (1.6 ± 0.1 and 26 ± 3 ms, n = 5) than those measured for the anomalous L-type channel during the predepolarization at +40 mV . A common feature of the different single L-type channels described so far is their voltage-dependent modal gating, whereby increasing voltage progressively drives the channels from a short-opening mode of activity (mode 1), prevailing at low voltages, to a long-opening mode (mode 2) prevailing at high positive voltages . Forti and Pietrobon proposed that the anomalous gating arises from the presence of a nonadsorbing closed state outside the activation pathway connected to the open state through a voltage-dependent transition within each mode, and used the simplified kinetic scheme of Fig. 6 to explain, at least qualitatively, the peculiar properties of cerebellar anomalous L-type channels. In this kinetic model, the individual open and closed states underlying the two modes within the two boxes are lumped together and connected by single voltage-dependent forward (k f ) and backward (k b ) rate constants. To account for the anomalous voltage dependence of both open and closed times , the open states within each mode (O, O*) are connected to a closed state outside the activation pathway (C b , C* b ), and the rate constants α, α* for entry into the closed states C b in mode 1 and C* b in mode 2 are assumed to increase with voltage, while the rate constants β, β* for exit from these closed states are assumed to decrease with voltage. Although the kinetic scheme in Fig. 6 is likely an oversimplification, we will use it here as a useful conceptual framework for the analysis and discussion of our data on anomalous L-type channels of motoneurons. A possible interpretation of the presence of both short and long reopenings at negative voltages is that they represent reopenings of the channel in either mode 1 (from C b ) or mode 2 (from C* b ), respectively. If this interpretation is correct, then any intervention that changes the probability of finding the channel in mode 2 at the end of the depolarization should change the relative proportion of long with respect to short reopenings, owing to the change in the relative probability of finding the channel in C* b with respect to C b when the membrane is repolarized. The probability of finding the channel in the long-opening mode at the end of the depolarization can be changed by either changing the length or the amplitude of the depolarization. One expects that if the depolarization is shortened the relative proportion of long with respect to short reopenings should decrease. Indeed, Fig. 5 shows that when the depolarization was shortened from 400 to 50 ms, most of the reopenings of the single anomalous channel in the patch became short. The long reopenings were too few to define the second slower component in the open time histogram of the reopenings. The histogram was best fitted by a single exponential with a time constant of 2 ms, quite similar to the time constant of the fast component best fitting the histogram of reopenings of the same channel after 400 ms. In three single channel patches, long reopenings were on average 40 ± 1% of the total number of reopenings after a 400-ms long depolarization, and decreased to 7 ± 4% of the total number of reopenings when the depolarization was shortened to 50 ms . In one single channel patch, after shortening the depolarization at +40 mV from 400 to 50 ms, the voltage was increased to +150 mV keeping the duration constant at 50 ms. The long reopenings decreased from 38 to 12% of the total number of reopenings when the moderate depolarization was shortened, and increased again to 36% of the total number of reopenings when the amplitude of the short depolarization was increased. The relative increase of long with respect to short reopenings with increasing length and amplitude of the previous depolarization is consistent with the interpretation that short and long reopenings are associated with two different gating modes of the channel (mode 1 and mode 2) and with the existence of a voltage-dependent equilibrium between the gating modes whereby the probability of the long-opening mode (mode 2) increases with increasing voltage. Consistent with this interpretation is also the finding that the probability of (long + short) reopenings, which depends on the probability of finding the channel in either C b or C* b at the end of the depolarization, was not much affected by changing the length of the depolarization. In three single-channel patches, the probability of reopenings changed from 63 ± 7% after 400 ms at +40 mV to 50 ± 5% after 50 ms at the same voltage , but the change was not statistically significant ( P < 0.2). The model in Fig. 6 predicts that changing the length of the depolarization should produce mirror changes in the probabilities of long and short reopenings, with a consequent unchanged probability of (long + short) reopenings, if the intrinsic probabilities of C b and C* b within the two individual modes were identical and as long as the rate constants of the transitions to C b and C* b were fast with respect to the duration of the depolarization. As a consequence of the relative increase of long with respect to short reopenings with increasing length of the depolarization, the peak ensemble average current at −60 mV was larger and decayed more slowly after the longer depolarization . In three single channel patches, the average time constant of decay of the ensemble current at −60 mV changed from 48 ± 1 ms after the long depolarization to 9.9 ± 5 ms after the short depolarization. On average, the ratio of the peak average currents at −60 mV after short and long predepolarizations was 0.49 ± 0.07. The ensemble averages at −60 mV after both long and short depolarizations showed a clear rising phase, which originates from the delay with which most reopenings occurred after the repolarization. The time constant of the rising phase after short depolarizations (1.5 ± 0.3 ms) was slightly smaller than that after long depolarizations (2.6 ± 0.5 ms), but the difference did not reach statistical significance ( P < 0.1). Fig. 8 shows directly that long moderate depolarizations are able to drive anomalous L-type channels from a short- into a long-opening mode. The unitary current recordings in Fig. 8 A were obtained from a patch containing a single anomalous L-type channel, in an experiment in which after 400 ms at +30 mV the membrane was repolarized at −10 mV, a voltage just above the threshold for channel activation. Control depolarizations to −10 mV for 800 ms were alternated with the prepulse protocol. In the large majority of control depolarizations at −10 mV (in 98 of 102 traces with openings), the unitary activity was characterized by relatively short openings and long closings and a low open probability, as shown by the first five representative traces in Fig. 8 A (right). The last trace represents a small minority of depolarizations (4 of 102 active traces) in which the channel shifted to a different mode of activity, characterized by long-lasting bursts with longer openings and shorter closings and by a much larger open probability. The representative traces and the ensemble average current of Fig. 8 A (left) show that a preceding depolarization to +30 mV for 400 ms increased the probability of observing the long opening mode, with a consequent potentiation of the average current at −10 mV. The fraction of active traces with the long opening mode increased from 4% in control depolarizations to 35% after 400 ms at +30 mV. These fractions were calculated using a discriminating open probability value of 0.1 to separate the traces with the long opening mode from those with activity similar to that in control. Fig. 8 , B–D, shows that, while the open time histogram of all traces at −10 mV after the prepulse required two exponential components with time constants of 1.7 and 9.1 ms for best fit, the open time histogram of the traces with P o < 0.1 could be best fitted by a single exponential with a time constant of 1.6 ms, similar to that of the fast component in the overall histogram and to the time constant obtained from the best fit of the open time histogram of control traces (excluding the four traces with the mode shift having P o > 0.1). The average current at −10 mV after the predepolarization shows a clear rising phase, and after reaching a maximum value, slowly decays towards the control value. On average, the time constant of decay was 177 ± 28 ms ( n = 4). The decay of the potentiated current should mainly reflect the kinetics of return of the channel from the long-opening mode to the short-opening mode prevailing in control. The slower decay of the average current at −10 mV with respect to that at −60 mV after the depolarization is consistent with the voltage dependence of k b , whereby k b decreases with increasing voltage . The rising phase of the current at −10 mV is clearly slower than the rising phase of the current at −60 mV after a similar prepulse , indicating that the rate of reopening from the closed states accessed during the depolarization (C b and C* b ) increases with more negative repolarization voltages. On average, the time constant of the rising phase increased from 2.6 ± 0.3 ms ( n = 6) at −60 mV to 11 ± 2 ms ( n = 4) at −10 mV. This finding is consistent with and supports the voltage dependence of the rate constants of exit from the closed states C b and C* b in the model in Fig. 6 , whereby β and β* decrease with increasing voltage. Consistent with this voltage dependence is also the peculiar lengthening of the closed times with increasing voltage of the depolarization . The two open time constants, obtained from the biexponential open time histogram at −10 mV were both smaller than those obtained for the reopenings at −60 mV after a similar depolarization . On the other hand, the open time constants at both −10 and −60 mV were larger than those obtained from the histogram of open times during the preceding depolarization . In the voltage range −60 to +40 mV, the open time constants decreased as shown in Fig. 9 A. This anomalous voltage dependence of the open times supports the existence, within each mode, of a closed state outside the activation pathway to which the open state is connected through a transition whose rate constant increases with increasing voltage. Accordingly, in Fig. 6 , the open states within each mode (O, O*) are connected to a closed state outside the activation pathway (C b , C* b ), and the rate constants α, α* for entry into these closed states increase with increasing voltage. Our interpretation that the fast and slow components in the open-time histograms reflect sojourns in the open states of modes 1 and 2, respectively, is further supported by the finding that, as already pointed out, the contribution of the slow exponential component in the open time histograms increases with increasing depolarization voltage, with a symmetrical decrease of the fast component . At voltages higher than +20 mV, the two open time constants become similar . At these positive voltages, the anomalous L-type channel in mode 2 opens only for brief times, thus explaining the absence of bursts of activity with long openings during depolarizations effective in inducing the change to the long-opening mode as seen on repolarization . One predicts that the potentiation of the anomalous L-type current by positive depolarizations should decrease with increasing repolarization voltage and there should be no potentiation of the current at repolarization voltages higher than +20 mV. Fig. 9 B shows that, in a single channel patch, the unitary activity of an anomalous L-type channel at +20 mV after a predepolarization to +40 mV for 400 ms was hardly distinguishable from that in control depolarizations at +20 mV. As a result, the same depolarization that produced a robust potentiation of the average current at −10 mV , hardly potentiated the current at +20 mV. Although Forti and Pietrobon did not speculate on the nature of C b and C* b , these states might correspond to either particular conformations of the channel or to open-pore blocked states, as proposed by Slesinger and Lansman . In the latter case, α, β and α*, β* would be the rate constants of voltage-dependent block and unblock of the channel in the short- and long-opening modes, respectively, and their voltage dependence would be consistent with block by a positively charged cytoplasmic particle . However, the result shown in Fig. 10 excludes any diffusible ion as the blocking particle. After excision in the K-gluconate/EGTA solution without divalents, the anomalous gating can persist unaltered for 40 min. Indeed, the data shown in Figs. 4 and 8 were derived after excision of the patch. The voltage-dependent induction of the long-opening mode in the inside-out patch in Fig. 8 shows that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels. In this study, we have shown that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range −60 to +40 mV, while closed times tend to increase with voltage at V > 20 mV. The open probability during depolarizing pulses is low at all voltages and has an anomalous bell-shaped voltage dependence. We have provided evidence that the two open states, leading to short and long reopenings, correspond to two gating modes of the channel, whose relative probability depends on voltage. Since the sojourn of the channel in both open states decreases with increasing voltage, the two open states must be connected to a closed state outside the activation pathway with a voltage-dependent transition whose rate constant increases with increasing voltage. The anomalous voltage dependence of the closed times suggests that the rate constant of reopening from this closed state decreases with increasing voltage. This voltage dependence leads to reopening of the channel upon repolarization and predicts a faster rate of reopening at more negative repolarization voltages, as found. According to our data, positive voltages favor both the transition from a short-opening gating mode (mode 1) to a long-opening mode (mode 2), and the occupancy of a closed state within each mode from which the channel reopens on repolarization, displaying short reopenings when it reopens from the closed state of mode 1 and long reopenings when it reopens from the closed state of mode 2 . The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of the closed states from which the channel reopens, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The properties of the first latency distribution of reopenings of anomalous L-type channels of mouse cerebellar granule cells, measured by Slesinger and Lansman as a function of repolarization voltage, are consistent with our conclusions. To explain their data, Slesinger and Lansman assumed the existence of a positively charged cytoplasmic blocking particle that may reversibly block the pore during the depolarization and be released upon repolarization at negative membrane potentials. According to this interpretation, C b and C* b would correspond to open-pore blocked states. Our finding that the anomalous gating persists after excision of the patch in divalent-free solution rules out block by a diffusible ion. It does not rule out block by a membrane-bound particle. The abrupt switch from the anomalous gating to the cardiac-type gating, observed by Forti and Pietrobon in one single channel patch, tends to exclude part of the channel as the blocking particle. An alternative interpretation, consistent with all the available data, is that C b and C* b represent conformational states of the channel and that voltage-dependent pore block is not involved in anomalous gating. Our data, both in motoneurons and cerebellar granule cells, do not show any apparent inactivation of single anomalous L-type channels during depolarizations effective in inducing reopenings, even though the kinetic scheme in Fig. 6 is clearly compatible with inactivation and actually might seem to be inconsistent with lack of inactivation. Simulations performed using the model in Fig. 6 show that, depending on the rate constants of the transitions between the states within each mode, the model can generate both noninactivating and inactivating currents during depolarizations effective in inducing reopenings (not shown). The extent of inactivation depends crucially on the ratio between forward and backward rate constants, and increases with increasing ratios above a certain value. Thus, the model predicts that macroscopic inactivation should become apparent at sufficiently high voltages. The fact that it was not apparent from our single channel ensemble averages in the range from +10 to +40 mV may signify that these voltages were not sufficiently high. Alternatively, a small extent of inactivation at high voltages might have been missed, due to the stochastic behavior in the records. Voltage-dependent potentiation is an interesting property shared by the different L-type channels described so far. This name refers to the ability of earlier depolarization to transiently increase macroscopic L-type current. In different cells, different voltage dependence, different time course, and different duration of voltage-dependent potentiation of L-type channels have been reported, reflecting different L-type channels and/or different modulatory mechanisms . The progressive shift towards a long-opening mode induced by increasing voltage can explain the voltage-dependent potentiation of L-type channels of cardiac and smooth muscle cells , as well as that of cardiac-type neuronal L-type channels . We have shown here and in our previous work that a voltage-dependent change in gating mode is also at the basis of voltage-dependent potentiation of anomalous L-type channels. The closed states giving rise to the anomalous gating confer to the voltage-dependent potentiation of anomalous L-type channels two specific properties. The transient increase of anomalous L-type current following a depolarization is delayed, and falls with increasing repolarization voltage to become almost zero at +20 mV, where the open probability in the two modes becomes similar. In similar experimental conditions, potentiation of cardiac L-type channels and brain α 1C is still observed at +20 mV, where the open probability of the two modes is still quite different. Anomalous L-type channels differ from cardiac-type channels also in the voltage range controlling the mode change, which is shifted to lower voltages for anomalous L-type channels . Furthermore, the potentiation lasts longer for anomalous L-type channels with respect to cardiac channels . An important specific property of anomalous L-type channels is that even very short or small depolarizations, insufficient to significantly shift the channels towards the long-opening mode, can induce short reopenings and thus transiently increase, for a brief time, the current upon repolarization. By delaying channel opening at resting potential, where the driving force is larger, the closed/blocked state from which anomalous L-type channels reopen provides a mechanism to maximize calcium influx after a transient membrane depolarization such as an action potential. With increasing duration and amplitude of the depolarization, the relative contribution of long reopenings, due to the mode shift, increases, leading to a potentiated and longer lasting transient increase of the current upon repolarization. The anomalous gating warrants maximal potentiation at resting potentials. One can predict that a presynaptic train of action potentials at high frequency may lead to a progressive increase of calcium influx through presynaptic anomalous L-type channels during the train, and/or may generate a large surge of calcium influx through postsynaptic anomalous L-type channels at the end of the train. Reopenings of L-type channels after single action potentials, with increasing open times at more negative repolarization voltages, and a progressive shift of the same channels to a long-opening gating mode during trains of action potentials have been observed in sensory neurons . Reopenings of calcium channels induced by single backpropagating action potentials have been observed also in dendrites of hippocampal cells . Most likely, anomalous L-type channels were involved in both cases. Kavalali and Plummer and Kavalali et al. have characterized L-type channels in hippocampal neurons with biophysical properties quite similar to those of anomalous L-type channels of cerebellar granule cells and motoneurons. The similarities include single channel current and conductance, low open probability, mean open times at +20 mV shorter than at −30 mV, potentiation after modest depolarizations (LVP) and lack of potentiation at repolarization voltages higher than +20 mV, slower decay of the potentiated current than that of cardiac L-type channels, and persistent activity in excised patches. Given their presence in cerebellar, hippocampal, sensory, and motor neurons, L-type channels with anomalous gating are probably widely expressed in the nervous system, while they are absent from cardiac and pituitary endocrine cells (our unpublished observations). The predicted capability of anomalous L-type channels to produce a delayed influx of calcium at resting potentials after previous neuronal electrical activity, whose extent depends on the duration and strength of such activity, makes them particularly suited to play a critical role in coupling transient neuronal activity with long-term changes in nervous system development and function. Indeed, several of these changes have been found to be specifically inhibited by dihydropyridine drugs (see introduction ). Calcium entry through anomalous L-type channels might play an important role also in neurotoxic pathogenesis, since these channels are probably still active under conditions of metabolic stress such as hypoxia, which would favor the voltage-dependent change in gating mode leading to potentiation of calcium influx. In addition, anomalous L-type channels may play a role in neuronal deterioration during aging . The molecular basis for the anomalous L-type channel is unknown. Its absence in pituitary endocrine cells, which express the α 1D subunit in large amounts , and, on the other hand, its abundance in rat cerebellar granule cells in primary culture, where the α 1D subunit is either not expressed or expressed in small amounts , suggest that, most likely, the pore-forming subunit of anomalous L-type channels is not α 1D . Since the α 1C subunit was detected as a major transcript in rat cerebellar granule cells in primary culture using degenerated oligonucleotide primer pairs under highly stringent conditions, and no new α 1 -related sequences were amplified under the same conditions , it appears likely that the anomalous L-type channel is related to the α 1C subunit. It remains to be established whether the anomalous behavior arises from an unknown splice variant of the α 1C subunit, from a particular subunit composition, or from an unknown type of modulation. | Other | biomedical | en | 0.999995 |
10228183 | P2X purinoceptors are ligand-gated cation channels that are activated by extracellular ATP and its analogues. These receptors exist in excitable and nonexcitable cells, including neurons, smooth and cardiac muscles, glands, astrocytes, microglia, and B lymphocytes . During the past few years, seven P2X purinoceptor subunits (P2X 1 –P2X 7 ) have been cloned . The P2X family has a distinctive motif for ligand-gated ion channels, with each subunit containing two hydrophobic transmembrane domains (M1 and M2) joined by a large intervening hydrophilic extracellular loop . The cDNA of each receptor is ∼2,000 bp in length and has a single open reading frame encoding ∼400 amino acids. A comparison of the amino acid sequences of the seven members shows an overall similarity of 35–50% . Dose–response analyses of the cloned receptors made with whole cell currents revealed a Hill coefficient larger than 1, suggesting that activation of the channel involves more than one agonist. This is consistent with experiments on the native receptors in PC12 cells and sensory neurons . Studies aimed at measuring the subunit stoichiometry predict that the naturally assembled form of P2X receptor channels contains three subunits . All of the P2X clones can be expressed in heterologous cells, such as HEK 293 cells and Xenopus oocytes. ATP is a potent agonist for all cloned P2X receptors, and the receptors are highly selective for ATP over most other adenosine derivatives. However, benzyl-ATP is 10-fold more potent than ATP in activating P2X 7 receptors . It is interesting to point out that α,β-methyl ATP is a poor agonist for the subtypes that do not show desensitization: P2X 2 , P2X 4 , P2X 5 , P2X 6 , and P2X 7 , but is a potent agonist for P2X 1 and P2X 3 receptors that do desensitize. Most studies of cloned P2X receptors have focused on the primary structure and pharmacology based on whole cell currents, while only a small amount of work has been done on the single channel properties. Single channel currents from P2X 1 receptors expressed in Xenopus oocytes were reported to have a mean amplitude of ∼2 pA at −140 mV and a chord conductance of 19 pS between −140 and −80 mV . The conductance for P2X 1 , P2X 2 , and P2X 4 channels expressed in Chinese hamster ovary cells were ∼18, 21, and 9 pS, respectively, at −100 mV with 150 mM extracellular NaCl, but the openings of P2X 3 were not resolved . To provide a firmer basis for further analysis of the P2X family, we have examined P2X 2 receptors at the single channel level. We have characterized the current–voltage (I–V) 1 relationships, cation selectivity of permeation, ATP sensitivity, proton modulation, and gating kinetics. P2X 2 receptors were expressed either in stably transfected human embryonic kidney 293 (HEK 293) cells or in Xenopus oocytes by mRNA injection . Since receptor expression is generally too high to obtain patches with only a single channel, we decreased the expression of the receptors in Xenopus oocytes by reducing the amount of mRNA to 25 ng, lowering the incubation temperature from 17° to 14°C, and shorting the incubation time to 16 h. For electrophysiological experiments, HEK 293 cells were cultured at 37°C for 1–2 d after passage. The medium for HEK 293 cells contained 90% DMEM/F12, 10% heat inactivated fetal calf serum, and 300 μg/ml geneticin (G418). The media were adjusted to pH 7.35 with NaOH and sterilized by filtration. The incubation medium (ND96) for Xenopus oocytes contained (mM): 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 , 5 HEPES, titrated to pH 7.5 with NaOH. All chemicals were purchased from Sigma Chemical Co. We made patch clamp recordings from HEK 293 cells 1–2 d after passage and from Xenopus oocytes 16 h after injecting mRNA. Single channel currents from outside-out patches and whole cell currents were recorded at room temperature . Recording pipettes, pulled from borosilicate glass (World Precision Instruments, Inc.) and coated with Sylgard, had resistances of 10–20 MΩ. For recording from HEK 293 cells, the pipette solution contained (mM): 140 NaF, 5 NaCl, 11 EGTA, 10 HEPES, pH 7.4. The bath solution and control perfusion solutions were the same and contained (mM): 145 NaCl, 2 KCl, 1 MgCl 2 , 1 CaCl 2 , 11 glucose, 10 HEPES, pH 7.4. For Xenopus oocytes, the pipette solution contained 90 mM NaF instead of 140 mM NaF and other components were the same as for HEK 293 cells; the bath and control perfusion solutions were the same as those used for HEK 293 cells except that they contained 100 instead of 145 mM NaCl. The patch perfusion solutions were the same as the bath solutions, except for modified divalent and ATP concentrations. Perfusate was driven by an ALA BPS-4 perfusion system ( ALA Scientific Instruments). To investigate the cation selectivity of the channels, we substituted different cations for Na + ion in the perfusate. To investigate the affinity of Na + for the channel pore, we varied the extracellular NaCl concentration without compensation by other ions, while the pipette solution was kept constant. The resulting change of ionic strength caused the development of small liquid junction potentials between the bulk solution and the perfusate. We calculated these potentials according to the Henderson equation . For solution exchanges from 100 to 150, 125, 100, 75, 50, and 25 mM NaCl, the junction potentials were −2.1, −1.2, 0, 2.5, 3.6, and 7.3 mV, respectively. Because these values are small compared with the holding potentials, we did not correct the membrane potential when we calculated the chord conductances. Currents were recorded with a patch clamp amplifier (AXOPATCH 200B; Axon Instruments ), and stored on videotape using a digital data recorder (VR-10A; Instrutech Corp .). The data were low-pass filtered at 5–20 kHz bandwidth (−3 dB) and digitized at sampling intervals of 0.025–0.1 ms using a LabView data acquisition program (National Instruments). The mean amplitudes of single channel currents were determined by all-points amplitude histograms that were fit to a sum of two Gaussian distributions. Chord conductances were calculated assuming a reversal potential of 0 mV. The excess open channel noise (σ ex ) was computed as the root mean square (rms) difference between the variances of the open channel current and the shut channel current . P o was defined as the ratio of open channel area ( A o ) to the total area ( A o + A c ) in the all-points amplitude histogram: 1 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{o}=\frac{A_{o}}{A_{o}+A_{c}}.\end{equation*}\end{document} This calculation is insensitive to short events. In our initial analysis, we treated the channel as having two amplitude classes, open and closed. All rapid gating events associated with the open channel were treated as noise. Essentially we defined open as “not closed.” Power spectra, S ( f ), were computed using the fast Fourier transform routine in LabView™. We used records of 50∼100-ms duration associated with open and closed states. The power spectrum of the excess noise was obtained by subtracting the spectrum of the closed state spectrum from that of the open state. The spectra were fit with the sum of a Lorentzian plus a constant: 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*}S(f)=\frac{S(0)}{1+(f/f_{c})^{2}}+S_{1}.\end{equation*}\end{document} The rms noise, σ L , from the Lorentzian component is: 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*}{\sigma}_{L}=\sqrt{\frac{{\pi}}{2}f_{c}S(0)}.\end{equation*}\end{document} The thermal (or Johnson) and shot noise contributions were calculated according to Defelice . The power spectra of thermal noise is white; i.e., it does not vary with frequency. Its value, S th , is given by: 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*}S_{th}=\frac{4k_{B}{\mathit{T}}}{R},\end{equation*}\end{document} where k B T is Boltzmann's constant times the absolute temperature, and R is the equivalent resistance of the open channel. The rms noise in a bandwidth f is given by: 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*}{\sigma}_{th}=\sqrt{\frac{4k_{B}Tf}{R}.}\end{equation*}\end{document} The power spectrum of shot noise, S sh , is also white and given by: 6 \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*}S_{sh}=2qi,\end{equation*}\end{document} where q is the charge of an elementary charge carrier, and i is the single channel current amplitude. The rms amplitude of shot noise over bandwidth f is: 7 \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*}{\sigma}_{sh}=\sqrt{2qif}.\end{equation*}\end{document} We tried to see if there were discrete subconductance levels making up the open channel “buzz mode” by using a maximum-point likelihood method (MPL; www.qub.buffalo.edu ) that use the Baum-Welch algorithm . While we did get convergence at four to six levels, these levels were not consistent among data from different patches, so at the present time we cannot confidently describe the substate structure. The single channel currents were idealized with a recursive Viterbi algorithm known as the “segmental k-means” algorithm . Idealization is dominated by the amplitude distribution, and therefore is essentially model independent. For simplicity, we used a two-state model for idealization: closed ↔ open (C ↔ O). The distributions of closed and open times were displayed as histograms with log distributed bin widths versus the square root of the event frequency . The mean open and closed times were simple averages from the idealized currents. The rate constants of state models were obtained by using the maximal interval likelihood method with corrections for missed events . We used two strategies to fit the data: (a) individual fitting (i.e., fitting the data sets from each experimental condition individually), and (b) global fitting (i.e., fitting a group of data sets obtained under different experimental conditions). The first method produces an independent set of rate constants for each condition, but suffers from poor identifiability: a given model may not have unique rate constants. Global fitting improves identifiability by using a model with fewer parameters. We did both kinds of analysis of the data, and the results were consistent between the two methods of analysis, but global fitting permitted fitting more complicated models. For simplicity, we will emphasize the results of global fitting across ATP concentrations at the same voltage, or across voltages at the same ATP concentration. When we globally fit data from different ATP concentrations, we assumed that the association rates were proportional to concentration [i.e., k ij = k ij (0)[ATP], where k ij (0) is an intrinsic rate constant at the specified voltage], while the other rates were assumed to be independent of ATP. When we performed global fitting on data from different voltages at same ATP concentration, the rates were assumed to be exponential functions of voltage; i.e., k ij = k ij (0)exp(− z δ ij V/ k B T ), where k ij (0) is the apparent rate constant at 0 mV and the specified ATP concentration, and z δ ij is the effective sensing charge. We used Akaike's asymptotic information criterion (AIC) to rank different kinetic models : 8 \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*}AIC=2[\;{ \,\substack{ ^{n} \\ {\sum} \\ _{j=1} }\, }\hspace{.5em}{\mathrm{log}}(maximum\;likelihood)-(number\;of\;free\;parameters\;of\;the\;model){\times}(number\;of\;data\;sets),]\end{equation*}\end{document} where j is the number of the data set. The model with a higher AIC is considered a better fit. We used Origin (Microcal Software, Inc.) and Scientist (MicroMath Scientific Software, Inc.) software to simulate and fit data. Fig. 1 A shows a typical single channel current, activated by 1.5 μM ATP at a membrane potential of −100 mV from a stably transfected HEK 293 cell. Channel openings appeared as flickery bursts with ill-defined conductance levels. There were a few clear closures and subconductance levels within a burst, but discrete levels could not be resolved from the all-points histogram . The spread of current levels was reflected by the much larger standard deviation of the open than the closed component, each of which could be fit reasonably well with a single Gaussian. In Fig. 1 B, the mean open current amplitude is 3.2 pA, equivalent to a chord conductance of 32 pS. The standard deviations of the open and closed histograms are 0.95 and 0.24 pA, respectively, so that the excess open channel noise σ ex is 0.92 pA; i.e., 29% of the mean current amplitude. Since the mean is clearly less than the peak current, we obtained a closer estimate of the maximal open channel current by measuring the mean of extreme values. Comparing the upper 5% of the two distributions, the peak amplitude and conductance were 4.3 pA and 43 pS, respectively, a closer estimate of the maximum ion flux. However, for convenience in discussing later results, unless specified otherwise, “channel current” and “conductance” will refer to the mean rather than the peak values. To further characterize the open channel noise, we followed the procedures of Sigworth for noise analysis using differential power spectra. We compared the power spectra of excess open channel fluctuations with the expected thermal and shot noise . The open channel spectrum was well fit with a Lorentzian with f c = 264 Hz, equivalent to a relaxation time of 0.62 ms, plus a constant. This noise is much larger than the expected thermal or shot noise, suggesting that the fluctuations most likely arise from rapid conformational changes in the channel. Fig. 2 A shows the single channel currents recorded from HEK 293 cells activated by 2 μM ATP at different holding potentials using outside-out patches with symmetrical Na + . The currents became small and noisy at positive holding potentials so that the unitary currents were not discernible. The single channel I–V curve exhibited a strong inward rectification similar to whole cell currents recorded under the same conditions . The rapid fluctuation of current in the “open” state was maintained at all holding potentials. We were unsuccessful in obtaining outside-out patches containing only a single P2X 2 channel when the receptor was expressed in HEK 293 cells. However, we were able to obtain patches with a single channel from Xenopus oocytes provided we carefully controlled the amount of mRNA, and the time and temperature of incubation. Fig. 4 A shows the single channel currents from an outside-out patch activated by different concentrations of ATP. As expected, increasing the concentration of ATP increased P o . The all-points histograms show that the average current and excess noise were independent of ATP concentration ( i = 3.5 pA and σ ex = 1.6 pA at −120 mV). Thus there is no indication that ATP is blocking the open channel. We calculated the probability of being open at each ATP concentration from the amplitude histograms using ratio of the open area to the total area . The open probability saturated when the ATP concentration reached 30 μM. The dose–response curve was fitted by the Hill equation with a Hill coefficient of 2.3, an EC 50 of 11.2 μM, and a maximal open probability of 0.61. The Hill coefficient and EC 50 are similar to those obtained from the dose–response curves of whole cell currents of our own data (not shown) and the literature , indicating that there are at least three subunits in a functional P2X 2 receptor ion channel. The data from this patch were very stable and used later in the comparison of kinetic models. To investigate the affinity of Na + for the open channel, we measured single channel amplitudes at holding potentials of −80, −100, −120, −140 mV for extracellular NaCl concentrations of 10, 25, 50, 75, 100, 125, and 150 mM. Fig. 5 shows single channel currents activated by 15 μM ATP at different extracellular Na + concentrations with a holding potential of −120 mV ( Xenopus oocyte). The amplitude increased with the concentration of NaCl but approached saturation at high Na + levels. Because the solutions were asymmetric across the patch, we calculated the conductance with the driving force as the difference between the holding potential and the Nernst potential. The single channel chord conductance, γ, calculated this way is plotted as a function of Na + concentration in Fig. 6 A. The conductance versus [NaCl] at each potential was well fit with the Michaelis-Menten equation : 9 \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*}{\gamma}=\frac{{\gamma}_{max}}{1+K_{s}/[NaCl]},\end{equation*}\end{document} yielding K s and γ max at each voltage. The equilibrium constant, K s , increased with depolarization . The relationship between K s and holding potential can be described by a Boltzmann equation for a single binding site: 10 \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_{s}(V)=K_{s}(0)exp\hspace{.167em}(-z{\delta}V/k_{B}T),\end{equation*}\end{document} where K s (0) is the dissociation constant at 0 mV, δ is the fractional electrical distance of the site from the extracellular surface, z is the valence of the permeating ion (1 in this case), and k B T is Boltzmann's constant times absolute temperature (∼25 mV at room temperature). The fitted values of K s (0) and δ were 148 mM and 0.21, respectively, so that a depolarization of 118 mV is required for an e-fold increase of K s . The Na + binding site appears to be ∼20% of the electrical distance from the extracellular surface, and is half saturated when exposed to 148 mM Na + at 0 mV. The maximal conductance, γ max , also increased with the potential as expected in a nearly linear part of the I–V curve . The P2X 2 receptor ion channel is a nonselective cation channel; however, the conductance is different for different cations. We measured the single channel currents at −120 mV from HEK 293 cells using outside-out patches with NaF as the intracellular solution, and LiCl, NaCl, KCl, CsCl, and RbCl as the extracellular solutions . From the currents obtained at −120 mV (Table I ), the selectivity was K + > Rb + > Cs + > Na + > Li + . Although currents carried by the different cations had the same flickering behavior, the excess open channel noise, σ ex , had a slightly different selectivity K + ≅ Rb + > Cs + > Na + > Li + . The relative noise, defined as σ ex / i , was Rb + ≅ Na + ≅ Cs + ≅ K + > Li + . The difference in selectivity of the relative noise for Li + suggests that it can affect the flickery kinetics. We compared σ ex with the thermal, σ th , and shot, σ sh , noise (Table I ). Again, σ th and σ sh were very small compared with σ ex , and the ratio (σ th + σ sh ) to σ ex ranged from 8 to 14% depending on the ions. The relative noise caused by the open channel fluctuations, when corrected for thermal and shot noise (σ 2 ex − σ 2 th − σ 2 sh ) 1/2 / i , followed the same cation sequence as σ ex / i . The effect of extracellular pH on the channel currents is illustrated in Fig. 8 A. Multiple-channel outside-out currents activated by 2 μM ATP increased ∼10-fold when pH was decreased from 8.3 to 6.8, and saturated with further decreases in pH . The fluctuations in these multichannel currents at higher pH appeared to be dominated by the overlap of independent channels, so that at pH 8.3, where the mean current is small, single channel events were visible. At pH 6.3, the current saturated and the frequency of fluctuations increased dramatically, apparently dominated by the open channel noise. As is clear from the rise time of the currents, the activation rate decreased with increasing pH, and the fall time remained constant . The potentiation of channel activity by protons is similar to the effect of increasing the ATP concentration, suggesting that protons may increase the affinity of the binding site for ATP. The pK a was ∼7.9 and the Hill coefficient was 2.5, again suggesting that there are more than two subunits in the channel. The results above and published studies on the effect of pH were based on whole cell or multi-channel recordings . To explore the possible effect of pH on gating and channel conductance, we examined the effect of pH on single channels. To obtain single channel activity from the stable cell lines, we exposed the patch to ATP for long times, so that run down reduced the number of active channels. Fig. 9 A shows these currents recorded at different values of extracellular pH. We measured the single channel amplitude and excess noise from the all-points amplitude histograms. The mean amplitude of the current was independent of pH, but the excess open channel noise increased with decreasing pH (Table II ). As visible in Fig. 9 A, the frequency of brief closures within open channel bursts appeared to increase as pH decreased. These interruptions were longer than the normal fast “flickery” behavior. It has been suggested that protons may block an open channel . To further characterize this phenomenon, we computed the power spectra of the open channel fluctuations , and fit them with a Lorentzian plus a constant (Eq. 3 ). The constant represents relaxations occurring at frequencies beyond our resolution. The fits are illustrated in Fig. 9 B as solid lines. The Lorentzian represents a two-state relaxation process whose characteristic time constant τ is related to the corner frequency, f c , by: 11 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}=\frac{1}{2{\pi}f_{c}}.\end{equation*}\end{document} Diffusional block of the open channel can be described as a two-state model (Scheme I), where O is the open state and C b is the protonated-blocked state. α and β are the blocking and unblocking rate constants. The relaxation time for this two-state process is related to the rate constants by: 12 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{1}{{\tau}}=2{\pi}f_{c}={\alpha}[H^{+}]+{\beta}.\end{equation*}\end{document} The prediction of proton block is that f c increases linearly with increasing proton concentration. However, our data show that f c decreased with increasing proton concentration . To further examine the possibility of proton block, we analyzed bursts kinetically using the maximum likelihood method with a two-state model. We fit the extracted α's and β's at different pH to an equation of the form: 13 \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*}{\alpha}={\alpha}_{0}[H^{+}]^{n},\end{equation*}\end{document} where α 0 = 224 μM −0.33 s −1 , n α = 0.33, β 0 = 1,493 μM −0.16 s −1 , and n β = 0.16. Since α and β are not directly proportional to the proton concentration, a single site model appears to be inappropriate. We speculate that we may be titrating several sites that display negative cooperativity. The effect of pH on the open channel is to modify the conformation of the channel rather than to provide a simple proton block. Table II summarizes the open channel properties at different pH. Remarkably, the effect of pH on the mean open channel current is negligible. To understand the kinetics of agonist binding and channel gating, we applied the maximum likelihood method to data from outside-out patches that were stable over time and ATP concentration . We began by fitting simple noncyclic models using the maximum-likelihood interval analysis and used AIC ranking to select a preferred model. The analysis was hierarchical in the sense that we fit portions of the reaction scheme under restricted conditions, and then merged these models to create a full description. The kinetic description required: (a) the number of closed and open states, (b) the connections between states, and (c) the values of the rate constants between the states and their dependence on concentration and voltage. The data was idealized into two classes: open and closed . We did not attempt to idealize the data making up the bulk of the flickery open channel activity since the amplitudes were uncertain, but instead defined open as a single conductance state possessing a lot of noise. The probability of a channel being open increased with ATP, as shown in Fig. 4 C. This could result from an increase in mean open time, a decrease in mean closed time, or a combination. Fig. 10 B shows the mean open and closed times calculated from idealized single channel currents, and plotted as a function of ATP concentration. The mean closed time dramatically decreased with the increase in ATP and saturated at 30 μM, while the mean open time was not affected by ATP. The results indicate that ATP controls the rate at which the channel opens, but not the rate at which it closes. Fig. 10 C shows the open- and closed-time histograms from idealized single channel currents induced by different ATP concentrations . The open-time histograms have two peaks and the closed-time histograms have at least three peaks at low concentration. When the ATP concentration was increased, the intermediate and long time constant peaks of the closed time distribution merged and only two peaks were visible. These results indicate that the channel has at least three closed and two open states. We made quantitative comparisons of various kinetic models to determine which model best described the behavior. The models were limited to three closed and two open states (of the same conductance), and at most 10 rate constants. These constraints proved necessary to obtain unique solutions during optimization. There are 98 unique models with that many states. Further constraints were imposed to simplify analysis. (a) We discarded models in which the unliganded states were open because we did not see any spontaneous openings in the absence of ATP. (b) Following traditional models for other ligand-gated channels, the closed states were connected so as to represent the binding of ATP. To evaluate the possible topologies, we used the program MSEARCH ( www.qub.buffalo.edu ) to compare the likelihood of all remaining models. The program evaluates all topologically unique models having a specified number of states of each conductance and optimizes the rate constants for each one. For this stage of the analysis, we used three data sets obtained at 5, 10, and 15 μM ATP. We calculated the likelihood of each model by adding the log-likelihoods from each concentration. This is more a test of the topology of the models than a test of the optimal values of the rate constants since the rate constants will change over concentration, but the connectivity won't. Fig. 11 shows the eight kinetic models that converged on all data sets within 100 iterations. They are listed in the order of AIC rank. To determine which model was best, we compared the log(maximum likelihood)s and AIC rankings (Table III ). The likelihoods of Models 1, 2, and 3 are the same, but Model 3 has two more parameters and, hence, a lower AIC rank. Models 1 and 2 have the same number of parameters, likelihood, and AIC rank, so we can not tell the difference between them. Model 7 has a larger likelihood than Models 1 and 2; however, its AIC ranking is much lower because of the increased number of parameters. Model 8 , which has a partially liganded open state, has the smallest likelihood and lowest AIC rank. When Models 1 and 2 were compared across concentration, they were indistinguishable and, for simplicity in what follows, we arbitrarily selected Model 1. In both models, state C 1 is unliganded, C 2 and C 3 are liganded, and O 4 and O 5 are open. k 12 and k 23 are the agonist association rates, k 21 and k 32 are the agonist dissociation rates, k 34 and k 35 are the channel opening rates, and k 43 and k 53 are the channel closing rates. The rate constants governing ATP binding and gating were solved by fitting across a range of ATP concentrations. Fig. 12 shows the rates from the model at bottom as a function of ATP concentration when the data from each concentration were fit independently. The association rates k 12 and k 23 showed a strong dependence on ATP concentration in the 5–20 μM range. However, when the ATP concentration was >20 μM, the rate constants appeared to saturate and the error limits on the parameters increased. A concentration-driven rate should not saturate, but there are a few explanations. First, there may be a concentration-independent state not contained in the model. Second, k 12 and k 23 approach k 35 at high ATP concentration, making k 35 rate limiting and rendering the optimizer incapable of properly solving the model. Third, if k 12 and k 23 are linearly proportional to concentration, then the intrinsic rate constants of both k 12 an k 23 are ∼2 × 10 7 M −1 s −1 , which is approaching the diffusion limit. We tested the first possibility by adding concentration-independent states to the model in Fig. 12 , but that did not prevent the association rates from saturating. We tested the identifiability of the model by simulating the model across concentrations (SIMU; www.qub.buffalo.edu ) and attempting to extract the rate constants using maximal interval likelihood. Fitting the simulated data, we found that the estimated rate constants also saturated (see below) so that the correct model is not identifiable with data from a single concentration. As far as the diffusion limit providing a true saturation, further experiments are required to test that prediction. However, we currently believe that the apparent saturation is an artifact caused by the lack of identifiability of the model at high concentrations. (Details of the test on the artifactual origin of saturating rates. We simulated data using Model 1 [Fig. 11 ], with the rate constants k 12 and k 23 increasing linearly with ATP in the 5–50 μM range. The intrinsic rate constants k 12 (0) and k 23 (0), obtained from the slope of k 12 and k 23 versus ATP from 5–30 μM , were 14 and 22 μM −1 s −1 , respectively. All other rate constants were made independent of ATP and set to values averaged across the data sets. We then analyzed the simulated data as if it were experimental data. The recovered rate constants were similar to the values used to simulate the data for ATP <20 μM. At higher ATP levels, however, the estimated values of k 12 saturated and k 21 even decreased. Large error limits also occurred in k 12 and k 21 at the high concentrations [Table IV ]. Thus, Model 1 [Fig. 11 ] cannot uniquely fit data at single high ATP concentration.) To improve identifiability, we fit the data simultaneously across all concentrations. Such global fitting makes the likelihood surface steeper . We assumed that the association rate constants were proportional to the ATP concentration, and the other rate constants were independent of ATP (see materials and methods ). This time, the rate constants derived from global fitting of simulated data were very close to the values used for simulation (Table V ). The results of global fitting to the experimental data are listed in Table VI . It is worth noting that the second ATP association rate constant, k 23 (0), is larger than the first, k 12 (0). This result shows that the binding sites are not independent, but that binding to one site modifies binding to the other. With independent sites, the association rate should decrease as the number of free sites decreases. The conclusion is quite model independent; for every model we tested, the association rates increased with proximity to the open state (see below). As ATP concentration increased, the three peaks in the closed time duration histograms became two , suggesting that at high ATP concentration, Model 1 could be simplified by removal of state C 1 . When we fit the kinetics of high concentrations of ATP by Model 1 and Model 1-1, the likelihoods were equal. Thus, at high ATP, k 12 gets so fast that C 1 is rarely occupied and Model 1-1 is sufficient to describe the kinetics. However, a large difference in maximum likelihoods arose when we fitted Models 1 and 1-1 to the data at low concentrations of ATP. Model 1-1 can well describe the kinetics of single channel currents of high ATP, but not low. Our model has only two binding steps. The fact that the P o curve has a Hill coefficient of 2.3 suggests that there are at least three binding sites in the P2X 2 channel. Since it is a homomer, this implies that three or more subunits are needed to form the channel. A more realistic model should have at least one additional partially liganded closed state . The rate constants from Model 1-2 are shown in Table VI . Again in this model, the first ATP binding step speeds up the second one. The transition rates near the open state are similar between Model 1 and Model 1-2. While Model 1-2 has two more free parameters than Model 1, it has 5.4 units higher likelihood so that Model 1-2 is preferred (see Table VIII ). The predicted P o as a function of ATP concentration is plotted in Fig. 4 C (⋄) and fit with the Hill equation with a Hill coefficient of 1.5, an EC 50 of 17.4 μM, and maximal P o of 0.74. However, compared with the experimental data, the EC 50 and maximal P o are too large and the Hill coefficient too small. These discrepancies can be reduced by connecting an ATP-independent closed state to the open states. Additional evidence for this closed state comes from the closed time histogram that has two components at saturating ATP . Adding a closed state to the right of the open states in Model 1-2 produces Model 1-4 . This modification corrects the prediction of the dose– response curve. Similarly, adding one more closed state to Model 1 produces Model 1-3 . Constraining Models 1-3 and 1-4 with detailed balance in the loops, and globally fitting the data from 5 to 50 μM ATP, we obtained rate constants with small error limits (Table VII ). The relative likelihoods and the AIC ranking of Model 1 and its expanded versions, Models 1-2, 1-3, and 1-4 are listed in Table VIII . Models 1-3 and 1-4, which contain loops, have much higher likelihoods than Model 1 or 1-2. Model 1-4 has the highest AIC rank, and therefore is the preferred model. The rate constants are listed in Table VI and VII. The transition rates near the open states for Models 1 and 1-2, and Models 1-3 and 1-4 are very similar, supporting the hierarchical approach. The predicted probability densities for the open and closed lifetimes of Model 1-4 are shown in Fig. 10 C and match the histograms reasonably well. Again, we found that the ATP association rate constants increased with proximity to the open states: k 12 (0) < k 23 (0) < k 34 (0). This is opposite to what would be expected from independent subunits. Each binding step makes the next faster. This cooperativity of binding appears model independent since all models tested had the same trend. From the Eyring model for the rates, the energy landscape for the whole reaction is shown in Fig. 14 . The kinetic model fits the single channel data quite well. In Fig. 4 C, the predicted P o (□) from Model 1-4 and its fit to the Hill equation (solid line) are plotted as a function of ATP. The maximal P o (0.64) and EC 50 (13.3 μM) are close to those of the experimental data, although the Hill coefficient (1.8) is slightly smaller. These values are much closer to experimental data than that from Model 1-2 , again suggesting that Model 1-4 is better than Model 1-2. We next tried to determine whether the rate constants were dependent on membrane potential using Model 1-4 . Fig. 15 A shows the single channel currents activated by 30 μM ATP at voltages from −120 to −80 mV. Fig. 15 B shows the voltage dependence of the mean open and closed times obtained from idealized currents, and Fig. 15 C shows P o as a function of voltage. The mean open time decreased with depolarization, while the mean closed time increased. The closing and opening rates are both voltage dependent, and the overall effect is to reduce the open probability with depolarization. P o values calculated from the all-points histogram were slightly larger than those calculated from the idealized currents, suggesting that some short lived events were missed, but the trend was the same; i.e., P o decreased with depolarization. To examine which rate constants vary with voltage, we globally fit the data between −80 and −120 mV with Model 1-4 . Each rate constant was taken to be of the form: 14 \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_{ij}(v)=k_{ij}(0)exp\hspace{.167em}(-z{\delta}_{ij}V/k_{B}T),\end{equation*}\end{document} where k ij (0) is the rate constant at 0 mV, k B T is Boltzmann's constant times absolute temperature, and z δ ij is the effective charge (in a lumped parameter model, a product of the sensing charge and the fraction of the total electric field felt at the location of the sensor). However, this model has many parameters and did not converge [with a detailed balance constraint in loop, there are 30 parameters, including 15 k ij (0) and 15 z δ ij ]. We had to apply further constraints to reduce the number of parameters. Since it is presumed that the ATP binding site is located in the extracellular loop , it is reasonable to assume that it is outside the electric field, and therefore the association and dissociation rates are voltage independent. We fixed them to the values obtained by global fitting based on Model 1-4 (Table VII ). The likelihood estimator converged, but with large error limits for k ij and z δ ij (Table IX ). Therefore, we could not make a firm conclusion regarding the voltage dependence for any individual rate constant. However, the predicted P o using the mean values of rate constants does decrease with depolarization and is similar to the P o from idealized currents . Fig. 15 D shows the open and closed interval histogram and the predicted probability densities (solid lines) from the rate constants. We predicted the shape of the whole cell I–V relationship by combining P o from outside-out patches and the single channel conductance. The whole cell current I is determined by the product of single channel current, i , the number of channels, n , and the open probability, P o : 15 \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=niP_{o}.\end{equation*}\end{document} P o obtained from histograms can be fit with the Boltzmann equation: 16 \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}P_{o}(V)=P_{o}(0)exp\hspace{.167em}(-z{\delta}V/k_{B}T),\end{equation*}\end{document} where P o (0) is P o at 0 mV, and is equal to 0.11. z δ is equal to 0.34, indicating that a hyperpolarization of 74 mV is needed for an e-fold increase of P o . If we presume that the voltage dependence of P o at different ATP concentrations is the same, we can use this result, together with the dose–response curve , to estimate P o (V) of a single channel. Multiplying P o (V) by the single channel current predicts the shape of the whole-cell I–V relationship. It is close to that predicted by Fig. 13 , Model 1-4 . In this study, we have characterized the single channel properties of cloned P2X 2 receptor ion channels. The characterization included general gating features, permeation properties, ATP concentration dependence, effects of pH, and kinetic analysis. The typical single channel Na + current has a chord conductance of ∼30 pS at −100 mV . The open channel current shows high frequency, high amplitude flickering with some apparent full closures. Because of the difficulty in resolving the fluctuations comprising this “buzz mode,” rather than build a substate model to characterize the open channel behavior, we characterized it as a single conductance with noise. The standard deviation of the excess open channel current is ∼30% of the mean. This is much larger than that of the acetylcholine receptor, for example, where the noise is only 2∼5% of the mean . The excess noise does not arise from thermal or shot noise nor from the voltage noise of the amplifier . The fluctuations appear to represent rapid conformational changes that modulate the open channel conductance. Occasionally, we saw relatively long-lived subconductance levels , but these were too infrequent to be evident in the all-points histograms. We attempted to estimate whether the fluctuations were to a discrete number of conducting states using a maximum-point likelihood approach. However, we could not find a consistent set of substate amplitudes between records from different patches, although it is clear that the flickers do not represent simple band-limited full closures of the channel. It is possible that there are actually a large number of states better described by a noise rather than a state model. The presence of these rapid fluctuations means that attempts to estimate the unitary channel current with noise analysis are prone to large, bandwidth-dependent errors. We do not think that the flickers arise from channel block by a diffusible agent, a mechanism that is often seen in other channels. Ca 2+ -activated K + channels can be blocked in a flickery manner by Na + , and cardiac Ca 2+ channels are discretely blocked by divalent ions . Since our currents were equally noisy with and without extracellular divalent ions, we do not believe that the excess noise comes from the block of divalent ions. ATP is not a candidate for blocking the channels since the mean amplitude and the excess noise are independent of ATP concentration . The voltage dependence of the excess noise is also not significant, suggesting that the flickers do not involve processes that sense the electric field. The simplest interpretation is that the excess noise arises from conformational transitions of the channel itself. The general features of the cloned P2X 2 receptors we have discussed are similar to data recorded from native receptors in rat sensory neurons and PC12 cells . In rat dorsal root ganglion (DRG) cells, single channel currents flickered much more rapidly than in PC12 cells—so rapidly that the lifetime of both states was almost always too short to be resolved by the recording system . The mean amplitude of the open state with 150 mM extracellular Na + was only 0.9 pA at −130 mV with an equivalent chord conductance of 7 pS and no obvious substates. In contrast, we observed a mean single channel current of 3.2 pA, much larger than in the rat DRG cells, with 145 mM extracellular Na + at −100 mV with an equivalent mean chord conductance of 32 pS. The true maximum conductance is even larger since the difference in amplitude of the upper 5% of closed and open distributions is 4.3 pA, ∼34% larger than the mean current. The single channel I–V relationship of the cloned P2X 2 receptors exhibited strong inward rectification . This result is consistent with the whole cell I–V relationship . Zhou and Hume studied the mechanisms of inward rectification of P2X 2 receptors. In their data, both gating and single channel conductance contributed to the inward rectification. They also reported that inward rectification did not require intracellular Mg 2+ or polyamines, and was present when the same solution was used on both sides of the patch. Our data supports these results. The currents in Fig. 2 were recorded in the presence of 1 mM extracellular Ca 2+ and Mg 2+ ; however, the single channel current I–V relation showed similar inward rectification when currents were recorded in the absence of divalent cations (data not shown). Since the mean open and closed times vary with voltage , the opening and closing rate constants are voltage dependent. Although our kinetic analysis was unable make a firm assignment of the voltage dependence to particular rate constants, P o did decrease with depolarization . The predicted whole cell currents based on Model 1-4 and single channel I–V curve matched reasonably well with the data. These results also suggest that both the instantaneous conductance and voltage-dependent gating contribute to the inward rectification. The dual mechanisms of inward rectification in this receptor are similar to the neuronal nicotinic acetylcholine receptor . The most important feature of the voltage dependence of P2X 2 kinetics is that it is minor. ATP is a potent agonist for cloned P2X receptors, except P2X 7 , and the receptors are highly selective for ATP over most other adenosine derivatives. Dose–response studies of whole cell currents reveal a Hill coefficient larger than 1 , suggesting that activation requires more than one agonist. This is reasonable since the channels are composed of multiple homomeric subunits. We studied P o over a wide range of ATP concentrations with outside-out patches and showed that the P o curve has a Hill coefficient of 2.3, an EC 50 of 11.2 μM, and a maximum of 0.61. A Hill coefficient of 2.3 suggests that there are at least three binding sites in the receptor. . Presumably, the cooperativity arises from the multimeric structure of the channel. Based on refolding studies of the P2X 2 extracellular domain (P2X 2 -ECD), Kim et al. predicted that the naturally assembled form of P2X 2 receptors may be tetrameric. Lewis et al. found that coexpression of P2X 2 and P2X 3 can form a new channel type by subunit heteropolymerization, providing further evidence that the P2X receptors are multimers. Recent experiments with chemical cross-linking of P2X 1 and P2X 3 receptors indicate that P2X receptor channels are trimeric. Since these results were obtained from native P2X receptors expressed in Xenopus oocytes, we expect that they are more representative than the studies on the isolated extracellular domains. The maximum P o of ∼0.6 indicates that the mean opening rates are slower than the closing rates. Our kinetic analysis based on Model 1-4 shows that the two opening rates k 46 and k 76 are much slower than corresponding closing rates k 64 and k 67 . The opening rate k 45 is faster than closing rate k 54 , while the opening rate k 75 is similar to the closing rate k 57 , so the overall opening rate is slower than the closing rate. The theory of independent ion passage predicts that the flux of a permeating ion should increase linearly with the ion concentration . However, most channels do not exhibit this behavior due to the competition for binding sites in the channel. Ion flux saturates when the binding–unbinding steps of permeation become rate limiting. This occurs at high ion concentrations when the rate of ion entry approaches the rate of unbinding. Conductances in the P2X 2 channels show clear deviations from independence . When the concentration of extracellular NaCl is raised, the single channel conductance saturates. In our data, the mean conductance versus Na + concentration was well fit by the Michaelis-Menten (MM) equation, with one binding site, X, in the pore (Scheme II). The rate constants are, in general, dependent on membrane potential. We tried to fit our conductance data with Scheme II, but could not obtain a unique set of rate constants: Scheme SII is over determined because the data does not have enough distinguishing features. If we assume that at high potentials the reverse flux is negligible, only the two forward rates are necessary and we can obtain solutions. We found that the equilibrium constant K s is voltage dependent , with the binding site located ∼20% of the way through the field relative to the extracellular face. It is interesting to speculate as to where the site may be relative to the primary sequence if one assumes that side chains form the selectivity filter rather than the backbone carbonyls . In their study of the ionic pores of P2X 2 receptors using the substituted cysteine accessibility method (SCAM), Rassendren et al. identified three residues: I328, N333, and T336 in the M2 domain that were located in the outer vestibule of the pore. Two of these are polar and might be part of a binding site for Na + . When the channel was open, D349C could be inhibited only by the small, positively charged MTSEA (2-aminoethyl-methanethiosulfonate), but not by MTSET {[2-(trimethylammonium) ethyl]methanethiosulfonate} or MTSES [sodium (2-sulfonatoethyl)methanethiosulfonate], implying that D349 is located near the middle of the channel. D349 is a negatively charged amino acid and is conserved among all seven P2X receptors. It is possible that D349 could be the site of permeant cation binding and is responsible for ionic selectivity. Our data shows P2X 2 is a nonselective cation channel. The ionic selectivity based on the conductance is: K + > Rb + > Cs + > Na + > Li + , Eisenman sequence IV . This sequence is different from free solution mobility and from the sequence of high field sites. This suggests the pore may be smaller than the nicotinic acetylcholine receptor with an interior having little charge in the selectivity filter. This is consistent with the results from substituted cysteine accessibility method experiments where only I328C, N333C, T336C, L338C, and D349C in the M2 domain were accessible to MTS reagents . Only D349 is negatively charged among these residues, and it may not be part of the selectivity filter. Based on whole cell currents of P2X 2 receptors, Brake et al. reported that the replacement of extracellular Na + by K + did not affect the reversal potential, suggesting that Na + and K + have a similar permeability near 0 mV. In our experiments, the currents carried by Na + were larger than the currents carried by K + at negative potentials. Similar results were reported for PC12 cells . The origin of the discrepancy between our results and theirs is masked by the lack of knowledge of the interplay between permeation and gating in the whole cell current. Different ionic environments may change the agonist binding and/or gating. In the nicotinic acetylcholine receptor , external monovalent ions compete with agonists for binding, changing the dose–response curves for reasons that have nothing to do with the permeation process itself. If the kinetics of ATP binding and/or gating is different for Na + and K + in the extracellular solution, P o will be different, changing the maximum conductance at a fixed agonist concentration. The excess open channel noise sequence is the same as the cation selectivity (i.e., K + ≅ Rb + > Cs + > Na + > Li + (Table I ), indicating that it is proportional to the single channel current amplitude, as expected if the noise arises from modulation of the normal flow. If the excess noise arose completely from simple conformational modulation of the pore, all ions would have the same relative selectivity. This is true for all alkali ions with the exception of Li + , which had ∼20% smaller relative fluctuations. This suggests a more specific interaction between Li + and the channel than for other permeant ions. The sensitivity of cloned P2X 2 receptors to ATP was affected by extracellular pH. King reported that with acidification, the ATP dose–response curve of whole cell currents shifted to the left without altering the maximal response. The effective receptor affinity for ATP was enhanced 5–10-fold by acidifying the bath solution (to pH 6.5), but was diminished four- to fivefold in an alkaline solution (pH 8.0). Different P2X receptors have different sensitivities to pH. Unlike P2X 2 receptors, P2X 1 , P2X 3 , and P2X 4 receptors decrease their apparent affinity with acidification . Our studies on outside-out patches showed that the mean current increased about an order of magnitude when the extracellular pH changed from 8.3 to 6.8, exhibiting a pK a of ∼7.9 . The Hill coefficient of 2.5 suggests that the channel has at least three binding sites, which is consistent with the stoichiometry study by Nicke et al. . In related experiments, extracellular protons potentiated adenosine binding to A 2A receptors, and this effect could be modified by mutagenesis or by chemically altering the strategic residues . In the extracellular loop of P2X 2 receptors, there are 9 histidine residues interspersed between 10 cysteine residues, the latter being conserved throughout the P2X 1–7 proteins. Both cysteine and histidine residues have been shown to be important for agonist and antagonist binding at the A 1 receptor, which is pH sensitive . It is reasonable to speculate that these two amino acids may play a similar role in ATP binding to P2X 2 receptors. Protonation of the histidine residues may account for the increase in P2X 2 current at low pH, but this seems unlikely because diethylpyrocarbonate, which irreversibly denatures histidyl residues, has no effect on the magnitude of the currents . The major effect of pH was on the kinetics of activation. The rate of activation increased as pH decreased , while the deactivation time constant was independent of pH. This suggests that the closing rates and the dissociation rates are not affected by protons. The simplest interpretation of the data is that in acidic environments, the binding site becomes more positive, increasing its affinity for ATP. However, since macroscopic kinetics is a function of all of the rate constants, many of which are not associated with binding, such an interpretation is not reliable. The single channel current amplitudes at different pH were similar, but the excess open channel noise increased when pH was lowered (Table II ). Comparing the single channel currents at different values of pH, as shown in Fig. 9 A, more brief closings can be seen at lower pH. The fluctuations caused by protons are slower than the fluctuations of the intrinsic channel flicker. While these results suggest that protons served as blockers, analysis of the power spectra and single channel kinetics contradict this interpretation. The blocking and unblocking rates were only weakly dependent on proton concentration (see Eq. 13 ). Power spectral analysis also showed that the corner frequency decreased with an increase in pH, opposite to the prediction for proton block. It appears that the brief closings at low pH are due to conformational changes produced by the titration of several sites. We used the maximum interval likelihood method to statistically compare kinetic models . The models were built hierarchically, beginning with one that described the transitions near the open states . The first model we chose had three closed states, C 1 , C 2 , and C 3 , representing unliganded, monoliganded, and biliganded closed states, and two open states O 4 and O 5 . Starting with this model, we expanded to Models 1-2, 1-3, and 1-4 to account for the ATP titration data. Comparing Model 1 with Model 1-2, and Model 1-3 with Model 1-4, we found that the opening and closing rate constants were surprisingly close (see Tables VI and VII ), suggesting that the transitions near the open state were well defined. This result supports our strategy of model development. Among the four models, Model 1-4 had the highest likelihood and AIC rank, and therefore is our preferred model (Table VIII ). In this model, there are five closed states, C 1 , C 2 , C 3 , C 4 , and C 7 , representing unliganded, monoliganded, biliganded, and triliganded closed states, and two open states, O 5 and O 6 . The three ATP binding steps require the channel to be at least a trimer. By constraining the ATP association rates to be proportional to concentration, reliable rate constants were obtained from global fitting with this model (Table VII ). The predicted probability density functions match reasonably well the open- and closed-time histograms at all concentrations, and the predicted P o is close to the experimental data so that our final preferred model is Model 1-4. From the results of the basic model and its expanded versions , we found that association and dissociation rate constants increased as they approached the open states (Table VI and VII). This means that the subunits are not independent and that the association rate for an incoming ATP is increased by the presence of bound ATPs. This trend was consistent among all models examined. The increase in association rates with consecutive binding is most surprising when one thinks about the opposing electrostatic factors. Bound ATP with a charge of −4 should repel the next incoming ATP (note: the actual valence of bound ATP is not known and is probably less than −4). Since the energy is proportional to the product of the charges, if the sites were independent, the second ATP would be repelled by a resident ATP with an energy proportional to 4*4 = 16, and the third by 8*4 = 32. We would thus expect the later binding rates to decrease by more than just the number of available sites. The trend of increasing association rates with occupancy must be caused by conformational changes in the channel that mask the electrostatic contribution. While the increasing rates of dissociation with occupancy fit the predicted electrostatic trend, given the conformational changes associated with binding, this trend may be coincidental. The binding of ATP causes the remaining unoccupied sites to open up in such a manner so as to increase the rates of ATP entry and exit without having a large effect on the equilibrium affinity of the site . If the channel were a tetramer, as predicted by Kim et al. , we might add one closed state to the left of C 1 as in Model 1-5 . We attempted to fit this model, but could not obtain unique rates. This may be because we had insufficient data at very low ATP concentrations, or because the channel really is a trimer . The predicted P o vs. ATP concentration from Model 1-4 fits well the P o , EC 50 , and Hill coefficient obtained from the dose–response curve , supporting the consistency of the model and the necessity of the last ATP-independent closed state. There have been only a few studies on the kinetics of P2X receptor ion channels based on single channel currents . Kinetic studies using whole cell currents showed that the rise time of current elicited by ATP was strongly concentration dependent, but the decay time was not . This is in accord with the pH experiments discussed above. There is some variability among reports regarding the rise and fall times. In rat sensory neurons, using fast solution exchange, the rise time was ∼10 ms at saturating ATP and the decay time was ∼100 ms . In smooth muscle and cloned P2X 1 receptors, the rise time was ∼5 ms, while in PC12 cells, cloned P2X 2 receptors, and rat superior cervical ganglion (SCG) cells, the rise time was ∼25 ms . In rat SCG cells, nodose, and guinea-pig coeliac neurons, the latency to the onset of whole cell currents was estimated to be ∼0.8–4 ms, and the 10–90% rise time at high ATP concentrations ranged from 5 to 20 ms . Since these experiments were done in the whole cell configuration, the rate of rise was likely limited by the speed of the solution exchange around the cells. Hess reported that the time resolution for solution exchange around a whole cell of this size is 2∼10 ms under maximal flow velocities and even slower for lower velocities. With outside-out patches, the rise times can be ∼250 μs . Moreover, because in the whole cell experiments there was no marker for the start of solution exchange, the latency to the onset of the currents could not be estimated accurately. Preliminary kinetic analysis of single channel currents from rat sensory neurons showed that the distribution of the open times could be approximately fit by two exponentials with time constants of 0.35 and 3.4 ms . The ratio between the fast component amplitude and the slow one varied between patches, with ratios of 47.4:1 to 4.8:1. Using the rise and fall time constants from whole cell currents at different concentrations of ATP, and based on kinetic models of the ACh receptor channel, Bean proposed a linear kinetic model with independent subunits for ATP activation . The association and dissociation rate constants k + = 1.2 × 10 7 M −1 s −1 and k − = 4 s −1 were chosen so that the simulations mimicked the kinetics seen in the bullfrog sensory neurons. Agreement between the model and the data suggested that the ATP binding sites could be independent. We applied the independent binding site assumption to Model 1-2; i.e., k 34 = 1/2k 23 = 1/3k 12 = 1/3k 10 = k + , and k 21 = 1/2k 32 = 1/3k 43 = k − . Although the estimation converged, the likelihood was much lower than our favored models. We also fit our data with Bean's model, which has only one open state , and again the fits were poor. In other ligand-gated channels, such as the Ca 2+ -activated potassium channel and ACh receptors , partially liganded channels can open. To explore whether these states were visible in our data, we compared the maximum likelihood of Model 8-1 that has partially liganded openings with Model 1-2 . Global fitting of Model 8-1 with data from 5, 10, 15, 20, 30, and 50 μM ATP produced a maximum likelihood 664 units lower than Model 1-2, suggesting the model is e 664 × less likely to produce the data. Apparently, P2X 2 channels do not open for a significant fraction of time in partially liganded states. In summary, Model 1-4 can well represent the channel gating processes. It is adequate to explain the behavior across concentration and is physically reasonable. Fig. 14 shows the calculated free energy barriers and wells at −120 mV referenced to 1 M ATP. The energies were calculated from the rate constants assuming an Eyring model. The free energies of all wells decreased with the reaction coordinate. State C 4 could go to either open state O 5 or O 6 with state O 5 being slightly more stable. O 5 and O 6 go to the same closed state C 7 that is the most stable in the reaction pathway. Although P o is not strongly dependent on membrane potential, it did decrease with depolarization , indicating that some of the rate constants are voltage dependent. To reduce the free parameters, we limited the voltage sensitivity to only the rate constants in the final loop and ended up with very large errors limits for k ij (0) and z δ ij . We have no confidence in the voltage dependence of any of the individual rate constants. Data from a wider voltage range will be required to adequately address this question. Despite the wide error limits, by lumping the kinetics into an equilibrium model to predict the probabilities of occupancy, the predicted P o and whole cell currents calculated from the mean values of k ij (0) and z δ ij were consistent with those obtained by idealization and with experimental data . The probability density functions match reasonably well with duration histograms . In conclusion, the currently optimal model, Model 1-4 , can be summarized as follows: (a) the channel proceeds through three ATP binding steps before opening; (b) the three ATP binding sites are not independent, but positively cooperative; (c) There are two open states, which connect to a common ATP-independent closed state; (d) activation and deactivation proceed along the same pathway; and (e) channels only open after being fully liganded. | Study | biomedical | en | 0.999997 |
10228184 | Reduction in junctional conductance (g j ) 1 by intracellular acidification has been reported for both vertebrate and invertebrate gap junctions (GJs). Unlike the reduction in g j to a residual (plateau) value with increasing transjunctional voltage (V j ), strong acidification reduces g j to undetectable levels; i.e., cells become completely electrically uncoupled. While a physiological role for acidification-induced uncoupling has not been established, possible roles may involve pathologies where there is cell injury. A lowered intracellular pH (pH i ) in response to injury may be of protective value in reducing GJ-mediated communication and limiting the spread of injury within a tissue. The reduction in g j by cytoplasmic acidification, in some cases, has been described with a simple titration curve . Wide differences in pH sensitivity have been reported for GJ-mediated cell communication, with apparent p K a s ranging from ∼pH 6 to 7.5. Also, both direct and indirect actions of H + have been proposed . Direct action by H + was suggested in early studies of cell pairs obtained from blastomeres of amphibian and teleost embryos . Evidence for indirect action through soluble cytoplasmic intermediaries has come from studies in both invertebrate and vertebrate GJs, but GJs in insects and nematodes, and perhaps invertebrates in general, appear to be formed by a family of proteins unrelated in primary sequence to connexins . In crayfish septate GJs and GJs between paired Xenopus oocytes, Ca 2+ /calmodulin was reported to be an essential intermediary . A requirement for Ca 2+ in acidification-induced uncoupling has been reported in rat ventricular myocytes and Novikoff hepatoma cells, cells that both predominantly express connexin (Cx)43 . In these cases, low pH i was reported to have no effect on g j without an accompanying increase in intracellular Ca 2+ . Mutations involving removal, substitution, and change in the position of His residues in the cytoplasmic loop of Cx43 were shown to significantly affect pH sensitivity, suggesting that protonation of these residues may be important in the pH dependence of GJs . More recently proposed molecular mechanisms of pH sensitivity involve both cytoplasmic loop (CL) and carboxy terminal (CT) domains. In Cx43, CT is thought to behave like a gating particle that, when bound to a receptor domain putatively localized in CL, closes the Cx43 channel . In Cx32, charge interactions within CL, as well as between CL and the proximal portion of CT, have been suggested to be responsible for pH sensitivity . Difficulties and differences in the methods of quantifying pH i together with multiple connexin expression in native cells may have contributed to wide differences in reported sensitivities . Also, studies of pH sensitivity in tissue or cell-pair preparations have been confounded by an inability to rapidly change pH i to determine kinetics and avoid slower secondary effects by nonuniform changes in pH i . Here we report the use of Cx46 hemichannels to investigate the effects of pH on GJ communication. When expressed in Xenopus oocytes, Cx46 hemichannels are functional and can be readily recorded in cell-attached and excised patch configurations . Single hemichannels in excised patches exposed to fast perfusion provide a means of examining the action of chemical modulators on connexins with millisecond time resolution. The coding region of Cx46 was cloned into the EcoR1 and Hind III sites of pGem-7Zf+ ( Promega Corp. ) from rat genomic DNA using PCR amplification with primers corresponding to amino- and carboxy-terminal sequences. Preparation of Xenopus oocytes and synthesis of RNA have been described previously . Each oocyte was injected with 50–100 nl of an aqueous solution of mRNA (2 mg/ml) together with DNA antisense to the endogenous Xen Cx38 (8 pmol/μl). We used the phosphorothioate antisense oligo 5′-GCT TTA GTA ATT CCC ATC CTG CCA TGT TTC-3′, which is complementary to Xen Cx38 commencing at NH 2 terminus–5 with respect to the ATG initiation codon. The EcoR1 and HindIII fragment cut from the Cx46-pGem construct was blunt-end ligated into pCl-Neo ( Promega Corp. ) at the EcoR1 site. Communication-deficient Neuro-2a cells (CCL-131; American Type Culture Collection) were transfected with this construct using lipofectin ( GIBCO BRL ) according to the manufacturer's protocol. Transfected cells were selected and maintained with 300 μg/ml (active) G418 ( GIBCO BRL ). Positive clones were screened by dual whole-cell patch recordings. These cells are referred to as N2a-Cx46. In macroscopic recordings of Cx46 hemichannel currents, Xenopus oocytes were bathed in a standard solution containing (mM): 88 NaCl, 1 KCl, 2 MgCl 2 , 1.8 CaCl 2 , 5 glucose, 5 HEPES, and 5 PIPES, pH 7.6. Both current-passing and voltage-recording pipettes contained 2 M KCl. A perspex recording chamber was designed for rapid solution exchange and consisted of a rectangular canal connecting inflow and outflow compartments. A suction tube was placed in the outflow compartment and a separate reservoir connected to the main chamber with an agar bridge was used for grounding. Bath volume was ∼0.5 ml, and total volume exchange was achieved in 1–2 s by application of test solutions to the inflow compartment. Flow rates in all experiments were consistent. Test solutions consisted of the standard bath solution pH adjusted over a range of 5.0–7.6 with HCl and NaOH. The pH of the solution in the inflow reservoir was monitored over the course of each experiment. For patch clamp recordings of Cx46 hemichannel currents, Xenopus oocytes were manually devitellinized in a hypertonic solution consisting of (mM): 220 Na aspartate, 10 KCl, 2 MgCl 2 , and 10 HEPES. Typically, patch pipettes were filled with (mM): 100 KCl, 30 NaCl, 2 MgCl 2 , 10 HEPES, 10 PIPES, 10 EGTA, and 1 CaCl 2 , pH adjusted to 7.5 with KOH. We refer to this solution as IPS-A (free [Ca 2+ ] ≈ 10 −8 M at pH 7.5). In experiments where the effect of Ca 2+ was examined, 2 mM BAPTA replaced EGTA. We refer to this pipette solution as IPS-B (free [Ca 2+ ] ≈ 10 −7 M with 0.7 mM added CaCl 2 at pH 7.5). To achieve the desired level of free [Ca 2+ ] at a given pH, an appropriate amount of CaCl 2 was added as computed using CHELATOR . The test solutions used to examine pH sensitivity in excised patches consisted of either IPS-A or IPS-B, with pH adjusted to the desired value with KOH and HCl. These solutions were applied to the patches from a theta tube mounted on a piezoelectric actuator . Throughout the course of an experiment, the bath was continuously perfused with a solution containing (mM): 88 NaCl, 1 KCl, 2 MgCl 2 , 5 glucose, and 10 HEPES, pH adjusted to 7.6. In whole cell patch experiments on N2a-Cx46 cells, the bath solution was a modified Krebs-Ringer consisting of (mM): 140 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 HEPES, 5 glucose, and 2 pyruvate, pH adjusted to 7.4 with NaOH. Patch pipette solutions consisted of (mM): 130 KCl, 10 NaCl, 1 MgCl 2 , 5 HEPES, 5 EGTA, and 1.4 CaCl 2 , pH adjusted to 7.2 with KOH. Free [Ca 2+ ] was calculated to be ∼5 × 10 −8 M. In some experiments, we used lower amounts of Ca 2+ with BAPTA in the patch pipette solutions to minimize the rise in Ca 2+ upon acidification. With intracellular acidification to pH 6.0 (by perfusing 100% CO 2 -equilibrated bath solution), free [Ca 2+ ] increased approximately fourfold (from 5 × 10 −8 to 1.7 × 10 −7 M) using 2 mM BAPTA with 0.38 mM added CaCl 2 . The test solutions used to acidify the cells were directly perfused onto patched cells. Voltage clamp recordings of macroscopic Cx46 hemichannel currents from single Xenopus oocytes were obtained with a two-electrode voltage clamp (GeneClamp 500; Axon Instruments, Inc. ). Data was acquired using pClamp 6.0 software and a Digidata 1200 interface ( Axon Instruments, Inc. ). Single Cx46 hemichannel currents in Xenopus oocytes were recorded from cell-attached and excised patches using an Axopatch 200B ( Axon Instruments, Inc. ) with the headstage in capacitive feedback mode. Cx46 cell–cell channel currents in N2a-Cx46 cells were recorded from cell pairs using the dual whole-cell patch clamp technique and two Axopatch 1-D patch clamp amplifiers ( Axon Instruments, Inc. ). g j was evaluated in the following manner. Both cells of a pair were maintained at the same holding potential from which voltage steps, ΔV j , were applied to one cell. The voltage steps were small and brief so as not to invoke V j - dependent changes in g j . Junctional current was measured directly as the change in current in the unstepped cell, ΔI. g j is thus given by ΔI/ΔV j . Data from both hemichannels and cell–cell channels were acquired with AT-MIO-16X D/A boards from National Instruments using our own acquisition software. The titration curve for inside-out patches was obtained from mean current analysis of multiple applications of low pH solutions to the exposed (cytoplasmic) face of each patch. For each patch, the leak-subtracted mean current during low pH application (after the first closure) was normalized to the leak-subtracted mean current during exposure to pH 7.5 to obtain I/I 7.5 for that application. This procedure allowed for comparison among patches with different numbers of hemichannels at different voltages. For analysis of closed durations in outside-out patches, single hemichannel currents were idealized with the Sublevel Hinkley Detector (SHD) to two levels, fully open and closed. When idealizing to two levels, the SHD acts as a half-height threshold algorithm. In Cx46 hemichannels, transitions between open and closed are characterized by transitions among multiple short-lived substates , leading to multiple crossings of the half height before entering a long lived open or closed state. The SHD was set at low sensitivity to avoid detection of these crossings. Dwell-time histograms were compiled and fitted in Origin (Microcal Software, Inc.). Ensemble currents were obtained by summing the current from each trace minus the leak current of the patch. Hence, 0 pA in the ensemble summations represents closure of all hemichannels in the patch. Fitted parameters to exponential functions were obtained in Origin. Single Neuro-2a cells were loaded with the indicator BCECF (Molecular Probes, Inc.) in one of two ways. In cells that were not patch clamped, loading was achieved by bathing cells in 10 μM BCECF-AM, the membrane permeable form. In cells that were patch clamped, loading was achieved by including the acidic form of BCECF in the patch pipette solution at a concentration of 10 μM. We found no significant differences with the two methods. Xenopus oocytes were injected with 50 nl of 2 mM BCECF (acidic form) to a achieve a final concentration of ∼10 μM. pH was determined from the ratio of emission values recorded with excitation wavelengths of 440 and 490 nm using a SPECTRAMASTER high speed monochromator and a MERLIN imaging system (Life Science Resources). Calibration was performed in Neuro-2a cells using 10 μM nigericin (Molecular Probes, Inc.) to equilibrate intracellular and extracellular pH. Calibration solutions ranged from pH 5.5 to 8.0. The 440/490 ratio for each pH was taken when the value had not changed for 3 min. A plot of the ratio value recorded for each tested pH was fitted to \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*}Ratio=A(pH^{2})+B(pH)+C.\end{equation*}\end{document} Ratios were converted to pH with the solution to the quadratic 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*}pH=\frac{-B+\sqrt{B^{2}-4(C-Ratio)A}}{2A}.\end{equation*}\end{document} For intracellular pH measurements in oocytes with BCECF, we used the calibration curve obtained using Neuro-2a cells. Although calibration curves for pH-sensitive dyes can shift depending on the type of cell , we merely make comparisons of relative changes in intracellular pH for applications of solutions equilibrated with 100% CO 2 and solutions acidified with HCl. Rapid solution switching in excised patches was achieved by using a theta tube mounted on a LSS 3100 piezoelectric driven actuator (Burleigh Instruments). The theta tube was pulled to an overall tip diameter of ∼150 μm. Flow rate through the theta tube was adjusted by changes in the heights of the reservoirs feeding each barrel to achieve a thin interface between the two streams. An excised patch was switched rapidly between the two streams by applying a voltage ramp waveform to the piezoelectric driven actuator. The waveform produced a 10-ms approach from the center of one stream towards the interface, followed by a 2-ms movement through the interface and again by a 10-ms approach to the center of the second stream. This waveform configuration minimized ringing and resonance of the piezo device and theta tube while achieving a rapid transition through the interface. The speed of solution switching was tested by measuring the change in the holding current due to a change in tip potential of a high-resistance patch pipette when switching between two solutions differing in salt composition . The 10– 90% difference in tip potential (measured in voltage clamp as shift in holding current) was typically traversed in ∼1 ms . Bath application of a modified Krebs-Ringer solution equilibrated with 100% CO 2 to pairs of Neuro-2a cells expressing Cx46 rapidly reduced g j to below detectable levels. Recovery depended on the time of exposure to CO 2 . Longer exposures caused recovery to be delayed and less complete. An example of a cell pair exposed to 100% CO 2 for ∼45 s is shown in Fig. 2 A, I. g j rapidly fell to below detectable levels, and no recovery of g j was detected for nearly 3 min after washout. Thereafter, g j slowly increased, but reached only a small fraction of the original value. Recovery was incomplete even as long as 30–40 min after washing (data not shown). In a different cell pair, effects of two consecutive shorter exposures to 100% CO 2 are illustrated. With an ∼25-s exposure , g j rapidly fell to below detectable levels, but remained undetectable for a shorter time (∼60 s) after washout. Thereafter, recovery proceeded relatively rapidly, reaching 50% of its original value within a few minutes. With a subsequent briefer exposure to this same cell pair, ∼15 s , g j again decreased but not completely before washout elicited a relatively rapid (3–4 min) and nearly full recovery to the level before this exposure. The dependence of recovery on the duration of exposure to CO 2 was the same in cell pairs that were exposed to CO 2 for the first time and in those that had prior exposures to CO 2 . Although equilibrating the Krebs-Ringer bath solution with 100% CO 2 reduced the extracellular pH from 7.2 to ∼5.5, CO 2 is known to cross cell membranes readily and cause rapid intracellular acidification . Thus, we examined whether reductions in g j occur by perfusing solutions acidified with HCl and buffered with combinations of HEPES and PIPES, presumably membrane impermeant buffers. Shown in Fig. 2 B are recordings of g j with applications of bath solutions with pH values adjusted to 6.5, 6.0, and 5.5. With application of a pH 6.5 solution, a modest reduction in g j occurred and essentially recovered fully upon washing. Applications of pH 6.0 and 5.5 solutions reduced g j increasingly, but not completely. Recovery of g j after washout of HCl acidified solutions was relatively faster than after washout of 100% CO 2 . For example, after perfusion of a pH 5.5 bath solution , g j recovered to ∼70% of control in <1 min, almost three times as fast as the decrease in g j . Recovery from prolonged exposure to pH 5.5 solutions was incomplete as with prolonged exposure to 100% CO 2 . In general, the reductions in g j with applications of HCl acidified solutions differed from those caused by 100% CO 2 by being smaller in magnitude and slower to develop even when the pH of the HCl acidified solution was ∼5.5, comparable with that of the 100% CO 2 -equilibrated solution. To examine the extent and time course of the changes in pH i that occurred with application of 100% CO 2 - and HCl-acidified solutions, we measured pH i using the pH indicator BCECF in separate N2A-Cx46 cells. Data from a representative N2A-Cx46 cell is shown in Fig. 3 . Changes in pH i occurred with both treatments, but differed in degree and time course. Typically with 100% CO 2 , pH i dropped rapidly from ∼7.25 to ∼6.0 within 25 s of application (∼0.05 pH U/s) . Upon washing, pH i recovered slowly, taking ∼5 min to reach control values (∼0.004 pH U/s). With HCl acidified solutions ranging from 5.5 to 6.5, pH i declined much more slowly, but recovered more rapidly. With an application of a pH 5.5 solution, pH i decreased from ∼7.25 to ∼6.0 over a period of 5 min (∼0.004 pH U/s) . pH i recovered to control values in ∼100 s (∼0.013 pH U/s). We presume that the faster rate of intracellular acidification achieved with 100% CO 2 is due to its high membrane permeability. Although we have no explanation for the difference in recovery rates, the recovery from bathing in HCl-acidified solutions may be faster because there is less acidification of intracellular compartments. The general agreement between the degree and time course of the reversible changes in g j and in pH i are consistent with the accepted view that GJ channels are closed by intracellular, but not extracellular acidification. The rapidity and reversibility of uncoupling with modest and/or brief acidification suggests that a component of the pH i effect may be a form of gating, perhaps by means of a direct action of H + on connexins. Delayed recovery and apparently irreversible loss of Cx46-mediated coupling with prolonged acidification indicates that there are additional long-lasting effects produced by acidification that are slower to develop. As a precursor to studies of single Cx46 hemichannels, we examined the effects of acidification on macroscopic currents of Cx46 hemichannels expressed in Xenopus oocytes. Cx46 hemichannels are typically activated by steps to positive voltages and mediate large, slowly rising outward currents . Fig. 4 illustrates the effects of applications of solutions acidified with 100% CO 2 and those acidified to pH 5.5 with HCl on the activation of Cx46 hemichannel currents. Currents were activated by 40-s depolarizations to +30 mV from a holding potential of −70 mV, spaced 50-s apart. During the fourth depolarization, application of 100% CO 2 equilibrated bath solution immediately reduced hemichannel currents. By the next depolarizing step, the slowly activating outward currents characteristic of Cx46 hemichannels were completely abolished, leaving a small endogenous current that is also seen in oocytes injected only with DNA antisense to Xen Cx38. With application of a pH 5.5 bath solution, currents decreased but, unlike with 100% CO 2 , were not completely abolished by the next depolarization. Washing with pH 7.5 bath solution led to full recovery for both treatments in these oocytes. Longer exposures to pH 5.5 solutions caused greater reductions in the Cx46 currents and prolonged exposure to either solution delayed recovery more (data not shown). However, unlike recovery of N2a-Cx46 junctional currents, the degree of recovery of hemichannel currents in Xenopus oocytes was erratic, sometimes recovering partially and other times recovering fully or even exceeding control values. Some of the erratic recovery was likely due to different rates and degrees of expression of new hemichannels. Electrophysiological monitoring of Cx46 expression in oocytes without acidification showed increases in hemichannel currents that were irregular over time and varied considerably among oocytes. When examining the time course of the changes in pH i using BCECF in single Xenopus oocytes, we observed a substantial and rapid acidification with application of 100% CO 2 . However, in the large oocytes, pH i did not change uniformly. Close to the cell periphery, pH dropped at an average rate of −0.02 pH U/s and thus could rapidly affect hemichannels from the inside. At increasing distances from the periphery, acidification was considerably slower, but eventually reached the same steady state value . An application of an HCl-acidified solution caused intracellular acidification that was considerably slower than that with CO 2 , falling at an average rate of −0.005 pH U/s at the cell periphery . As with 100% CO 2 , the rate of acidification slowed with increasing distance from the surface. With HCl-acidified solutions, equilibration of internal and external pH was not achieved during a 3-min application. Similar to the results in Neuro-2a cells, applications of 100% CO 2 solutions and HCl-acidified solutions to Xenopus oocytes induced intracellular acidification to different degrees that correlated with the degree to which Cx46 hemichannel currents were reduced. These results are consistent with the sensitivity exhibited by Cx46 cell–cell channels in Neuro-2a cells to intracellular acidification. The more rapid effect on hemichannel currents seen with application of HCl-acidified pH 5.5 solution contrasts the slow decrease in g j seen with cell–cell channels . These data suggest that either there is an extracellular site of Cx46 pH sensitivity exposed in the unapposed hemichannel configuration or that changes in external pH can rapidly act internally by permeation of H + through the open hemichannel pore. To determine whether soluble cytoplasmic factors mediate pH sensitivity and to permit more rapid changes in pH, patches containing Cx46 hemichannels were excised from Xenopus oocytes in inside-out configurations. Patches were switched between high and low pH solutions by means of a piezoelectric actuator (see materials and methods ). Fig. 6 A shows a recording from a patch containing four active hemichannels placed in a stream composed of IPS-A at pH 7.5. Switching to IPS-A at pH 6.0 led to rapid and complete closure of all the hemichannels. Upon returning to pH 7.5, the hemichannels reopened promptly. Multiple applications to the same patch show the robustness of the low pH effect; rapid closure occurred each time the pH 6.0 solution was applied. In an expanded view of one of these applications, all the hemichannels closed rapidly. Although there were occasional brief, low amplitude bursts (perhaps due to partial openings), no full openings occurred for the duration of this pH 6.0 application. Upon switching back to pH 7.5, the hemichannels returned to normal gating behavior. This result clearly demonstrates that no soluble cytoplasmic factors are required for pH sensitivity in Cx46 hemichannels. Furthermore, the effect of pH 6.0 application was essentially the same in solutions buffered with concentrations of HEPES and/or PIPES ranging from 0.25 to 25 mM (data not shown), suggesting no dependence on buffer composition or strength, . The titration curve of the reduction in mean current by acidification is fit by the Hill equation with an apparent pK a of 6.4 and an n of 2.3 . H + and Ca 2+ have been shown to act synergistically to modulate coupling between neonatal and adult rat myocytes. To examine pH sensitivity from the cytoplasmic side with Ca 2+ concentrations fixed at high and low levels, we used inside-out patches and exposed them to solutions containing 2 mM BAPTA (IPS-B) with appropriate amounts of added Ca 2+ at pH 7.5 and 6.0 according to Schoenmakers et al. . Fig. 7 shows applications of pH 6.0 solutions with the Ca 2+ concentration fixed at (A) 10 −7 and (B) 10 −5 M to inside-out patches each containing three Cx46 hemichannels. At either Ca 2+ concentration, the mean current rapidly decreased ∼90% upon switching to pH 6.0. Furthermore, changing Ca 2+ from 10 −7 to 10 −5 M while maintaining pH fixed at 7.5 did not alter hemichannel open probability (data not shown). These data indicate that Ca 2+ up to 10 −5 M does not appreciably affect Cx46 hemichannel open probability and that Ca 2+ concentrations over a physiologically significant range (10 −7 to 10 −5 M) have little or no qualitative effect on pH sensitivity. These results are in agreement with those we obtained for Cx46 cell–cell channels in pairs of N2a-Cx46 cells in which application of 100% CO 2 uncoupled cell pairs equally well when the patch pipettes were filled with IPS containing BAPTA or EGTA (data not shown). We did not examine the effects of elevated extracellular Ca 2+ on pH sensitivity since increases in extracellular Ca 2+ by themselves close Cx46 hemichannels and substantially shift voltage-dependent activation . To better examine the time course of hemichannel closure with acidification, we performed an ensemble analysis of currents from inside-out patches. In Fig. 8 A, 20 sequential recordings from a representative patch containing a single Cx46 hemichannel demonstrate the reproducibility of the effect of a 2-s pH 6.0 application to the cytoplasmic side; the pH on the extracellular side (i.e., the pipette solution) was maintained at 7.5. On switching to pH 6.0, unitary conductance was lowered by ∼15% and open probability was markedly reduced. Few full reopenings occurred during the pH 6.0 applications, but those that did occur had reduced conductance. The sum of these 20 recordings and 112 more from four other patches containing multiple hemichannels is shown in Fig. 8 B. The decay in current was fit well by two exponentials (time constants of 110 and 935 ms), as shown in Fig. 8 C. The steady state value obtained from the fit was −282 pA. The average total current before pH 6.0 application was −4,032 pA. Thus, ∼7% of the original current remained in pH 6.0. This value is in good agreement with the average value of 10% obtained from analysis of mean currents through individual patches . As shown in Fig. 8 D, on switching to pH 6.0 (dashed line), the onset of the ensemble current decrease had no measurable delay. The rapidity of the reduction in current with acidification suggests that protons are acting directly on Cx46 hemichannels. As evident in the ensemble analysis , Cx46 hemichannel currents fully recovered from each successive 2-s acidification. However, less recovery was seen in patches exposed to pH 6.0 for longer times. With applications of 5–10 s in duration, the degree of hemichannel loss in multichannel patches ranged from 0 to 50%. In the example shown in Fig. 9 A, two successive 5-s applications of pH 6.0 were applied to an inside-out patch containing multiple hemichannels. After the first application, recovery was delayed, and the number of active hemichannels was reduced by roughly 50%. The second application reduced the number of active hemichannels even more. In Fig. 9 B, ensemble currents normalized to the mean current at pH 7.5 for 1-, 2-, and 5-s applications of pH 6.0 solutions are superimposed. The initial current decay of all three followed nearly the same time course, but the degree of recovery from 5-s applications was substantially reduced; ∼20% of the channels did not recover. These data show that the degree of recovery is dependent on the duration of acidification, qualitatively similar to the observations with macroscopic recordings of g j in Neuro-2a cells expressing Cx46 . At voltages at which hemichannel open probability is high and closed durations are relatively short (e.g., −30 mV) application of a pH 6.0 solution to the cytoplasmic face of an open hemichannel induced closure that usually lasted for the duration of the application, although occasional reopenings occurred. These closed durations at pH 6.0 were substantially longer than those associated with gating at pH 7.5. This is evident in the 20 sequential pH 6.0 applications shown in Fig. 8 A. Likewise, if a pH 6.0 solution were applied to a closed hemichannel, the hemichannel also often remained closed for the duration of the application. In the example shown in Fig. 10 A, a pH 6.0 application occurred >300 ms after both hemichannels in the patch had fully closed. No full reopenings were seen for the duration of the application. These results indicate that the site of pH i sensitivity is accessible from the cytoplasm when the pH or voltage gate fully closes a Cx46 hemichannel. To test whether the cytoplasmic site of pH sensitivity is in the electric field induced by an applied voltage, we examined voltage dependence of pH i sensitivity. If H + interacts with a site in the voltage-induced field, positive voltages would be expected to increase the effectiveness of low pH applied to the cytoplasmic face by driving H + into the membrane and pore. Fig. 10 B is an example of an application of a pH 6.0 solution to the cytoplasmic face of a single Cx46 hemichannel in an inside-out patch. At +40 mV, H + is less effective at causing closure than at −40 mV, opposite that expected for an increased entry of H + into the membrane or pore. These data suggest that H + acts on a cytoplasmic site whose accessibility and/or efficacy in inducing closure is voltage dependent. At negative voltages, the affinity of the hemichannel for H + is increased or the open/ closed equilibrium for H + -induced gating is shifted towards closed. The polarity of voltage dependence together with no need for an open hemichannel to effect acidification-induced closure from the inside indicate that the H + binding site (pH sensor) for cytoplasmic pH sensitivity is exposed in both closed and open conformations of the hemichannel and that block by H + or some H + - induced factor within the pore is unlikely. To examine the effects of extracellular acidification at the single hemichannel level, we excised patches containing Cx46 hemichannels in outside-out configurations and rapidly switched between pH 7.5 and 6.0 solutions. The pH on the intracellular side (i.e., the pipette solution) was maintained at 7.5. 20 sequential recordings of 2-s pH 6.0 applications from a patch containing a single active hemichannel are shown in Fig. 11 . During the pH 6.0 application to the extracellular side, open probability was reduced, but not nearly as much as with pH 6.0 applications to the cytoplasmic side. The sum of the leak-subtracted currents from a total of 50 applications to this patch is displayed in Fig. 11 B. The ensemble current decayed with a single exponential time course to a steady state value of −336 pA from an initial average of −502 pA, a 33% decrease. This is in contrast to the >90% reduction for pH 6.0 applications to the cytoplasmic face of the hemichannel. As for low pH applications to the cytoplasmic side, the effect is rapid. The extracellular pH sensitivity in excised patches suggests that a lower-affinity extracellular site may exist that closes Cx46 hemichannels. However, it is possible that low pH, applied extracellularly, acts rapidly at the cytoplasmic site by permeation of H + and/or buffer. We examined this possibility by comparing the closed time distribution of outside-out patches exposed to extracellular pH 6.0 (bulk cytoplasmic pH maintained at 7.5) to the distribution of opening latencies upon rapidly switching cytoplasmic pH in inside-out patches from 6.0 to 7.5. Assuming that protonation/deprotonation is rapid compared with channel gating kinetics and that closed hemichannels do not conduct H + and/ or buffer, hemichannel closure with extracellular pH at 6.0 should prevent the entry of H + /buffer, thereby causing the cytoplasmic site to reequilibrate with the bulk cytoplasmic pH of 7.5. Hemichannel closure under this condition would lead to reopening and is analogous to opening of closed hemichannels exposed to pH 6.0 on the cytoplasmic side upon rapid switching to pH 7.5. As seen in Fig. 12 , B and C, the mean closed time of 600 ms of hemichannels in outside-out patches at pH 6.0 is essentially the same as the mean latency to opening of hemichannels in inside-out patches when switched from 6.0 to 7.5 (636 ms). These values are more than three times longer than the mean closed time at pH 7.5 , indicating that low cytoplasmic pH favors the hemichannel to adopt a closed state with a mean dwell time of ∼600 ms. Dwell time analysis of single hemichannels at pH 7.5 indicate that this closed state is a small but visible component of a complex set of closed states (data not shown). These data argue that closure of hemichannels by low extracellular pH can be explained by H + acting intracellularly. If there is no extracellular site for H + , and H + action requires entry through the pore to access the cytoplasmic site, then the expectation would be that extracellular acidification would also have the same polarity of voltage dependence as internal acidification due to the conformational sensitivity. As evidenced in Fig. 12 E with applications of pH 6.0 solutions to the extracellular face of a hemichannel, the voltage dependence indeed has the same polarity. There is a tendency of H + to close hemichannels more effectively at inside negative voltages, consistent both with the conformational dependence of the cytoplasmic site and with an increased flux of H + through the hemichannel to access the cytoplasmic site. The combination of reduced H + influx and conformational sensitivity makes Cx46 hemichannels appear nearly insensitive to low extracellular pH at large positive voltages. At inside negative voltages, gating transitions of Cx46 hemichannels between fully open and fully closed states are relatively slow, and appear to involve transitions among numerous, short-lived subconductance levels . We have termed these slow transitions “loop gating” transitions . Acidification-induced closures are similarly slow for Cx46 hemichannels, as demonstrated in Fig. 13 B. Approximately 100 ms after rapidly switching to pH 6.0 IPS, a widely fluctuating closing transition began and took ∼100 ms to complete. After closing, there were additional small fluctuations that may represent partial reopenings; similar fluctuations are seen with voltage-dependent loop gating at pH 7.5 as well. Although the time course of the acidification-induced transitions varied from ∼10 to 100 ms, all hemichannel closures at low pH in both inside-out and outside-out configurations exhibited this slow, sloppy gating between fully open and closed states. Removal of a large portion of the CT domain of Cx43 was shown to render Cx43 cell–cell channels nearly insensitive to acidification . In the same connexin, H95, which is highly conserved among connexins, was found to be important in pH gating, possibly as a site of protonation with intracellular acidification . We tested whether truncation of CT or mutation of H95 affected pH gating in Cx46 hemichannels expressed in Xenopus oocytes. Fig. 14 shows currents in Xenopus oocytes expressing wild-type and mutant Cx46 hemichannels elicited by depolarization to various potentials (+5 to +15 mV). Oocytes were exposed to 100% CO 2 -equilibrated bath solutions for ∼140 s. BCECF monitoring of an oocyte exposed to 100% CO 2 for the same amount of time revealed that pH i would have dropped to 6.0 uniformly within the oocyte by the end of the application and would have recovered to pH 7.5 by the fourth depolarization after washout (data not shown). For wild-type Cx46 , currents decreased rapidly with CO 2 and recovered quickly upon washing (the current is near control amplitude by the next trace). Cx46 hemichannels truncated at residue 259, Cx46ΔCT 259 , responded with no noticeable difference compared with wild type . This mutant lacks 82% of the CT. These data indicate that, unlike in Cx43, most of CT is not necessary for pH gating in Cx46. We substituted Asp and Cys for His95. The side chain pK a should be significantly shifted in Asp (∼3.9 for Asp, compared with 6.1 for His), decreasing the likelihood that position 95 is protonated with acidification to pH 6.0. The side chain pK a of Cys is more basic, 8.1, increasing the likelihood that position 95 is protonated even at neutral pH. Both Cx46*H95D and Cx46*H95C functionally expressed as hemichannels. With 100% CO 2 application, both Cx46*H95D and Cx46*H95C hemichannel currents were nearly abolished , as with wild-type currents. The recovery of Cx46*H95C currents from acidification was markedly slower than for the other hemichannels, requiring more than twice the time to recover. The similarity of the closing kinetics and degree of current reduction for both mutants compared with wild type suggests that the apparent pK a of Cx46 pH sensitivity does not reflect the pK a of the side chain at position 95. The delayed recovery of Cx46*H95C currents after acidification with 100% CO 2 is not likely due to a shift in the pH sensitivity to more basic values, based on the BCECF data indicating that pH i would have recovered to control values by the fourth trace after washout. Inspection of single H95 mutant hemichannels was difficult, as their expression was consistently lower than that of wild type. In one successful cell-attached patch of Cx46*H95D hemichannels, unitary conductance was reduced by ∼60% and voltage gating appeared flickery (data not shown). These data suggest that H95 is not required for pH gating in Cx46, but moderate shifts in pH sensitivity with H95 substitutions could have gone undetected by our procedure. Also, H95 substitutions can affect gating and conductance, and possibly structure, which confounds interpretations of their effects on pH gating. That H95 (H94 in Group I connexins) is highly conserved in connexins and that a number of H95 substitutions in Cx43 are nonfunctional suggest that this residue may play an important structural role . The importance of this residue in GJ channel function is also indicated by the naturally occurring H94N and H94Y substitutions in humCx32 that causes CMTX (X-linked form of Charcot-Marie-Tooth disease), a peripheral neuropathy that leads to axonopathy and demyelination . Cx46 hemichannels and cell–cell channels share a high sensitivity to cytoplasmic pH. They also both exhibit an incomplete recovery from prolonged acidification. The lack of complete recovery was particularly evident for electrical coupling between Neuro-2a cell pairs exposed to 100% CO 2 for ∼45 s . Junctional conductance was completely abolished and remained undetectable for several minutes after washout, long after pH i would have returned to normal values. The recovery that did occur was much slower than the relatively rapid recovery with shorter CO 2 exposures (e.g., 15 s) and never reached the original conductance. We did not quantify the degree of recovery because it slowly increased with time and took so long that maintenance of the whole-cell recording was compromised. Recovery of macroscopic Cx46 hemichannel currents from prolonged acidification in single Xenopus oocytes was faster and often more complete. However, we often observed increases in expression over time and did not try to control temperature or adjust the amount of mRNA injected in the oocytes to stabilize expression. Excised patches containing hemichannels also showed incomplete recovery from prolonged acidification on the cytoplasmic side, indicating that soluble cytoplasmic factors are not necessary. When hemichannel activity in excised patches was lost during prolonged acidification, we did not see recovery even after times as long as 10 min. Reports of recovery of g j from acidification in Xenopus oocyte cell pairs expressing other connexins and mutants range from poor to good . It is possible that there are differences among connexins and/or cells' abilities to recover from prolonged acidification. From the hemichannel data obtained from patches, it appears that prolonged acidification causes a population of Cx46 channels and hemichannels to enter a nonconducting state from which there is slow or no recovery. We provisionally term the process of entering this state pH inactivation. The fraction of channels and hemichannels entering the inactivated state increases with the duration of acidification. The remaining fraction recovers rapidly upon washing. We consider this reversible form of pH regulation to be pH gating. We cannot distinguish whether pH gating and pH inactivation are mediated by the same or separate sites of H + action. We have not determined whether recovery from the pH-inactivated state is absolutely prohibited. The very slow recovery of Cx46-mediated coupling in Neuro-2a cells after prolonged acidification may represent exit from the pH-inactivated state or may represent another process such as formation of new channels. Although we have no mechanistic explanation for the long-term pH inactivation of connexin channels and hemichannels, this form of regulation may be important in delaying the recoupling of cells that are recovering from acidosis, such as that which can accompany ischemia . We have observed incomplete recovery of g j with prolonged exposure to CO 2 in other connexins expressed in HeLa cells, such as Cx32 and Cx43 (Bukauskas, F.F., unpublished results), suggesting that pH inactivation may be widespread among connexins. If this were the case, the titration curves obtained during experiments in which cells are slowly acidified over a period of 30–60 min would reflect both the pH gating and inactivation processes . Inactivation by pH could decrease the number of active channels with time, thus shifting the apparent pK a of pH gating to more alkaline values. An obvious and simple conclusion than can be made from our excised patch experiments is that pH gating of Cx46 hemichannels does not require a cytoplasmic intermediate. The possibility that Ca 2+ acts as an intermediary in Cx46, at least over a physiologically relevant concentration range of 10 −7 to 10 −5 M, was also ruled out, as were the buffers we used, HEPES and PIPES. These data contrast the key role of Ca 2+ proposed by Peracchia et al. and Cotrina et al. . Lowering of intracellular pH without a concomitant rise in intracellular Ca 2+ was reported to have no effect on g j in Xenopus oocytes expressing the endogenous Cx38 and in Novikoff hepatoma cells, which express Cx43 . Thus, acidification was proposed to have no role except to raise intracellular Ca 2+ , which in turn closed GJ channels by a Ca 2+ /calmodulin-mediated mechanism. While we did not quantitatively assess the effects of Ca 2+ on pH sensitivity over the full range of pH in Cx46 hemichannels, closure induced by application of pH 6.0 to excised patches was as robust in 10 −7 M [Ca 2+ ] as in 100-fold higher concentrations. Furthermore, the use of BAPTA-containing pipettes in Neuro-2a cell pairs expressing Cx46 did not prevent uncoupling with application of CO 2 or acidified solutions. At least for Cx46, a rise in Ca 2+ does not appear to mediate or be necessary for acidification-induced closure. We did not examine pH sensitivity in Ca 2+ concentrations <10 −7 M and, therefore, cannot rule out the possibility that some amount of Ca 2+ is necessary to express sensitivity to pH. With no need for a soluble intermediate for Cx46, one is left with either a direct action of H + on the connexin itself or an indirect action via a membrane-associated protein. Direct effects of H + on Cx46 may be distinguished from indirect effects via membrane-associated intermediates by examining the speed of H + action in excised patches subjected to rapid solution switching. Although modulation by means of soluble cytoplasmic second messengers is impossible in excised patches, membrane-delimited pathways, in which indirect effects on channels are mediated by close proximity to a membrane-bound intermediate, can be quite rapid. For example, muscarinic responses of I K(ACh) channels via G-proteins can develop with average latencies of ∼100 ms; effects could be seen in individual channels as early as ∼30–40 ms . We observed a minimal latency to the onset of closure near zero in ensemble currents with fast application of pH 6.0 to the cytoplasmic side of an open hemichannel. Thus, the effects of H + on Cx46 hemichannels appears to be too fast to result from a membrane-delimited process that would involve activation, lateral diffusion, and contact of an intermediate with the connexin. Recently, Cx32 has been shown to contain two cytoplasmic calmodulin-binding domains . However, bound calmodulin is unlikely to have a role as mediator of the rapid and reversible form of H + -induced closure in Cx46 as we have shown this effect to be quite Ca 2+ insensitive. Mutagenesis, in principle, should also provide a means of distinguishing direct from indirect actions by identifying titratable residues. However, mutational studies of connexins performed thus far indicate that this approach is not likely to be straightforward. Mutations of the putative titratable His residue at position 95 in Cx43 have not clearly established the role of H95 in pH gating . We qualitatively examined the pH sensitivity of mutants of Cx46 similar to those examined in Cx43. We replaced H95 with an acidic residue, aspartate, and a more basic residue, cysteine. The lack of an obvious effect of His95 substitutions on susceptibility to acidification suggests that this residue may not be responsible for pH gating in Cx46. Moreover, removal of 82% of CT did not appear to severely impair acidification-induced closure in Cx46; a similar degree of truncation of CT in Cx43 did reduce pH sensitivity . These data suggest that the mechanisms of pH dependence in Cx46 and Cx43 differ. The inside-out excised patch permitted rapid, uniform acidification, and recovery was complete with brief exposures; thus, it was reasonable to determine a titration curve, which is an equilibrium measure that assumes complete reversibility. We could have obtained a titration curve when acidifying outside-out patches, but we established that H + applied extracellularly acts intracellularly at the same site as intracellular acidification. Given that the same site is acted on in both excised patch configurations and that we could not control intracellular pH during extracellular application, it made little sense to obtain a titration curve for extracellular H + in outside-out patches. We could not obtain accurate titration curves in intact cells because during the relatively slow cytoplasmic acidification and recovery, what we term pH inactivation makes hemichannel closure incompletely reversible. Thus, a change in pH sensitivity upon excision of patches remains a possibility. However, comparison of the effect of acidification with CO 2 and with HCl on oocytes and the titration curve for acidification of the intracellular side of inside-out patches suggests that patch excision does not reduce pH sensitivity. By applying solutions with similar pH values that induced different rates of intracellular acidification (i.e., media equilibrated with 100% CO 2 and media adjusted to pH 5.5 with HCl), we demonstrated that the decrease in g j is similar in time course to the decrease in pH i . This result suggests that Cx46 cell–cell channels are sensitive only to intracellular acidification, which would place the pH sensor on the cytoplasmic side. We were surprised that acidified HEPES/PIPES buffered solutions, lacking CO 2 or a membrane permeable weak acid, were still able to substantially lower intracellular pH. For the case of a whole-cell patch of a Neuro-2a cell, application of pH 5.5 medium decreased pH i from 7.25 to 6.0 in 300 s . For calculation purposes, we considered a 20-μm diameter cell filled with a solution buffered with 10 mM HEPES (pK a of 7.55). At pH 7.25, the ratio of deprotonated to protonated buffer is 0.5. At pH 6.0, that ratio drops to 0.028. Thus, the concentration of protonated buffer increases from 6.67 mM at pH 7.25 to 9.73 mM at pH 6.0. To produce this change, ∼7.7 × 10 9 H + ions need to cross the membrane at an average rate of ∼2.6 × 10 7 /s. Perhaps protons are diffusing through ion channels in the Neuro-2a cell membrane. Zhou and Jones were able to block intracellular acidification in bullfrog sympathetic neurons with 1 μM tetrodotoxin, suggesting that Na + channels are the pathway for H + entry. We did not attempt to block intracellular acidification by blocking ion channels in Neuro-2a cells or Xenopus oocytes. Unlike Cx46 junctional currents in Neuro-2a cells, Cx46 hemichannel currents recorded macroscopically from Xenopus oocytes or from excised patches are rapidly affected by both intracellular and extracellular acidification. However, the sensitivity to extracellular acidification appears to be explainable by H + entry through the pore to reach the cytoplasmic site. The primary evidence that extracellular H + acts intracellularly is the similarity of the mean closed time in extracellular pH 6.0 and the mean latency to opening of hemichannels rapidly switched from pH 6.0 to 7.5 on the cytoplasmic side. These two time intervals should be equal if extracellular and intracellular H + ions act at the same intracellular site, and the following reasonable assumptions hold: closed hemichannels do not conduct H + and pH at the sensor after hemichannel closure rapidly reaches the value in the solution bathing the inner face. These analyses used multiple low-pH applications 2 s in duration so as not to evoke pH inactivation. The agreement between closed time and latency to open also argues against the possibility that protons or buffer permeate through the seal between the membrane and the patch pipette glass to decrease intrapipette pH enough to affect channel open probability. Also in support of extracellular H + acting intracellularly is the conservation of the polarity of voltage dependence of low pH action from either side. The polarity of voltage dependence with H + added to the cytoplasmic side is opposite that expected for H + movement to a pH sensor or binding site located deep in the pore, and thus the voltage dependence is likely to result from a charge movement associated with the conformational change of gating. The polarity of voltage dependence is the same with H + applied externally, when the field would increase flux to an intracellular site. Since the pH gradient at the cytoplasmic mouth of an open hemichannel is unlikely to extend very far, the H + binding site is likely to be near the entrance to the pore. The apparent lack of extracellular pH sensitivity of Cx46 cell–cell channels in Neuro-2a cells is readily explained by the removal of the critical pathway for H + entry from the outside, namely through the open hemichannel. Cell–cell channels, once docked, establish a tight electrical seal as evidenced by the absence of a leakage current during de novo channel opening . Thus, for Cx46 cell– cell channels exposed to low extracellular pH, titration of the pH sensor would require sufficient acidification of the cytoplasm, presumably through other pathways not in such close proximity to the pH sensor as that through the hemichannel. Such a mechanism would take time and is consistent with the slower time course of uncoupling in response to application of solutions acidified with HCl. Likewise, the faster time course of uncoupling in response to application of 100% CO 2 equilibrated solution is consistent with the faster time course of cytoplasmic acidification by means of CO 2 permeating through the membrane. With a minimal latency to the onset of closure near zero, as demonstrated by the ensemble currents, we conclude that the pH sensor is located on Cx46 itself. The same pH sensor is likely to mediate pH sensitivity both in Cx46 cell–cell channels and Cx46 hemichannels. It is certainly possible that the conformations of docked and undocked Cx46 hemichannels differ so that the pK(s) of titratable residue(s) differ or even that different titratable residue(s) are exposed. While we have not identified a critical residue for pH sensitivity, we would expect that a substitution that greatly altered pH sensitivity would do so both in cell–cell channels and hemichannels. At the single channel level, cell–cell channels both in vertebrate and invertebrate tissues show two types of gating transitions: (a) fast transitions between the fully open state and a long-lived substate termed the residual conductance state, and (b) “slow” transitions (tens of milliseconds) between the fully open state and a fully closed state . The fast transitions to and from the residual subconductance state are evoked by transjunctional voltages of either polarity. V j gating generally does not close the GJ channel completely, which can largely explain the macroscopic minimum (residual) conductance characteristic of V j dependence . In contrast, the slow transitions close the channel completely. These transitions are infrequent in most types of cell– cell channels, although their frequency may be increased at large V j s, but are readily observed in response to a variety of chemical agents known to markedly decrease g j ; e.g., CO 2 , volatile anesthetics, alkanols, and arachidonic acid . Bukauskas and Peracchia proposed that the fast V j gating transitions and the chemically induced slow transitions represent two distinct forms of gating. Interestingly, de novo channel formation also involves slow transitions, which were described as formation currents . We demonstrated similar slow gating transitions to the fully closed state and fast transitions to a substate in unapposed Cx46 hemichannels and proposed the existence of two distinct voltage gates, a V j gate and a “loop gate” . The name “loop gate” was assigned because of the resemblance of the slow hemichannel gating transitions to the formation currents, which presumably involve the extracellular loops. Recent studies of Cx46 hemichannels using substituted cysteine accessibility measurement indicate that the loop gates are located extracellular to the 35 position in the first transmembrane domain . In this study, we demonstrate that acidification closes Cx46 hemichannels fully with transitions that resemble those of loop gating and CO 2 -induced gating in cell– cell channels. This similarity suggests that the same mechanism may operate to close cell–cell channels and hemichannels in response to intracellular acidification. While we have not quantitatively analyzed the complex transitions induced by acidification in Cx46 hemichannels, they appear to differ from voltage induced loop gating transitions in that they are more noisy and less resolvable into discrete sub-conductance levels . This difference suggests that loop and pH gating, while sharing common elements, may also use discrete elements. Nevertheless, the voltage dependence of pH gating suggests that both voltage and pH act on the loop gate. The use of functional, unapposed hemichannels represents an approach complementary to the study of cell– cell channels. A major advantage of hemichannels demonstrated here and in an earlier paper is the possibility of obtaining cell-attached and excised membrane patches that allow for direct, stable, and low capacitance recordings at single and multiple channel levels. Recording of cell–cell channels requires double whole-cell patch clamping, which introduces whole cell capacitance and a limited prospect of recording from a single channel. While direct “attached patches” on junctional membranes have been reported in earthworm septate axons , this technique has not proved widely applicable to vertebrate cell pairs. The slow rate and lack of uniform solution exchange in cell pairs has also hindered studies of modulation of cell–cell channels. Excision of patches in inside-out or outside-out configurations allowed us to change pH rapidly and uniformly at either face of Cx46 hemichannels. It also allowed us to examine the effects of H + in a cell-free environment and directly address the issue of cytoplasmic intermediates. While hemichannels in isolation facilitate study of connexin modulation by H + and other factors, they are not cell–cell channels and their properties as components in cell–cell channels may differ. However, knowledge of the properties of hemichannels in isolation is likely to reduce the measurements required to characterize cell–cell channels satisfactorily. Moreover, comparison of hemichannels and cell–cell channels should clarify the role of hemichannel interactions in channel function. | Study | biomedical | en | 0.999996 |
10228185 | The inherited disease cystic fibrosis (CF) 1 is characterized by secretion of a thick viscous mucus that plugs the submucosal glands and small airways. This leads to chronic airway infections and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) . The predominant site of CFTR expression in the human lung is the serous cells of the submucosal glands . Serous cells account for 60% of the cellular volume of the submucosal gland in human airways . Stimulation of an isotonic fluid secretion from the serous cells contributes to the hydration of the secretions from the mucous cells, thereby forming the low viscosity mucus that lines the conducting airways. Serous cells are also a major source of antimicrobial enzymes and peptides that help maintain an aseptic environment in the lungs . Salt concentration can influence the activity of these antimicrobial agents and it was recently suggested that altered salt concentration in the airway surface fluid may contribute to chronic airway infection in CF . Thus, the serous cells make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions and represent a potentially important target in CF therapy. These considerations indicate the importance in understanding the mechanisms of fluid and electrolyte transport by serous cells. Shen et al. screened 12 cell lines derived from lung adenocarcinomas in an attempt to identify a cell line that displayed electrophysiological properties consistent with human airway serous cells. They identified the Calu-3 cell line as being serous cell in nature, forming a monolayer with a transepithelial resistance of ∼100 Ω · cm 2 , expressing high levels of CFTR and responding to both cAMP- and Ca 2+ -mediated agonists with changes in net transepithelial ion transport as measured by short circuit current (I sc ) . Several studies have produced variable results in the basal and stimulated transport properties of the Calu-3 cells and the ionic basis of the responses to secretory agonists remains unsettled . In this report, we present studies with Calu-3 cells that displayed a low basal I sc (13 μA cm −2 ) and robust sustained responses to secretory agonists enabling the measurement of isotopic fluxes. The results demonstrate that Calu-3 cells, when stimulated by forskolin, secrete HCO − 3 by a Cl − -independent, Na + -dependent, 4,4′-dinitrostilben-2,2′-disulfonic acid (DNDS)–sensitive, electrogenic mechanism. Secondly, when stimulated by 1-ethyl-2 benzimidazolinone (1-EBIO), an activator of the basolateral membrane Ca 2+ -activated K + channels (K Ca ) , HCO − 3 secretion is reduced and the Calu-3 cells secrete predominately Cl − by a bumetanide-sensitive, electrogenic mechanism. Calu-3 cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 (1:1) supplemented with 15% fetal bovine serum and 2 mM glutamine. The cells were incubated in a humidified atmosphere containing 5% CO 2 at 37°C. For measurements of short-circuit current (I sc ), Calu-3 cells were seeded onto Costar Transwell cell culture inserts (0.33 cm 2 ) or Snapwell inserts (1.1 cm 2 ). Both the Transwell and Snapwell inserts were collagen-coated overnight with 0.01% human placenta collagen type VI ( Sigma Chemical Co. ). On day one, the medium bathing the apical surface was removed to establish an air interface. Apical medium was removed and the cells fed every 48 h. After ∼7–14 d, the cells formed a confluent monolayer that held back fluid, thus maintaining an apical air interface. Short circuit current measurements were performed after an additional 14–28 d in culture. Patch-clamp experiments were performed on single cells plated onto glass cover slips 18–48 h before use. For measurements of I sc , the bath solution contained (mM): 120 NaCl, 25 NaHCO 3 , 3.3 KH 2 PO 4 , 0.8 K 2 HPO 4 , 1.2 MgCl 2 , 1.2 CaCl 2 , and 10 glucose. Mannitol was substituted for glucose in the mucosal solution to eliminate the contribution of Na + glucose cotransport to I SC as previously reported by Singh et al. . The pH of this solution was 7.4 when gassed with a mixture of 95% O 2 –5% CO 2 at 37°C. For the Cl − -free solution, equimolar Na-gluconate replaced NaCl, 1 mM Mg-gluconate replaced MgCl 2 , and 4 mM Ca-gluconate replaced CaCl 2 . Calcium was increased to 4 mM to compensate for the Ca 2+ buffering capacity of the gluconate. The HCO − 3 -free buffer consisted of (mM): 145 NaCl, 3.3 KH 2 PO 4 , 0.8 K 2 HPO 4 1.2 MgCl 2 , 1.2 CaCl 2 , 10 HEPES, pH adjusted with NaOH, 10 glucose or mannitol and was gassed with air. For the Na + -free Cl − -free solution, equimolar N -methyl- d -glucamine–gluconate replaced NaCl, choline-HCO 3 replaced NaHCO 3 , 1 mM Mg-gluconate replaced MgCl 2 , and 4 mM Ca-gluconate replaced CaCl 2 . This solution contained 10 μM atropine to block the cholinergic effect of choline . The effects of forskolin and 1-EBIO on apical membrane Cl − currents (I Cl ) were assessed after permeabilization of the serosal membrane with nystatin (360 μg/ml), and the establishment of a mucosa-to-serosa Cl − concentration gradient. Serosal NaCl was replaced by equimolar Na-gluconate and Ca 2+ was increased to 4 mM with Ca-gluconate. Nystatin was added to the serosal membrane 15–30 min before the addition of drugs. Successful permeabilization of the basolateral membrane was based upon the recording of a current consistent with the mucosal-to-serosal flow of negative charge. The effect of 1-EBIO on basolateral membrane K + currents (I K ) was assessed after permeabilization of the apical membrane with nystatin (180 μg/ml) for 15–30 min, and establishment of a mucosa-to-serosa K + concentration gradient. For measurements of I K , mucosal NaCl was replaced by equimolar K-gluconate, while serosal NaCl was substituted with equimolar Na-gluconate. Calcium and Mg 2+ salts were replaced as above. During inside-out patch-clamp recordings, the bath contained (mM): 145 K-gluconate, 5 KCl, 1 MgCl 2 , 1 EGTA, 0.78 CaCl 2 , (free Ca 2+ = 400 nM), and 10 HEPES, pH adjusted to 7.2 with KOH. The pipette solution contained (mM): 140 K-gluconate, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , and 10 HEPES, pH adjusted to 7.2 with KOH. For outside-out recordings, the bath contained 1 mM CaCl 2 in the absence of any added EGTA, while the pipette solution Ca 2+ was buffered to 200 nM with EGTA (0.71 mM Ca 2+ , 1 mM EGTA). Transwell inserts were mounted in an Ussing chamber (Jim's Instruments). Snapwell inserts were mounted in Ussing chambers (NaviCyte), and the monolayers were continuously short-circuited after fluid resistance compensation using automatic voltage clamps (558C-5; Iowa Bioengineering). Transepithelial resistance (R T ) was measured by open-circuiting the monolayer, or with a 2-mV bipolar pulse and the resistance calculated by Ohm's law. Forskolin, 1-EBIO, clotrimazole, 293B, and acetazolamide were added to both sides of the monolayers at the indicated concentrations. Bumetanide and charybdotoxin (CTX) were added only to the serosal bathing solution. 20 min after the Snapwell filters were mounted in Ussing chambers, isotopes ( 36 Cl, 22 Na, or 86 Rb) were added to the bath solution on one side of the monolayers. After an additional 20 min, by which time isotopic fluxes had reached a steady state, two 0.4-ml samples were taken from the unlabeled side and fresh unlabeled solution of equal volume was added. This time was considered time = 0 (T 0 ), and samples were taken thereafter at 15-min intervals for the next 75 min. When the effects of forskolin, 1-EBIO, or forskolin plus 1-EBIO were studied, the drugs were added to the serosal and mucosal sides at T 30 and fluxes before (T 0 − T 30 ) and 15 min after the drug additions (T 45 − T 75 ) were compared. Isotope activities were determined in a Packard liquid scintillation counter. All samples were weighed and these volumes were used to correct the chamber volume and to calculate the unidirectional ion fluxes using standard equations . The net residual ion flux (J R net ) was calculated from the difference in I sc and the net fluxes of Cl − , JCl net ; Na + , J Na net ; and Rb − , J Rb net , where J R net = I sc − (J Na net + Ξ Rb net − Ξ Cl net ). Single channel currents were recorded in the inside-out and outside-out patch-clamp recording configuration using a List EPC-7 amplifier (Medical Systems) and recorded on videotape for later analysis as described previously . Pipettes were fabricated from KG-12 glass (Willmad Glass Co.). All recordings were done at a holding voltage of −100 mV. The voltage is referenced to the extracellular compartment as the standard method for membrane potentials. Inward currents are defined as the movement of positive charge from the extracellular compartment to the intracellular compartment, and are presented as downward deflections from baseline in all recording configurations. Single channel analysis was performed on records sampled after low-pass filtering at 400 Hz. Data records for all experimental conditions were at least 60-s long. The nP o (the product of the number of channels, n , and the channel open probability, P o ) of the channels was determined using Biopatch software (3.11; Molecular Kinetics). nP o was calculated from the mean total current ( I ) divided by the single channel current amplitude ( i ), such that nP o = I / i . i was determined from the amplitude histogram of the current record. Nystatin was a generous gift from Dr. S. Lucania (Bristol Meyers-Squibb). 293B (trans-6-cyano-4-( N -ethylsulfonyl- N -methylamino)- 3-hydroxy-2,2-dimethyl-chroman) was a generous gift from Dr. Rainer Greger (Albert-Ludwigs-Universtat, Freiberg, Germany). 1-EBIO was obtained from Aldrich Chemical Co. Acetazolamide, clotrimazole, and bumetanide were obtained from Sigma Chemical Co. Forskolin was obtained from Calbiochem . DNDS was from Pfaltz and Bauer. Charybdotoxin was obtained from Accurate Chemical and Scientific Corp. and made as a 10 μM stock solution in standard bath solution. 1-EBIO, 293B, and clotrimazole were made as >1,000-fold stock solutions in DMSO. Nystatin was made as a 180 mg/ml stock solution in DMSO and sonicated for 30 s just before use. Forskolin and bumetanide were made as 1,000-fold stock solutions in ethanol. Cell culture medium was obtained from GIBCO BRL . All data are presented as means ± SEM, where n indicates the number of experiments. In total, we evaluated 216 filters with standard bath solutions on the mucosal and serosal membrane surfaces. The basal I sc and R T under these conditions averaged 13 ± 0.8 μA · cm −2 (range 2–21 μA · cm −2 ) and 353 ± 14 Ωcm 2 (range 187–667 Ωcm 2 ), respectively. Forskolin (2–10 μM) induced, in all filters tested ( n = 109), a damped oscillatory response that became stable and sustained after 5–10 min at a plateau value of 66 ± 4 μA · cm −2 (range 50–103 μA · cm −2 ). A representative current trace is shown in Fig. 1 A. The increase in I sc caused by forskolin was accompanied by a decrease in R T to an average of 189 ± 7 Ωcm −2 (range 111–333 Ωcm −2 ). Bumetanide (20 μM), an inhibitor of the NaK2Cl cotransporter, caused only a small inhibition of the forskolin stimulated I sc (Δ −4.9 ± 1.3 μA · cm −2 , n = 11). The failure of bumetanide to inhibit the forskolin-stimulated increase in I sc suggests that the NaK2Cl cotransporter does not contribute to the I sc , and this raised the question whether the I sc was due to Cl − secretion. Additional experiments were performed to establish the ionic basis of the forskolin-stimulated I sc . To help elucidate the ionic basis of the forskolin- induced increase in I sc , we performed unidirectional ion flux measurements with 36 Cl, 22 Na, or 86 Rb; the latter was used as a measure of K + movements. The Cl − flux studies are shown in Fig. 1 B and are summarized together with Na + and Rb + fluxes in Table I . As in the previous experiments, there was a small basal I sc under control conditions of ∼8 μA · cm −2 (i.e., 0.3 μEq · cm −2 · h −1 ) that was stimulated 6–10-fold by forskolin in the subset of 36 filters used for the flux studies. Under control conditions, there was no net movement of Cl − or Rb + and a small net absorption of Na + . Forskolin increased both unidirectional fluxes of Cl − four- to fivefold . Both Rb + fluxes were increased 1.5-fold, but forskolin had no effect on the fluxes of Na + (Table I ). Because both unidirectional fluxes of Cl − and Rb + were increased to a similar extent, there was no net flux of Cl − or Rb + caused by forskolin. The difference between I sc and the net flux of each ion was calculated and is given in Table I as J R net . Because there was no net flux of Cl − or Rb + under control or forskolin conditions, neither of these ions account for the basal or forskolin-stimulated I sc . However, the net absorption of Na + fully accounts for the control, basal I sc , and a small portion (15%) of the I sc in the forskolin-stimulated cells. When the flux studies for Cl − , Na + , and Rb + were combined to calculate theJ R net using the mean I sc (control 0.31 ± 0.053 μEq · cm −2 · h −1 ; forskolin 2.60 ± 0.144 μEq · cm −2 · h −1 , n = 36) for the studies in Table I , the control J R net was −0.12 ± 0.11 μEq · cm −2 · h −1 and the forskolin J R net was 2.37 ± 0.189 μEq · cm −2 · h −1 . These results demonstrate that the forskolin-induced increase in I sc cannot be accounted for by the net transepithelial secretion of Cl − or the absorption of Na + or K + . Rather, the increase in I sc caused by forskolin must be attributed to the net movement of an unmeasured ion, often referred to as the net residual ion flux, J R net . Because HCO − 3 is the only remaining ion of significant concentration, J R net is likely to be due to the net secretion of HCO − 3 and additional experiments were performed to test this hypothesis. Ion substitution experiments were performed to help further establish the ionic basis of the forskolin-stimulated I sc . Consistent with the failure of bumetanide to inhibit the forskolin-stimulated I sc and the J Cl net of only 0.09 ± 0.257 μEq · cm −2 · h −1 , substitution of Cl − with gluconate caused only a partial reduction of the response to forskolin . Similar to the control response, the I sc response to forskolin in Cl − -free solution was rapid in onset with a transient peak and a sustained plateau of 46 ± 1.6 μA · cm −2 ( n = 24) . The subsequent addition of Cl − (30–60 mM) to the mucosal or serosal solution did not cause a further increase in I sc (data not shown). As in the Cl − containing solution, bumetanide (20 μM serosal) had no effect on the forskolin-stimulated I sc (Δ 0.15 ± 0.76 μA · cm −2 , n = 6) . In contrast, removal of HCO − 3 from the mucosal and serosal bathing solutions resulted in a greatly diminished response to forskolin . After a transient response, I sc was increased by only 4 ± 1 μA · cm −2 ( n = 10) in HCO − 3 -free solutions. Substitution of Na + with N -methyl- d -glucamine, Cl − with gluconate, and NaHCO 3 with choline HCO 3 also resulted in a greatly reduced response to forskolin. Forskolin caused a transient increase in I sc without a sustained plateau in the Na + -free, Cl − -free, HCO − 3 - containing solution , which resembles the response in HCO − 3 -free media. However, the subsequent addition of Na + (30 mM) to the serosal but not the mucosal solution caused a sustained increase in I sc of 24 ± 1.0 μA · cm −2 ( n = 12) in forskolin-stimulated cells . Addition of Na + (30 mM) to the serosal solution be-fore forskolin caused a small decrease in I sc Δ −7.6 ± 0.2 μA · cm −2 ( n = 12) as expected for the serosal-to-mucosal diffusion of a cation. This decrease in I sc was reversed and I sc rose to a sustained level of 23 ± 0.8 μA · cm −2 ( n = 12) with the subsequent addition of forskolin. Thus, the forskolin-stimulated increase in the I sc was Cl − independent but Na + and HCO − 3 dependent. The above results are consistent with forskolin-stimulated net secretion of HCO − 3 . To further test this hypothesis, the pharmacological sensitivity to various inhibitors of HCO − 3 transport were evaluated. The carbonic anhydrase inhibitor, acetazolamide (1 mM mucosal and serosal), caused a 27% decrease (a reduction of 13 ± 1 μA cm 2 , n = 6) in the forskolin-stimulated I sc in Cl − -free solutions . DNDS (3 mM), an inhibitor of Cl − /HCO − 3 exchangers and Na + :HCO − 3 cotransporters, was without effect when added to the mucosal solution ( Δ = 0.2 μA · cm −2 , n = 6), but caused an inhibition of 56% (Δ −26 ± 1 μA · cm −2 , n = 6) when added to the serosal side in Cl − -free solutions. Similar results were obtained in Cl − -containing solutions (Δ −2.5 ± 1.3 μA · cm −2 , n = 6 mucosal; Δ −27 ± 2 μA · cm −2 , n = 6 serosal). The half maximal inhibitory concentration ( K i ) for serosal DNDS was 300 μM. The inhibitory effects of serosal DNDS and acetazolamide were additive, together causing a 75% decrease in I sc . The Na + -K + -ATPase inhibitor, ouabain (100 μM), caused an immediate and complete inhibition of the forskolin-stimulated I sc . Neither CTX (50 nM), a blocker of Ca 2+ activated K + channels , nor 293B (100 μM), a blocker of the cAMP/ PKA activated K + channel inhibited the forskolin-stimulated I sc . The nonselective K + channel blocker, Ba 2+ (5 mM serosal side), inhibited the forskolin-stimulated I sc by only 10 ± 2 μA · cm −2 ( n = 6). The requirement for serosal Na + , the inhibition by ouabain, and the partial inhibition by serosal DNDS suggests some of the secreted HCO − 3 is mediated by the uptake of HCO − 3 across the basolateral membrane on a Na + :HCO − 3 cotransporter. 2 The partial inhibition of I sc by acetazolamide suggests some of the secreted HCO − 3 originates from a metabolic source. The Cl − independence and the failure of mucosal DNDS to inhibit I sc suggests the exit of HCO − 3 across the apical membrane is not mediated by a Cl − /HCO − 3 exchanger. The above results are consistent with the conclusion that forskolin stimulation causes the electrogenic secretion of HCO − 3 . To further test this hypothesis, we performed experiments to determine whether forskolin caused an alkalinization of the apical solution. Calu-3 cells were studied under open circuit conditions with a small volume of fluid (100 μl) on the apical surface (1.1 cm 2 ) and 5 ml of continuously gassed (95% O 2 / 5% CO 2 ) NaCl, NaHCO 3 buffer, pH 7.4, on the serosal side. Cells were incubated without or with forskolin (2 μM) and the apical solution collected after 90 min. The apical sample was thoroughly gassed before measuring its pH with a miniature pH electrode. Studied in this manner, we found forskolin caused an alkalinization of the apical solution to a pH of 7.8 ± 0.06 ( n = 6), whereas control untreated filters showed a small acidification of the apical solution, pH 7.3 ± 0.05 ( n = 6). The forskolin-stimulated alkalinization of Δ0.5 pH over a 90-min period corresponds to the net movement of HCO − 3 of 1.7 μeq · cm −2 · h −1 or 46 μA · cm −2 , a value in good agreement with the forskolin-stimulated increase in I sc of 53 μA · cm −2 under short circuit conditions. 3 Based on these pH measurements, the ion flux measurements, the ion substitution studies, and the pharmacology studies, we conclude that the forskolin-induced I sc response in Calu-3 cells is due to the net secretion of HCO − 3 by a Cl − -independent Na + -dependent, and DNDS-sensitive electrogenic mechanism. We previously demonstrated that the novel benzimidazolinone, 1-EBIO, induced a sustained transepithelial Cl − secretory response in rat colonic mucosa, human colonic T84 cells, and murine airway epithelia . CTX and clotrimazole inhibited the 1-EBIO– stimulated Cl − secretion consistent with the activation of basolateral membrane K + channels that was confirmed in permeabilized monolayers . Moreover, patch clamp studies demonstrated 1-EBIO activates an inwardly rectifying, calcium activated, CTX, and clotrimazole-sensitive K + channel . Permeabilized monolayers revealed 1-EBIO also activates an apical membrane Cl − conductance . The studies reported here were performed to determine if 1-EBIO would have similar effects on Calu-3 cells. In 46 experiments, 1-EBIO (1 mM) increased I sc from a basal value of 8 ± 0.8 to 62 ± 4 μA · cm −2 with only a modest decrease in R T (control 397 ± 21 Ωcm 2 vs. 1-EBIO 336 ± 20 Ωcm 2 ). A current trace of a typical I sc response to 1-EBIO is shown in Fig. 5 A. The response was rapid in onset and sustained over a long period. Dose–response studies revealed the half maximal effective concentration of 1-EBIO was ∼500 μM. Consistent with the activation of the K Ca channels, CTX (50 nM) inhibited 47% of the 1-EBIO–stimulated I sc . The half maximal effective concentration of CTX was 3.2 nM ( n = 4). Clotrimazole (10 μM), a nonpeptide inhibitor of K Ca , also inhibited 87.6 ± 1.9% ( n = 5) of the response to 1-EBIO with a K i of 1.2 μM ( n = 5). Bumetanide (20 μM) inhibited ∼50% of the 1-EBIO–stimulated I sc (Table II ). DNDS and acetazolamide caused only small (<10%) decreases in the 1-EBIO–stimulated I sc . Unidirectional fluxes of 36 Cl revealed that 1-EBIO caused the net secretion of Cl − . As in previous experiments , there was no net secretion of Cl − in control monolayers. 1-EBIO caused a sixfold increase in the serosal-to-mucosal flux of Cl − without altering the mucosal-to-serosal flux leading to net Cl − secretion. Moreover, the net secretion of Cl − fully accounted for the increase in I sc caused by 1-EBIO, leaving a small J R net of only 0.25 ± 0.263 μEq · cm −2 · h −1 . Bumetanide inhibited the serosal-to-mucosal flux of Cl − and thereby caused a 70% inhibition in J Cl− net in 1-EBIO–stimulated monolayers. The above results demonstrate Calu-3 cells secrete HCO − 3 when stimulated by forskolin and Cl − when stimulated by 1-EBIO. In the next series of experiments, we evaluated the effects of 1-EBIO on forskolin stimulated monolayers. As in the previous experiments, forskolin increased I sc from a control value of 6.8 ± 0.7 to 67 ± 4.3 μA · cm −2 ( n = 12) without causing the net secretion of Cl − and leaving a J R net nearly equal to the change in I sc . 1-EBIO further increased I sc to 114 ± 5 μA · cm −2 . Similar results were obtained if the order of the addition of forskolin and 1-EBIO were reversed. CTX inhibited 79 ± 2% ( n = 8) and bumetanide inhibited 80 ± 1% ( n = 5) of the forskolin plus 1-EBIO–stimulated I sc . When added to the forskolin-stimulated cells, 1-EBIO caused a twofold increase in the serosal-to-mucosal flux of Cl − and a J Cl− net that was nearly equal to the I sc . Thus, 1-EBIO caused a 70% decrease in the forskolin-stimulated J R net . These results suggest 1-EBIO can switch the forskolin-stimulated Calu-3 cells from HCO − 3 - to Cl − -secreting cells. One hypothesis to explain the effects of 1-EBIO on Calu-3 cells is the activation of basolateral membrane K + channels that would tend to hyperpolarize the membrane potential. The inhibition of the 1-EBIO response by CTX and clotrimazole support this hypothesis. Hyperpolarization of the membrane potential would increase the driving force for anion exit of both HCO − 3 and Cl − across the apical membrane. However, hyperpolarization of the basolateral membrane potential would also tend to decrease the driving force for basolateral membrane HCO − 3 entry on the Na + :HCO − 3 cotransporter, whose Na + to HCO − 3 stoichiometry is reported to be 1:2 or 1:3 in various cell types . A second hypothesis, and one that is not mutually exclusive with the former hypothesis, is that 1-EBIO activates apical membrane anion channels that were not activated by forskolin and that the 1-EBIO-activated channels allow for the preferential exit of Cl − over HCO − 3 . To test these hypotheses, we performed studies on permeabilized monolayers. The pore forming antibiotic nystatin was used to permeabilize the apical membrane and a transepithelial mucosal-to-serosal K + gradient was established. After permeabilization, 1-EBIO increased I K , and this was inhibited by both CTX and clotrimazole (B). In 17 experiments, 1-EBIO (1 mM) increased I K an average of 91 ± 9 μA · cm −2 and this was inhibited 66 ± 2% by CTX (50 nM, n = 10) and 95 ± 2% by clotrimazole (10 μM, n = 7). Thus, 1-EBIO does activate basolateral membrane K + channels. In contrast, forskolin (2 μM) failed to cause an increase in I K . After the establishment of a mucosal-to-serosal Cl − gradient, the addition of nystatin to the serosal membrane elicited an absorptive I Cl of 58 ± 9 μA · cm −2 . Thus, in contrast to the measurements of I K , treatment of the monolayers with nystatin appears to uncover or activate a substantial basal I Cl . Similar results were observed in T84 cells studied under the same experimental conditions . Therefore, this effect of nystatin is not unique to Calu-3 cells. The mechanisms involved in this nystatin induced increase in I Cl are unknown. The subsequent addition of forskolin (10 μM) to the nystatin-treated monolayers increased I Cl by an additional 186 ± 15 μA · cm −2 ( n = 7) . 1-EBIO failed to cause any further increase in I Cl in the forskolin treated monolayers. However, 1-EBIO alone when added to the nystatin-treated monolayers increased I Cl by an additional 74 ± 11 μA · cm −2 ( n = 6) and forskolin further increased I Cl by an additional 110 ± 12 μA · cm −2 . Thus, both forskolin and 1-EBIO when added alone can activate an apical membrane Cl − conductance in nystatin-treated Calu-3 monolayers. Forskolin caused a 2.5-fold greater increase in I Cl compared with the 1-EBIO response. The lack of specific Cl − channel blockers prevents us from determining whether the same channel or different Cl − channels are activated by forskolin and 1-EBIO. However, when forskolin and then 1-EBIO was added, the effects on I Cl were not additive, suggesting that forskolin alone can maximally activate the apical Cl − conductance. Therefore, the effect of 1-EBIO in causing the switch from HCO − 3 secretion to Cl − secretion appears to result from the activation of basolateral membrane K + channels and decreased driving force for HCO − 3 entry across the basolateral membrane. This hypothesis will be considered further in the discussion. The above results indicate that Calu-3 cells express K + channels with similar pharmacological characteristics to the K + channels we described previously in T84 cells and that this conductance may be important in altering the driving force for HCO − 3 entry across the basolateral membrane that elicits Cl − secretion in Calu-3 cells. Thus, we wished to characterize this K + channel at the single channel level. Inward and outward single-channel currents observed on excision of membrane patches into a symmetric K + bath containing 400 nM free Ca 2+ are shown in Fig. 9 A. Channel activity showed no obvious voltage dependence and required Ca 2+ in the bath (data not shown). The average current–voltage for four such patches is shown in Fig. 9 B (•). Single channel currents were inwardly rectified with average chord conductance values of 31 ± 2 pS at −100 mV and 9 ± 0.2 pS at +100 mV. The K + -to-Na + selectivity of this channel was assessed by replacing 100 mEq pipette K + with Na + ; P K /P Na was calculated from the Goldman-Hodgkin-Katz relation. Replacing pipette K + with Na + shifted the reversal potential by −20 mV . A shift of −27 mV is predicted for a perfectly K + selective electrode. From these data, the calculated K + -to-Na + selectivity ratio is 5.5:1. This conductance and K + :Na + selectivity values are similar to what has been previously reported for a Ca 2+ -activated K + channel in T84 cells as well as primary cultures of canine tracheal epithelial cells . We previously demonstrated that 1-EBIO directly activated the K Ca of T84 cells in excised patch-clamp recordings . Thus, we determined whether 1-EBIO would similarly activate K Ca in excised, inside-out single channel patch-clamp recordings from Calu-3 cells. The effect of 1-EBIO (200 μM) on one patch is shown in Fig. 10 . Under control conditions (400 nM free Ca 2+ in the bath), minimal K Ca channel activity was observed. 1-EBIO produced a large increase in channel activity that was readily reversible after washout of the 1-EBIO. In 14 inside-out recordings, 1-EBIO increased nP o from 0.08 ± 0.02 to 1.68 ± 0.39. These results indicate that this channel, as in T84 cells, is responsible for the increase in the basolateral membrane K + conductance and I sc during an 1-EBIO–mediated secretory response. We demonstrate above that the 1-EBIO–induced basolateral membrane K + conductance is sensitive to block by CTX and clotrimazole . We therefore determined whether these inhibitors would block the channel in excised outside-out and inside-out patches. The effect of CTX (50 nM) on K Ca in an outside-out patch is shown in Fig. 11 A. When holding the patch at −100 mV, addition of CTX to the outside of the channel resulted in a complete inhibition of channel activity. This block was voltage dependent and was partially relieved by voltage clamping the patch to +100 mV. The inhibition by CTX was completely reversible. Similar results were obtained in three additional outside-out patches. Clotrimazole (10 μM) also completely inhibited K Ca activity, reducing nP o from 1.59 ± 0.24 to 0.05 ± 0.02 . Thus, results from these K + channel blocker experiments further indicate that 1-EBIO is activating this inwardly rectifying Ca 2+ -activated K + conductance in Calu-3 monolayers resulting in the stimulation of Cl − secretion and the inhibition of HCO − 3 secretion. The results of our studies with Calu-3 cells demonstrate that forskolin stimulates the net secretion of HCO − 3 . Forskolin consistently caused an increase in I sc to a new sustained plateau. Ion flux studies revealed that this increase in I sc could not be explained by the net transport of Na + , Rb + , or Cl − , leaving HCO − 3 secretion as the likely basis for the increase in I sc . Ion substitution experiments demonstrated HCO − 3 , but not Cl − , was required to elicit a sustained increase in I sc with forskolin. In addition, Na + was required in the serosal bath to elicit a forskolin response. Inhibitor studies revealed that the forskolin response was sensitive to ouabain, indicating a role for the Na + /K + -ATPase. The forskolin response was also sensitive to DNDS on the serosal side but not the mucosal side, indicating a role for a basolateral membrane Na + :HCO − 3 cotransporter or Cl − : HCO − 3 exchanger. However, because Cl − was not required and serosal Na + was, the effects of DNDS are likely to result from the inhibition of a basolateral membrane Na + :HCO − 3 cotransporter. Acetazolamide caused a partial inhibition of the forskolin response, consistent with some of the secreted HCO − 3 arising from metabolic sources. The ion flux studies failed to show evidence of net secretion of Cl − in response to forskolin, and bumetanide did not inhibit the I sc response. Thus, forskolin did not cause the net secretion of Cl − across Calu-3 cells under short circuit conditions. Rather, we conclude forskolin causes the net secretion of HCO − 3 by a Cl − -independent, Na + -dependent, and DNDS-sensitive electrogenic mechanism in Calu-3 cells. The forskolin-stimulated alkalinization of the mucosal bathing solution of Calu-3 cells, studied under open circuit conditions, lends further support to this conclusion. Although forskolin did not stimulate the net secretion of Cl − , it did cause a fivefold increase in both unidirectional fluxes of Cl − and it is of interest to understand the mechanisms that underly these changes. Our first interpretation was that forskolin increased the transcellular passage of Cl − in both directions. Thus, the opening of CFTR would allow for both the exit and entry of Cl − across the apical membrane. The NaK2Cl cotransporter in the basolateral membrane would allow the entry of Cl − leaving one to explain how Cl − exits the cell in the serosal-to-mucosal direction. However, bumetanide did not alter the unidirectional fluxes, consistent with the lack of change in the forskolin-stimulated I sc . Thus, the NaK2Cl cotransporter does not appear to mediate the entry of Cl − across the basolateral membrane in the forskolin-stimulated monolayers. We next entertained the possibility that Cl − may move across the basolateral membrane on a Cl − :HCO − 3 exchanger. However, the increases in both unidirectional fluxes in response to forskolin were still observed in HCO − 3 -free buffer. Thus, the increased fluxes do not depend on extracellular HCO − 3 . Because this experiment does not exclude the possibility that a basolateral membrane anion exchanger is operating in a Cl − :Cl − exchange mode, we examined the effects of serosal DNDS (1 mM) on the Cl − fluxes. DNDS cause a 70% decrease in both unidirectional fluxes in the forskolin-stimulated monolayers. Therefore, the increase in Cl − fluxes caused by forskolin can largely be accounted for by a Cl − :Cl − exchange across the basolateral membrane and the exit and entry of Cl − via CFTR across the apical membrane. The studies with 1-EBIO demonstrated the Calu-3 cells are not limited to the secretion of HCO − 3 , but rather they can also be stimulated to secrete Cl − . 1-EBIO, like forskolin, consistently caused a sustained increase in I sc . 36 Cl flux studies showed the 1-EBIO–stimulated increase in I sc could be fully accounted for by the net secretion of Cl − . In addition, both the increase in I sc and the net secretion of Cl − were inhibited by bumetanide. Studies on permeabilized Calu-3 monolayers revealed 1-EBIO activates both a basolateral membrane K + conductance and an apical membrane Cl − conductance as previously shown in studies on T84 cells . CTX and clotrimozole both inhibited the 1-EBIO I sc response as well as the 1-EBIO–activated K + current in permeabilized monolayers. Patch-clamp studies demonstrated the presence of an intermediate conductance, inwardly rectified, Ca + -activated K + channel in Calu-3 cells that was activated by 1-EBIO and blocked by CTX and clotrimozole. We and others have also identified a Ca + -activated K + channel with identical biophysical properties and pharmacological profile in T84 cells . Moreover, Welsh and McCann and McCann et al. have already shown that this channel is expressed in native airway epithelial cells and is therefore not just in epithelial cell lines. Recently, three different groups have cloned the same K + channel, variously referred to as hIK-1, hSK4, and hIK . These channels have identical biophysical properties and pharmacological profile to the channel observed in canine tracheocytes, T84 cells, and Calu-3 cells. Northern blot analysis has confirmed the presence of the mRNA for hIK-1 in T84 and Calu-3 cells (Devor, D.C., unpublished results). Thus, we conclude that one site of action of 1-EBIO is the activation of hIK-1 in the basolateral membrane of Calu-3 cells. Permeabilization of monolayers demonstrated 1-EBIO also activates an apical membrane Cl − channel; however, the identity of the apical membrane Cl − channel that is activated by 1-EBIO is less certain. Haws et al. have reported the predominant Cl − channel observed in Calu-3 cells is a low conductance channel with properties consistent with those of CFTR. 1-EBIO is a benzimidazolinone and other benzimidazolinones have been reported to activate CFTR . Thus, it is possible that the Cl − channel activated by 1-EBIO in Calu-3 cells is CFTR. However, further studies will be necessary to confirm this hypothesis. Calu-3 cells secrete HCO − 3 in response to forskolin and Cl − in response to 1-EBIO. However, when the two agonists are added together, anion secretion is dominated by Cl − secretion and there is a decrease in the net secretion of HCO − 3 . Studies with primary cultures of human bronchial epithelial cells lead Smith and Welsh to suggest that airway epithelia may also switch between HCO − 3 and Cl − secretion. Ashton et al. have also suggested that pancreatic ductal epithelial cells can be differentially stimulated to secrete HCO − 3 or Cl − . The mechanisms that underlie the switch between HCO − 3 and Cl − secretion are largely unknown. Our results with Calu-3 cells offer some insight and suggest a model to explain how the same cell can secrete HCO − 3 when stimulated by forskolin and Cl − when stimulated by 1-EBIO or 1-EBIO plus forskolin. The first tenet of the model is the presence of an anion channel in the apical membrane that can conduct both HCO − 3 and Cl − . Whether there are two separate channel types, one favoring HCO − 3 and activated by forskolin and one favoring Cl − and activated by 1-EBIO, or a single channel type that conducts both HCO − 3 and Cl − is not clear at this time. Nonselective anion channels have been reported but to our knowledge an epithelial anion channel that favors HCO − 3 over Cl − has not yet been described in the literature. Because HCO − 3 secretion is stimulated by forskolin, the anion channel mediating the secretion of HCO − 3 is likely to be activated by cAMP and PKA, as is CFTR. CFTR is highly expressed in Calu-3 cells and activated by forskolin when measured by anion efflux methods and patch clamp analysis . Preliminary studies using impedance analysis have shown forskolin does activate an apical membrane anion conductance in Calu-3 cells (Bridges, R.J., unpublished observations). Patch-clamp anion selectivity studies have shown CFTR can conduct HCO − 3 , although at a fraction (0.15–0.25) of the Cl − conductance . Heterologous expression of wt-CFTR but not ΔF508-CFTR in NIH3T3 fibroblasts and C127 mammary cells was shown to confer the cells with a Na + -independent, HCO − 3 -dependent, forskolin-regulated intracellular pH recovery mechanism . Illek et al. have shown, in α-toxin– permeabilized monolayers of Calu-3 cells, the activation of a HCO − 3 current by cAMP with a similar HCO − 3 to Cl − selectivity as observed in the patch-clamp studies. In addition, Smith and Welsh demonstrated cAMP-stimulated HCO − 3 secretion across normal but not CF airway epithelia and they suggested HCO − 3 exit across the apical membrane is through the Cl − channel that is defectively regulated in CF. Thus, we propose that CFTR mediates the exit of HCO − 3 across the apical membrane of Calu-3 cells. The involvement of an anion channel in HCO − 3 secretion is not a new concept. However, previous models have proposed the anion channel acts as a shunt pathway mediating the exit of Cl − from the cell . Luminal Cl − is then thought to be used by an apical membrane Cl − :HCO − 3 exchanger that mediates the exit of HCO − 3 from the cell. Thus, this model for HCO − 3 secretion necessitates the presence of luminal Cl − for the apical membrane exit of HCO − 3 . The studies with Calu-3 cells demonstrate Cl − is not required for the secretion of HCO − 3 . Ishiguro et al. have recently reported results on HCO − 3 secretion in interlobular ducts from guinea pig pancreas that demonstrate agonist-stimulated HCO − 3 efflux at low (7 mM) luminal Cl − concentrations. These authors suggest their results are not easily reconciled with HCO − 3 transport across the luminal membrane being mediated by a Cl − :HCO − 3 exchanger in parallel with a Cl − conductance. Rather, they too argue for a conductive, channel mediated, exit of HCO − 3 across the apical membrane . Our findings are consistent with this hypothesis, and they suggest the Calu-3 cells will be a useful cell line to help further test this hypothesis as well as to determine the role of CFTR in apical HCO − 3 exit. The second tenet of the model is the presence of an electrogenic Na + :HCO − 3 cotransporter (NBC) in the basolateral membrane that mediates the entry of HCO − 3 into the cell. Boron and Boulpaep were the first to describe an electrogenic NBC with Na + :HCO − 3 stoichiometry of 1:3 that mediates the exit of HCO − 3 across the basolateral membrane in the proximal tubule of the tiger salamander Ambystoma tigrinum . Romero et al. using mRNA from the tiger salamander kidney have recently expression cloned this NBC. The cloning of a human homologue of the renal NBC has also recently been reported , as has a unique human pancreatic isoform . The stoichiometries of the cloned NBCs have not yet been established but Xenopus oocyte expression studies have shown the renal NBC is electrogenic, Na + - and HCO − 3 -dependent, Cl − -independent, and disulfonic stilbene–sensitive . These characteristics are shared by NBCs studied in kidney, glial, liver, pancreas, and colon . Our studies with Calu-3 cells demonstrate that forskolin-stimulated HCO − 3 secretion also shares these characteristics, consistent with the presence of a NBC in the basolateral membrane. Preliminary reverse transcription–PCR and sequencing studies have shown Calu-3 cells express a NBC (Gangopadhyay and Bridges, unpublished observations) lending further support to this notion. Studies in progress are focused on ascertaining which of the NBC isoforms is expressed in Calu-3 cells as well as the membrane localization, apical versus basolateral, of the cotransporter. According to Fig. 12 , we predict a basolateral membrane NBC with a Na + :HCO − 3 stoichiometry that favors the entry of HCO − 3 when Calu-3 cells are stimulated by forskolin. Both the pancreatic and renal isoforms of the NBCs have consensus phosphorylation sites for protein kinase A and therefore may be regulated by cAMP-mediated agonists . Thus, in addition to the activation of an apical membrane anion channel (CFTR?), forskolin may also activate HCO − 3 entry on the NBC. Whether a NBC mediates entry or exit of HCO − 3 depends on the stoichiometry of the transporter, the membrane potential, and the concentrations of Na + and HCO − 3 inside and outside the cell. Sodium: HCO − 3 stoichiometries of 1:2 and 1:3 have been reported , indicating that turnover of the NBC may result in the transfer of one or two negative charges across the membrane at usual membrane voltages. The 1:2 stoichiometry is associated with NBC-mediated HCO − 3 entry, whereas a 1:3 stoichiometry is consistent with HCO − 3 exit. If one assumes typical ion concentrations of 145 mM Na + , 25 mM HCO − 3 outside, and 15 mM Na + and 15 mM HCO − 3 inside, then HCO − 3 will enter a cell on the NBC at membrane potentials less hyperpolarized than −85 mV when the Na + :HCO − 3 stoichiometry is 1:2 and −49 mV when it is 1:3. Membrane potentials more hyperpolarized than these valves will lead to HCO − 3 exit from the cells. Thus, the activation of basolateral membrane K + channels by 1-EBIO is expected to hyperpolarize the membrane potential, and this will inhibit the entry of HCO − 3 on the NBC. If the hyperpolarization is of sufficient magnitude, this change in driving force may drive HCO − 3 out of the cell across the basolateral membrane. Hyperpolarization will also tend to drive anions (HCO − 3 and Cl − ) out of the cell across the apical membrane. However, because basolateral membrane entry of HCO − 3 becomes inhibited, this apical membrane hyperpolarization will favor Cl − secretion. Therefore, we propose that the switch between HCO − 3 secretion and Cl − secretion is determined by the basolateral membrane potential. Differential regulation of the basolateral membrane potential by secretory agonists would provide a means of stimulating HCO − 3 or Cl − secretion. As shown in Fig. 12 , CFTR could serve as both a HCO − 3 and a Cl − channel mediating the apical membrane exit of either anion depending on the nature of the anion provided by the basolateral membrane cotransporter mechanisms. Why does forskolin fail to stimulate Cl − secretion in Calu-3 monolayers? Cyclic AMP–stimulated Cl − secretion is known to require the activation of both an apical membrane Cl − conductance and a basolateral membrane K + conductance; the former depolarizes and the latter repolarizes the membrane voltage to maintain a driving force for Cl − exit . Permeabilization studies demonstrated forskolin does activate an apical membrane Cl − conductance , but that it fails to activate a basolateral membrane K + conductance . Thus, unless the basal K + conductance can maintain the apical voltage above the Cl − equilibrium potential (E Cl < −35 mV, assuming intracellular Cl − = 30 mM), Cl − can not be secreted. Indeed, the expected high Cl − conductance of the apical membrane of forskolin-stimulated Calu-3 cells would set the apical membrane voltage at E Cl and this would provide the driving force for HCO − 3 exit since E HCO3 is –13 mV (assuming intracellular HCO − 3 = 15 mM and extracellular = 25 mM). 4 This electrical coupling may explain the apparent Cl − dependence of HCO − 3 secretion in some epithelia and further emphasizes the importance of CFTR in Cl − and HCO − 3 secretion. If the results we have obtained with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO − 3 secretion in the human airways warrants greater attention. Calu-3 cell HCO − 3 secretion in response to cAMP-mediated agonists is quite similar to that observed in pancreatic duct cells where mutations in CFTR have profound pathological effects. Pancreatic function in CF patients is characterized by impaired fluid, HCO − 3 , and Cl − secretion by the ductal epithelial cells, the site of CFTR expression . Impaired secretion ultimately leads to destruction of the pancreas by digestive enzymes in the obstructed ducts. The principle secreted ion by the ductal cells is HCO − 3 , which drives Na + and water into the lumen by electrical and osmotic coupling. The secreted alkaline fluid serves to regulate the activities of the digestive enzymes and to flush them into the duodenal lumen. Secreted HCO − 3 is also thought to have an osmotic advantage . With the aid of carbonic anhydrase, HCO − 3 can quickly combine with protons to make CO 2 and H 2 O, and thereby tend to make the fluid hypoosmotic. If the airway submucosal glands and surface epithelium function in an analogous manner, potential roles for HCO − 3 in the airways may include the processing, regulation, and clearance of submucosal gland–derived enzymes, mucus, and antimicrobial agents. Early studies have suggested mucus undergoes a transition from gel to sol at alkaline pH and HCO − 3 secretion could therefore aid in the clearance of mucus from the submucosal glands, a process that is impaired in CF. Airway serous cells also express abundant amounts of carbonic anhydrase , some of which may be of the type IV membrane-associated isoform that could convert the secreted HCO − 3 to CO 2 and H 2 O in the lumen of the gland or in the airway surface fluid. The rapid loss of CO 2 during ventilation of the airways would favor a shift in the enyzmatic equilibrium toward the conversion of HCO − 3 to H 2 O. The volatility of the HCO − 3 /CO 2 buffer system, especially at an air–liquid interface, while having potential physiological significance, will also make the investigation of HCO − 3 secretion in the airways a formidable challenge to the experimentalist. Studies with Calu-3 cells will provide a means to further investigate the mechanisms involved in serous cell HCO − 3 secretion, and perhaps with this knowledge how to better study HCO − 3 secretion in the intact airways. | Study | biomedical | en | 0.999997 |
10323162 | The arrival of laparoscopic techniques for hysterectomy renewed the interest in partial hysterectomy. Laparoscopic hysterectomy has some advantages such as shorter hospital stays and quicker postoperative recovery. 1 However, the decision to remove or to retain the cervix is still a matter of controversy. Advocates claim that retention of the cervix has advantages regarding urinary, bowel and sexual function. 2 In our private practice, laparoscopic supracervical hysterectomy (LASH) has been done since 1993. The purpose of this study was to describe our early experience with LASH for uterine non-malignant diseases and to assess short- and medium-term outcome, including the patient's view about the operation. Between January 1993 and April 1996, 41 patients underwent LASH in our clinic. All operations were done by the same gynecologist. All patients had negative preoperative cervical cytologic smears and had no history of cervical intramuscosal neoplasia (CIN). Our approach had been to do LASH in all patients with benign diseases, when vaginal surgery or hormonal treatments were not possible. Different surgical techniques were used in the LASH procedures. The surgical methods used were either bipolar coagulation and scissors or harmonic scissors and coagulation. In one case, we used endoscopic stapling devices. Following amputation of the uterus at the level of the internal ostium of the cervix, the epithelium lining the cervical canal was coagulated. Patient demographic data, surgery characteristics and complication data were determined by retrospective chart review. Patient satisfaction was evaluated by a questionnaire, which was sent to all patients in 1997. Patients were asked about symptoms of vaginal bleeding, discharge, sexual function and their overall satisfaction with the procedure. Forty-one patients underwent LASH, and all procedures were successful. The patients' ages ranged from 32 to 53 years, with an average age of 45. The uterine weights varied from 63 to 860 grams, with an average weight of 192 grams. The most common preoperative indications for LASH were dysfunctional uterine bleeding, fibroids and endometriosis. Operative complications included one bladder perforation, which was corrected laparoscopically, and one case of bleeding at the trocar site. Postoperative complications were bladder atony requiring temporary catheterization (1) and paralytic ileus (1). Hospital readmission occurred in two patients: One patient was readmitted at the 11th day because of a pulmonary embolism, and the other required readmission for several hours on the sixth day because of vaginal bleeding from the colpotomy incision. Intercourse was denied. At examination, some bleeding was observed at the stitches of the colpotomy. Ultrasound did not reveal a hematoma. After a vaginal tamponade for a few hours, further recovery was uneventful. Forty patients completed the questionnaire, giving an overall response rate of 98%. The length of follow-up ranged from 10 to 51 months, with an average length of 27 months. Ten patients (25%) reported occurrence of some periodic vaginal bleeding after their operation. For this, two of them were treated with a vaginal cervical stumpectomy. Four more patients (10%) mentioned complaints of discharge. One patient had a second laparoscopy because of abdominal pain due to foreign-body reaction to endo-GIA staples. At the top of the cervical stump, a cavity had been formed. The two endo-GIA staples that had been used for clipping the adnexae were removed from this cavity, which was excised from the top. Histology showed a foreign body reaction around the metal parts of the staples. The resected top did not show endometrium or endometriosis. Following this procedure, the patient no longer complained of abdominal pain. Eleven patients reported sexual problems before operation. Six patients had dyspareunia, two had lack of sexual desire due to irregular bleeding, and three mentioned lack of desire without further specification. Ten of the eleven patients reported an improvement of sexual functioning, mostly because of less pain and absence of bleeding. One patient with lack of desire reported a worsening of sexual functioning afterwards. Twenty-nine patients reported no problems before operation and noticed no difference afterwards. Overall, 39 women were satisfied with their operation, and one patient was not satisfied because of persistent vaginal bleeding without need for further treatment. When new technology is introduced in a clinic, evaluating the new procedure is important. In our clinic, we performed LASH in all patients with benign diseases, when vaginal surgery or hormonal treatments were not possible—so being a therapeutic alternative to abdominal hysterectomy. Preservation of the cervix implicates the risk of development of a cervical stump carcinoma; however, the availability of accurate screening possibilities for detecting early cervical malignancy lessens the need to remove the cervix at hysterectomy. Furthermore, the prognosis of a carcinoma of the cervical stump was similar to that of cervical cancer in patients with an intact uterus in the study of Kovalic et al. in 1991. 3 In addition, Kilkku et al. 4 – 6 showed certain advantages of partial hysterectomy. Their studies showed decreased urinary tract and bowel symptoms and improved sexual function. In our study, preservation of the cervix seemed to have some disadvantages. Ten percent of our patients had symptoms of discharge, and 25% of our patients experienced some regular vaginal bleeding. Two of them were treated by a vaginal cervical stumpectomy. The other patients accepted the condition, and all except one were satisfied in spite of the light menses. In the literature, the reported rate of bleeding of the retained cervix is 4.8%. 7 Our higher rate might be due to our technique, although we coagulated the endocervical canal. It is described that coring of the cervix to include the anatomical endocervical canal rather than amputation at the uterine isthmus may be better in preventing bleeding. 8 Another explanation for our high rate may be that we detect more cases because our length of follow-up is longer than in most studies. Only 3 out of 10 of our patients with recurrence of vaginal bleeding consulted their gynecologist with this symptom; therefore, we would have missed the other seven cases when only reviewing the patients' charts. Our high rate of bleeding was not a reflection of our learning curve, since these patients were equally spread over the studied period. In preoperative counseling of patients, discussing the disadvantage of possibly recurrent bleeding is important. In the literature, an acceptable complication rate for laparoscopic hysterectomy has not yet been established. There are some reports of complications, but comparing them is difficult because many different descriptions are used. Most data on complications come from retrospective studies of surgeons with a particular expertise in laparoscopic surgery, and this may underestimate the problem. A recently published prospective multi-center observational study in the Netherlands provided a complication rate for laparoscopic hysterectomy of 9%. 9 This study included a wide variety of experience in laparoscopic surgery. Our questionnaire also contained questions about the patient's sexual function and overall satisfaction. Despite the problems discussed, patient satisfaction was high. To interpret these results properly, comparative prospective studies are needed. Although preservation of the cervix at laparoscopic hysterectomy for benign diseases will be satisfactory in most cases, some women have complained of complications related to the remaining cervix when asked in a questionnaire. After amputation of the uterus, special attention should be paid to the careful coring of the endocervical canal. This study underlines also the importance of follow-up on patients who undergo a new surgical technique to detect long-term complications. Further prospective studies with prolonged follow-up are needed to evaluate the risks and benefits of retaining the cervix at laparoscopic hysterectomy. | Study | biomedical | en | 0.999993 |
10323163 | Laparoscopic cholecystectomy was initially performed in Germany by Muhe and in France by Mouret. 1 – 2 Soon after this initial report, McKernan and Saye and Reddick and Olsen performed the first laparoscopic cholecystectomies in the United States in 1988. 1 , 3 During the next ten years, laparoscopic cholecystectomy evolved into the accepted approach to manage symptomatic gallbladder disease. Multiple prospective randomized trials and retrospective reviews have shown that laparoscopic cholecystectomy allows for shorter hospitalizations, decreased analgesic requirements, earlier return to full activity and decreased total costs. 4 – 8 The same studies have also demonstrated that laparoscopic cholecystectomy can be performed with minimal morbidity and an infrequent but slightly higher incidence of bile duct injuries than for open cholecystectomy. Perioperative mortality has been reported to be uncommon. Although cholecystectomy is the most commonly performed laparoscopic procedure by general surgeons, there have been few published reports of large series performed by a single surgeon. 9 We retrospectively reviewed the last 1025 consecutive laparoscopic cholecystectomies performed by a single attending surgeon over a 54-month period. This series was compared to the previously published initial series of 600 consecutive laparoscopic cholecystectomies performed by the same attending surgeon over a 28-month period. 10 Factors evaluated included conversion rates to open cholecystectomy, time to discharge, intraoperative and postoperative complications, mortality, success and outcomes of intraoperative cholangiography and the role of the surgical residents in perioperative outcomes. One thousand and twenty-five laparoscopic cholecystectomies were attempted by a single attending surgeon from September 1992 to February 1997. The initial 600 laparoscopic cholecystectomies attempted by the same attending surgeon from May 1990 to August 1992 were previously published and used for comparison with this most recent series. All surgeries were performed at a teaching hospital by a single attending surgeon or under his direct supervision by a surgical resident. Preoperative evaluation in all patients from both series consisted of an abdominal ultrasound and liver function tests (direct and indirect bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and albumin). Biliary scintigraphy, oral cholecystography, abdominal CT scans, endoscopic retrograde cholangiopancreatography (ERCP) and serum amylase and lipase were obtained on a selective basis. Those patients with symptoms suggestive of peptic ulcer disease or gastrointestinal reflux disease were evaluated preoperatively by either endoscopy or upper gastrointestinal contrast series. Laparoscopic cholecystectomy was performed using the standard four puncture technique. Pneumoperitoneum was established by the placement of a Veress needle umbilically or by the “open” Hasson technique in selected patients. Patients having prior upper abdominal surgery had the Veress needle inserted into the right or left upper abdomen with the placement of a 5 mm trocar and laparoscope for initial inspection. During the initial 600 laparoscopic cholecystectomies, 5 mm trocars were placed in the right anterior axillary line and the right midclavicular line, and 10 mm trocars were placed in the epigastrium and the umbilicus. In the most recent series, the 10 mm epigastric trocar was replaced by a 5 mm trocar. Blunt dissection and limited electrocautery was used to identify the cystic duct and artery. Anterior and lateral traction were applied to the infundibulum of the gallbladder during this dissection. Intraoperative cholangiograms were performed by digital fluoroscopy in this series of patients and by “static” x-rays in the initial series. The cystic duct and artery were ligated with hemoclips and transected. The gallbladder was dissected from the liver bed in a retrograde direction using electrocautery. The gallbladder was removed through the epigastric incision in the first series of patients. In the most recent series of patients, a 5 mm, 0° laparoscope was placed in the epigastric port for visualization, and the gallbladder was removed through the umbilical incision. Closed suction drains were used infrequently and at the discretion of the attending surgeon. Fascial closure of the umbilical trocar site with an absorbable suture was performed in the majority of patients. All skin incisions were reapproximated with an absorbable subcuticular suture. A laparoscopic cholecystectomy was attempted in 1025 patients, 775 (75.6%) females and 250 (24.4%) males, from September 1992 to February 1997. Mean age for the patients was 49.8 years and ranged from 14 to 91 years. Patient demographic from this series and the initial series of 600 patients is demonstrated in Table 1 . A laparoscopic cholecystectomy was performed electively in 816 (79.6%) patients with 209 (20.4%) patients admitted prior to surgery. The indications for laparoscopic cholecystectomy for both series are summarized in Table 2 . A surgery resident was the operating surgeon under the direct supervision of the attending surgeon in 838 (81.8%) of the cases in this series and in 52.0% of the cases in the initial series of 600 patients. Intraoperative cholangiography was selectively attempted in 235 (22.9%) patients. Only 11 (4.7%) of the intraoperative cholangiograms could not be completed. Choledocholithiasis was diagnosed by intraoperative cholangiograms in 38 (3.7%) patients. Management of the common bile duct stones is summarized in Table 3 . Twenty-seven (2.6%) patients overall and 5 (3.0%) patients with acute cholecystitis had to be converted to an open cholecystectomy. The indications for conversion to open cholecystectomy in these patients are shown in Table 4 . The conversion rate overall and for acute cholecystitis was 4.0% and 29.0%, respectively, in the initial series of 600 patients. There were 13 (1.3%) intraoperative complications and only 1 (0.1%) bile duct injury. The intraoperative complication rate was 1.0%, and the bile duct injury rate was 0.5% in the initial series of 600 patients. Table 5 summarizes the intraoperative complications that occurred in the series of 1025 patients. There were two major complications: a common bile duct laceration and a duodenal injury. The anterior common bile duct laceration was repaired primarily. A T-tube was placed after the repair. This patient recovered uneventfully. The duodenal injury went unrecognized until the third postoperative day. The injury was most likely secondary to electro-cautery. This complication was managed by duodenal exclusion and drainage. He died of multisystem organ failure on the sixty-sixth postoperative day. Major postoperative complications occurred in 28 (2.7%) patients and are summarized in Table 6 . The postoperative complication rate was 1.2% in the initial series of 600 patients. Nine patients required secondary procedure related to their postoperative complications. Endoscopic retrograde cholangiopancreatography was performed in three patients, one for a cystic duct stump leak and two for symptomatic choledocholithiasis. They were managed successfully with a common bile duct stent in the patient with a cystic duct stump lead and with sphincterotomy and stone extraction in the latter two. The two patients with subhepatic abscess were managed by laparotomy and closed suction drainage and percutaneous CT-guided drainage, respectively. All four patients with umbilical hernias underwent repair, one emergently for an incarcerated hernia. Three patients (0.3%) unexpectedly had adenocarcinoma of the gallbladder. The diagnosis was made by the pathologist and not intraoperatively in two of the patients. Both had mucosal-based lesions and are alive at 18 and 23 months, respectively. The third patient was converted to an open procedure, but had unresectable disease. She is alive at six months. Fourteen (1.4%) patients were readmitted to the hospital within 30 days of their laparoscopic procedure. There were 5 (5.0%) postoperative deaths ( Table 7 ) and only one, a duodenal injury, was directly related to a complication of the procedure itself. The initial series of 600 patients had 1 (0.2%) mortality, resulting from a post-operative myocardial infarction. Ninety-two (9.0%) patients were discharged on the day of surgery, 896 (87.8%) within 24 hours of surgery, and 948 (92.9%) with 48 hours surgery. In the initial series of 600 patients, no patients were discharged on the day of surgery, 537 (89.5%) were discharged within 24 hours of surgery, and 564 (94.0%) within 48 hours of surgery. Although symptomatic cholelithiasis remained to be the most common diagnosis in both groups, there was an increased incidence of laparoscopic Cholecystectomy for biliary dyskinesia and acute cholecystitis in the second group of patients (p<0.001). The total number of cholecystectomies performed has increased during the laparoscopic era. 11 – 13 The increased willingness to recommend elective Cholecystectomy for biliary dyskinesia can be attributed to the implementation and widespread use of cholecystikinin-augmented hepatobiliary scanning to document a poorly contracting gallbladder as much as it can be attributed to the laparoscopic technique. 14 Before the cholecytokinin-augmented hepatobiliary scans became available in 1989, surgeons were less willing to subject patients to an open Cholecystectomy when gall-stones were not documented. Since the laparoscopic era, the threshold to subject a patient to Cholecystectomy who doesn't have gallstones has decreased when he/she has a positive hepatobiliary scan. Whether patients are benefiting from this is to be determined. 15 Unfortunately, the outcomes in this subset of patients has not been clearly defined. Initially, laparoscopic cholecystectomy for acute cholecystitis was considered a relative contraindication because surgeons reported increased perioperative morbidity and conversion rates to open cholecystectomy in as many as 33% of the cases. 16 Not surprisingly, our conversion rate in the initial 600 patients for acute cholecystitis was 29.0%, and this accounted for the highest percentage of conversion to open cholecystectomy. Recently, conversion rates have been reported to be 10%, and perioperative complication rates have been reported to be comparable to those of an open cholecystectomy for acute cholecystitis. 17 , 18 We were able to perform a laparoscopic cholecystectomy in 97% (161/166) of the patients with acute cholecystitis in the last 54 months, and common bile duct exploration became the most common indication for conversion to open cholecystectomy. Our review supports what others have demonstrated—that although laparoscopic cholecystectomy for acute cholecystitis is more technically challenging, it can be performed safely by an experienced laparoscopist. 19 It should also be noted that our low conversion rate persisted despite the fact that a surgery resident was the operating surgeon in 68.7% of the acute cholecystitis cases. The conversion rate of laparoscopic cholecystectomy for acute cholecystitis performed by surgery residents had been reported to be 30.5%. 20 The most common complications of laparoscopic surgery are related to Veress needle and trocar insertion. 21 Although intraoperative complications occurred at a similar rate (1.0% to 1.3%) between the two groups, the latter series had more complications related to Veress needle placement. Most of these injuries occurred in patients having prior abdominal surgery. A more liberal use of an alternative entry site Veress needle puncture or an open technique (Hasson) in patients having prior abdominal surgery would have been a safer means in preventing these injuries. 22 , 23 As expected, the peak incidence of bile duct injuries happened during the initial series in the first 75 patients. Since that time, there has been only one bile duct injury in 1550 patients. This was significant in the fact that a surgery resident has performed nearly 82% of all laparoscopic cholecystectomies in the last 1025 patients. A similar low incidence of bile duct injuries in laparoscopic cholecystectomies performed by surgery residents was reported by Wu et al. 24 Postoperative complications occurred in only 1.5% of the cases in the initial series, but in 2.7% in this series. The increase was attributed to a higher incidence of cardiopulmonary complications. The alterations in cardiopulmonary physiology are well tolerated in most patients undergoing laparoscopic cholecystectomy but may not be tolerated in those patients with poor cardiopulmonary reserve. The increased intra-abdominal pressure may cause acid-base disturbances, decreased regional and systemic oxygen delivery, and a reduced cardiac preload with an increased cardiac afterload. 25 – 27 However, Tangle et al. reported that the perioperative morbidity rate for laparoscopic cholecystectomy in the elderly population was not different from that reported for patients less than 65 years of age, and Maxwell et al. reported similar results in octogenarians. 28 , 29 Two patients in this series and in the initial series developed deep venous thrombosis in their lower extremities, and one of them (in each series) had a fatal pulmonary embolus. The incidence of deep venous thrombosis and pulmonary embolus after laparoscopic cholecystectomy has been reported to be higher than for open cholecystectomy, but the true incidence after laparoscopic cholecystectomy is not known. 30 Our routine is to use sequential compression devices on all patients during laparoscopic cholecystectomy. Sequential compression of the lower extremities has been shown to effectively neutralize venous stasis during laparoscopic cholecystectomy. 31 But whether this decreases the risk of postoperative thromboembolic complications after laparoscopic cholecystectomy has not been determined. Another postoperative complication that occurred in two patients was a subhepatic abscess. This complication may be the result of spilled gallstones, and it is reported with increasing frequency. 32 Perforation of an acutely inflamed or emphysematous gallbladder during laparoscopic cholecystectomy was not associated with an increased incidence of subhepatic abscess compared to those patients with unperforated gallbladders when patients were given appropriate antibiotics and their abdominal cavities were properly irrigated. 33 The mortality rate did increase from 0.2% to 0.5% (p = 0.66). The mortality rate for the 82-month period was 0.25%, and this is similar to other reported series. 6 – 8 No classification system to assess preoperative comorbidities was calculated as part of the study for either group. Only one mortality was directly related to an intraoperative complication. This patient had a duodenal injury, probably the result of thermal damage from electrocautery that went unrecognized until the third postoperative day. A pyloric exclusion was performed, but the patient succumbed to multisystem organ failure. Patients were discharged from the hospital at relatively the same time postoperatively between the two series ( Table 7 ) . A small percentage of patients in this series were discharged on the same day as their surgery. Other institutions have had more success performing laparoscopic cholecystectomies on an outpatient basis. 34 , 35 It is difficult to pinpoint the reason for our institution's inability to match these results. Perhaps we are not properly educating our patients on the safety of outpatient laparoscopic cholecystectomy and the expected postoperative events. We are currently implementing a clinical pathway for outpatient laparoscopic cholecystectomy. This is intended to uniformly educate all health care employees involved in the perioperative care of patients undergoing laparoscopic cholecystectomy in order to improve our same day discharge rate. The results of this study will be reported in a couple of years. Nevertheless, the average length of stay for all patients, including those that were converted to an open procedure, was only 1.25 days for the 82-month period. Intraoperative cholangiograms (IOC) were performed selectively. Patients in this series with an elevated alka-line phosphatase, total bilirubin or serum transaminases, a history of jaundice or gallstone pancreatitis or a radiographically documented dilated common bile duct underwent IOC. Any difficulty recognizing the biliary tract anatomy was an absolute indication for IOC. An increased percentage of IOC were attempted in this series (22.9% compared to 18.5%). This increase likely represents a greater percentage of laparoscopic cholecystectomies performed in patients with acute cholecystitis. In this series, only 11.5% of the patients having an elective laparoscopic cholecystectomy underwent IOC. Yet, 48.8% of the patients with acute cholecystitis had IOC. When inflammatory changes were encountered in the triangle of Calot, IOC was used liberally. The success rates were similar at 95.3% for this series and at 95.5% for the initial series. Digital fluoroscopy was used during the last 54 months. This technique has been reported to be less time consuming and more accurate than static IOC. 36 , 37 Laparoscopic ultrasound was not used to evaluate the common bile duct in any of the patients in either series. Choledocholithiasis was identified during IOC in 17.0% (38/224) of the patients in this series and in 6.6% (7/106) in the initial series. Initially, IOC was used more frequently in patients that did not have clinical suspicion for choledocholithiasis. This was during the learning phase of the procedure. As our proficiency with the procedure increased and as prospective randomized studies suggested its more selective use in patients with clinical criteria suggesting choledocholithiasis, our percentage of positive IOC increased. 38 , 39 In our entire series of 1625 patients, 1253 did not have their biliary tract imaged by IOC or ERCP. Only 2 (0.16%) have returned with symptomatic choledocholithiasis. This is much less than other series have reported. 40 Laparoscopic cholecystectomy can be performed by surgery residents under the direct supervision of an experienced laparoscopist with minimal intraoperative and postoperative morbidity. The indications for laparoscopic cholecystectomy over the last 82 months has evolved to include a greater percentage of patients with biliary dyskinesia and acute cholecystitis. Despite an increased number of cases being performed for acute cholecystitis, conversion rates to open cholecystectomy and biliary tract injury rates have decreased, and the perioperative morbidity has remained the same. | Review | biomedical | en | 0.999996 |
10323164 | The provision of basic operative skills training in general surgery has traditionally relied heavily on the two most common abdominal procedures, appendicectomy and cholecystectomy. Performance of both of these operations under supervision gave surgical residents the opportunity to learn the principles of tissue handling and instrument technique, which they could subsequently apply to more complex procedures. One of the consequences of the widespread adoption of minimal access surgical techniques for these and other common abdominal operations was that a whole generation of trained surgeons was forced to re-evaluate and re-learn these procedures. This threatened to reduce the pool of suitable training operations for surgical residents. The aim of this study was to compare the outcome of this procedure when performed by trainees compared with qualified surgeons and to evaluate the impact of the introduction of the laparoscopic method on the status of cholecystectomy as a training operation in a university clinic. Laparoscopic cholecystectomy was first introduced to this unit in 1992. The first 80 cases were performed by qualified surgeons only, mainly by a group of three surgeons who developed a special interest in laparoscopic and endoscopic surgery. From January 1995 onwards, surgical residents in their third or higher year of training were allowed to perform laparoscopic cholecystectomy under the supervision of a trained surgeon who acted as cameraman. Residents also attended an animal laboratory course and were encouraged to practice on a laparoscopic training rig. Between January 1992 and December 1996, a further 252 cases were performed, and these form the basis of this study. There were 57 male and 195 female patients with a mean age of 53 years (range 16-89 years). One hundred and ninety-nine procedures (79%) were elective operations for biliary colic or chronic cholecystitis, and the remaining 53 (21%) were performed following urgent admission with acute cholecystitis. Acute cholecystitis was defined by the presence of acute localized right upper quadrant pain with temperature greater than 37.5°C and leukocytosis. Confirmation of the diagnosis was by ultrasound with demonstration of gallstones, gallbladder wall thickening or edema, and peri-cholecystic fluid. For patients admitted with acute cholecystitis, operation was scheduled within 72 hours of hospitalization. Over the complete two-year period covered by this study, trainee surgeons performed 37% of all elective laparoscopic cholecystectomies and 29% of all procedures done for acute cholecystitis ( Table 1 ) . However, the percentage of cases performed by residents increased progressively as the combined experience of all the surgeons in the unit expanded. During the first 50 cases “available” to residents, just 6% were operated on by trainees, whereas after a further 200 cases, residents operated on 58% of patients. The change in the percentage of patients operated on by residents compared to qualified surgeons over the study period is shown in Figure 1 . The duration of the procedure (skin-to-skin time) was affected by the grade of the operator ( Table 1 ) . Residents required more time (119 ± 33 minutes) compared to qualified surgeons (97 ± 42 minutes) for elective cases, although this did not reach statistical significance. However, when the operation was performed for acute cholecystitis, the duration of the operation was significantly longer for residents (145 ± 50 minutes) than trained surgeons (111 ± 54 minutes, p<0.05, chi-squared test). Conversion rate and complications according to operator grade are shown in Table 2 . The rate of conversion to open cholecystectomy was not significantly different between trainee and qualified surgeons [resident: 2 cases (2%); surgeon: 11 cases: (6%)]. There was only one bile duct injury in a patient operated on by a staff surgeon, which was recognized at the time of injury and converted to laparotomy for primary repair. Bile leak due to cystic duct stump insufficiency was seen in four patients, two of whom had been operated on by resi-dents. All four cases were managed successfully by endoscopic stent insertion. Other complications included subhepatic abscess and port-site bleeding, which occurred in one patient each, respectively, and minor wound infections in three patients. Mean postoperative stay is shown in Table 3 . Postoperative stay was not influenced by the grade of operator or by the presence of acute cholecystitis. The widespread adoption of minimal access techniques raised concerns about the possibly detrimental effects this might have on operative experience of surgical residents. 1 , 2 Certainly, most residents in training viewed the period during which their mentors re-learned many standard abdominal procedures with a degree of dissatisfaction, since they were deprived of access to many cases that they would previously have performed themselves, especially appendicectomies, inguinal herniorraphies, and cholecystectomies. These three common general surgical procedures had traditionally provided ample opportunity for residents to acquire basic surgical operative skills training at a relatively early stage in their careers. In this study, the introduction of laparoscopic cholecystectomy was associated with a serious hiatus in exposure of residents to biliary surgical procedures as primary operator. However, once the learning phase had been passed, cholecystectomy became readily reintegrated into the resident training schedule with no increase in complication rate or hospital stay. The average length of stay documented in this study, in common with European experience in general, is longer than that reported from North American centers. Economic considerations will continue to force a decrease in postoperative hospital stay, although the period under review in this study was too short to detect any change in length of stay. Because of the major socio-political changes in this region associated with German reunification, it is difficult to obtain a valid comparison of resident training experience in this clinic prior to the introduction of laparoscopic techniques, but the proportion of cases currently performed by residents is in line with the numbers reported from other centers. These other studies have also shown a similar dip in residents' exposure to biliary surgery during the development phase of laparoscopic surgical services, which recovers once institutional expertise with the procedure increases. 3 , 4 One important trend that has emerged is that laparoscopic cholecystectomy tends to be performed by residents at a more senior level, whereas, previously, residents were introduced to open cholecystectomy at a more junior grade. 1 It has been suggested that residents need a higher level of skill and should be more advanced in their training before being allowed to perform cholecystectomy laparoscopically. 5 In addition, many centers advocate the use of training rigs and animal models as a means of acquiring laparoscopic skills before operating on patients. 6 , 7 The trainees in this clinic are encouraged to practice laparoscopic dissection techniques using a training rig and attend an animal laboratory laparoscopic training course. Interestingly, our residents regarded the training gained in the animal laboratory as being of limited benefit compared to the value of proctored operative experience. Although laparoscopic skills can be measurably improved by bench training, the need for animal laboratory training is less clear-cut now that sufficient experience has accumulated throughout the surgical community 5 , 8 – 10 Although undoubtedly of great value in the early stages of dissemination of these operative skills and in the evaluation of new techniques, routine animal workshop training may be a luxury that many centers feel is no longer affordable or necessary. These teaching modalities can certainly never replace the absolute requirement for supervision of the trainee by an experienced laparoscopic surgeon during the learning curve phase. The exact number of procedures that should be performed under supervision has been the subject of some debate. Davidoff and colleagues reported that the risk of bile duct injury was highest during the first 13 cases. 11 Analysis of the Southern Surgeons' Club series in the South-Eastern United States showed that 90% of bile duct injuries occurred within the first 30 cases of the operating surgeon. 12 The European Association of Endoscopic Surgery has recommended that surgeons who have not graduated from a residency program that provides structured experience in laparoscopic surgery should attend an approved EAES laparoscopic cholecystectomy course and perform 3-5 preceptor-assisted cases, as well as providing documentation of their subsequent ten cases before being granted privileges in laparoscopic cholecystectomy. 13 The accompanying decrease in the number of open cholecystectomies also has important implications for residency training. As with other centers, we have found that the number of open cholecystectomies has dwindled dramatically. These cases tend to be technically difficult and, therefore, usually are performed by a senior resident or staff surgeon. 1 Although some reports contend that residents still receive adequate exposure to open biliary procedures in the era of laparoscopic cholecystectomy, we would suggest that it is becoming increasingly difficult to achieve this aim. 14 Organizers of residency programs must continue to monitor these changing trends to ensure that the needs of their trainees are met in the future. Appendicectomy, whether open or laparoscopic, will remain an important “starter” operation, allowing junior residents to develop laparoscopic skills at an early stage. 15 As a higher percentage of laparoscopic cholecystectomies are performed by residents, and junior residents learn laparoscopic technique from appendicectomy, some of the more straightforward cases should “filter down” to the more junior residents. With the exception of numbers of open biliary cases, the situation is likely to continue to improve rather than worsen for surgical residents. | Other | biomedical | en | 0.999996 |
10323165 | A variety of investigations are available for imaging the common bile ducts (CBD) before laparoscopic cholecystectomy (LC), and none has been accepted as gold standard. The guidelines were reasonably well established regarding the management of common bile duct stones in the era of open cholecystectomy. With the development of preoperative ERCP, when many bile duct stones can be confirmed and treated, the situation has changed, and there is ongoing debate about a rational method of investigation of the bile ducts before laparoscopic cholecystectomy. There are arguments for and against each modality of investigations, eg, preoperative ERCP, intravenous cholangiogram (IVC), ultrasonography (USS), and routine use of peroperative cholangiogram (POC). Routine POC has been advocated to demonstrate the biliary anatomy and to demonstrate unsuspected bile duct stones. Selective ERCP has reduced the incidence of unsuspected stones. We present a personal series of 700 laparoscopic cholecystectomies in the 7-year period between 1991-1998 where we have adopted a policy of not performing routine peroperative cholangiography. Seven hundred patients have undergone laparoscopic cholecystectomy during the 7-year period. Prospective analysis of all the patients was carried out with the help of meticulously filled proformas and clinical notes. There were 544 females and 156 males. Diagnosis of symptomatic gallstone disease was made by ultrasound examination (USS). All the patients had estimation of liver function tests (LFTs). Investigation of bile ducts was by combination of history of jaundice, cholangitis and gallstone pancreatitis, LFTs and ultrasound evidence of dilated CBD. The first 215 patients underwent routine preoperative IVC in the initial period of the series. The remaining 485 patients had only LFTs and ultrasound examination. Preoperative ERCP was performed in 78 patients based on clinical his-tory, abnormal LFTs and/or dilatation of biliary tree on ultrasound. Selective peroperative cholangiogram (POC) was performed in 42 patients because of failed ERCP or slightly abnormal LFTs. We have presented our results with regard to conversion rate, morbidity, mortality, management of complications and incidence of retained stones. Results are shown in Table 1 and ( Table 2 . The total number of operative complications was 12 (1.71%), and the total number of laparoscopic cholecystectomy related complications was 15 (2.14%). Out of 13 patients who had bile leak, four needed laparotomy and drainage, and two were treated conservatively. The remaining seven patients who had localized bile collection were treated by ultrasound-guided insertion of a pigtail catheter. Two of these patients also required ERCP and temporary stenting of the CBD. ERCP was also required to extract the stone in one patient with retained stone. A single case of biliary stricture was referred to a hepatobiliary unit where Roux-en-Y hepaticojejunostomy was performed. Conversion to open procedure was performed in 36 patients (5.1%). The total mortality in the series was 0.43% (n=3). One of them was after a bile leak, and the other two were due to associated severe cardiorespiratory diseases. Selective POC was performed in 42 patients (6%). Bile duct stones were demonstrated in four patients, and in two patients they were retrieved laparoscopically. Another two patients required open bile duct exploration, and four patients who had an operative cholangiogram developed bile leak (9.5%). Postoperative ERCP was required in only four patients. In one patient, it was carried out to retrieve the stone from the CBD, and this was the only case in a series of 700 patients. In two patients, it was performed to place the stent in the CBD following bile leak. In the remaining one patient, an ERCP was performed as there were abnormal LFTs in the postoperative period, but the ERCP did not demonstrate any abnormality. Standard method of investigation of bile ducts before laparoscopic cholecystectomy is still controversial. In the early days of our experience, it was a routine practice to perform intravenous cholangiogram in all the patients before laparoscopic cholecystectomy. This was mainly due to the fact that insufficient data was available about the investigation of bile ducts before laparoscopic cholecystectomy. It also seemed IVC was helpful in delineating the anatomy of the biliary tree and indicating the possibility of CBD stones. After initial experience, we found that a large majority of patients had normal IVC's if there was a negative clinical history and normal LFT's and USS. This made us abandon the IVC as preoperative investigation of the biliary tree and adopt the policy of selective peroperative cholangiogram (POC) if there was minor alteration in the criteria of common bile duct stones. The other argument in favor of routine POC is that it provides a radiological picture of the anatomy of the biliary tree. But there are reports suggesting that the majority of biliary injuries occur due to the surgeon misidentifying the anatomy. 1 With the policy of extremely careful dissection of Calot's triangle and adherence to the rule ‘Not to ligate or divide any structures until the anatomy of Calot's triangle is clearly defined,’ 1 we believe that we can avoid relying on routine radiological imaging as a guide. The number of preoperative ERCP's is 78 (11.14%), similar to other reported series, which is between 7 and 12%. 2 – 5 This was based on the criteria of clinical history of jaundice, cholangitis and gallstone pancreatitis, abnormal LFT's suggesting biliary obstruction and/or dilatation of the biliary tree on ultrasound. Selective peroperative cholangiography under fluoroscopy control was performed in 6% of patients, which is low compared to other series where an aggressive policy is adopted and routine POC is carried out in all laparoscopic cholecystectomies. We performed POC's only in those patients where ERCP was unsuccessful or where there were minor abnormalities in the above-mentioned criteria. There was a 9.5% incidence of complications in this series, highlighting the risks involved in routine POC's. 6 Routine POC does not appear to decrease the absolute incidence of biliary injuries 6 , 7 but it reduces the sequelae. It definitely increases the cost of the procedure. 8 There are also reports that in comparison to selective POC's, routine POC did not increase the detection of common bile duct stones. 8 If POC is not performed, the incidence of unsuspected CBD stones during cholecystectomy is 4% to 6%. 9 , 10 With the selective use of preoperative ERCP and POC, the majority of these patients are identifiable, and our series has shown that there was only one patient with retained stone. The other contributing factor for the very low incidence of retained stones in this series may be due to either spontaneous passage of CBD stones in the postoperative period or they have remained asymptomatic. There are no incidences of further recurrent stones to date. In laparoscopic cholecystectomy, a policy of selective preoperative ERCP and not routine peroperative cholangiogram has been found to be cost effective, reduce the time of operations and is associated with low morbidity, mortality and is not associated with a significant incidence of retained bile duct stones or bile duct injuries. | Study | biomedical | en | 0.999998 |
10323166 | Minimally invasive surgical techniques have revolutionized the treatment of many of the problems seen by the general surgeon. Although the impact has been greatest in the treatment of cholelithiasis, many of the same advantages achieved with laparoscopic cholecystectomy can be realized with advanced laparoscopic procedures. Since the first initial reports of laparoscopic right hemicolectomy in February 1990 1 and sigmoid resection in October 1990, 2 laparoscopic-assisted colectomy (LAC) has been found to have numerous advantages when compared to open colectomy. Among these advantages are less blood loss, fewer wound complications, more rapid return of intestinal function, less pain, shorter hospitalization and quicker return to work. 3 – 7 But LAC has not been widely accepted as the surgical treatment of choice for patients requiring colon resection. There are two main factors which have prevented the widespread use of LAC techniques. First, the procedure is technically much more difficult and, second, although some have reported good results, 8 – 11 LAC has not yet been proven to yield equal or better results for the treatment of colon cancer when compared with open colectomy. Indeed, much concern has been raised about the possibility of increased recurrence rates, port site metastasis, and the possibility that LAC will not prove to be an adequate resection for cure of cancer. 12 – 14 Prospective randomized multicenter trials are currently investigating these concerns. Even though LAC for benign disease has yielded good results, only a small percentage of surgeons offer LAC for the treatment of benign disease when discussing options with their patients. Overall, the biggest impediment to the widespread adoption of LAC for benign disease remains the difficulty of the procedure. In our experience, colon mobilization and division of the mesentery have been the most difficult parts of the procedure for the surgeon to learn. The anastomosis is usually completed extracorporeally or with the transanal circular stapler, much as would be done during open surgery. The development of the ultrasonically activated shears (Laparosonic Coagulating Shears [LCS], Ethicon Endo-Surgery/Ultracision, Smithfield, RI) has provided an alternative technology for mobilization of the colon and division of the mesentery. To evaluate the efficacy, safety, and efficiency of this new energy source, we retrospectively reviewed a portion of our series of LAC cases. From October 1990, to May 1997, 118 laparoscopic colon resections were completed for a variety of indications. Thirty-three of these patients had a colectomy other than a right hemicolectomy or a sigmoid resection and were eliminated from the study. The charts of the remaining 85 patients who underwent either laparoscopic-assisted right hemicolectomy or laparoscopic-assisted sigmoidectomy were reviewed retrospectively by the authors. Fifty patients had benign disease, and 35 had malignant disease. Patients who underwent curative resection for carcinoma of the colon were entered in an Institutional Review Board (IRB) approved prospective study. The operative notes were reviewed to determine the method in which the colon was mobilized and the mesentery divided. From this review, two groups were identified: one in which this dissection was done without the LCS (no LCS group), and one in which this dissection was done with the LCS (LCS group). The age, sex, indication for surgery, operative times, estimated blood loss (EBL), and length of stay (LOS) were documented for each group. Statistical analysis was performed on all variables utilizing t-test methodology. All surgical procedures were performed in a similar fashion by the senior author or by the laparoscopic surgery fellow under the direct supervision of the senior author. In all patients the colon was mobilized and the mesentery was divided with a totally laparoscopic technique. In cases completed without the LCS, hemostasis was obtained with a combination of clips, endoscopie linear cutting staplers, and monopolar cautery delivered with scissors. Pre-tied ligating loops or manually tied ligatures were not used. The technique using the LCS varied according to whether there was benign or malignant disease. In most cases of benign disease, the mesentery was divided completely with the LCS. Other methods for hemostasis were usually not necessary unless the benign disease was so extensive that it required a wide dissection of the mesentery. For cases of malignancy, a high ligation of the vascular pedicle (ileocolic artery for right colectomy and inferior mesenteric or superior sigmoid artery for sigmoid resection) was accomplished with an endoscopie linear cutting stapler. The remainder of the mesentery was divided exclusively with the LCS. After mesenteric dissection, a mini-laparotomy was made for specimen extraction. A plastic wound protector was routinely placed in the mini-laparotomy incision during specimen extraction. The anastomosis after right hemicolectomy was completed extracorporeally via the minilaparotomy, and the anastomosis after sigmoid resection was created intracorporeally using the circular stapler passed transanally. Of the 85 patients, 36 had their procedures completed without the LCS and were in the no LCS group, while 49 had use of the LCS and were in the LCS group. The female:male ratio was 2:1 in the no LCS group and 1.6:1 in the LCS group. The average age was 67.9 years (range 28-101) in the no LCS group and 62.6 years (range 25-91) in the LCS group. These small sex and age differences were not significant. Right hemicolectomy was indicated for carcinoma 74% of the time in both groups ( Table 1 ) . Large adenomas, arteriovenous malformations, and, in one case, lymphoma were the other indications. Sigmoid colectomy was indicated for diverticulitis in 58% of the no LCS group and 79% of the LCS group ( Table 2 ) . Carcinoma was the indication in 28% and 21% of the groups, respectively. The other indications for sigmoid colectomy in the no LCS group were sigmoid stricture and sigmoid volvulus. Hence, the majority of the sigmoid resections were completed for diverticulitis, and the majority of the right hemicolectomies were for carcinoma. Fifteen of 36 patients (42%) in the no LCS group and 22 of 49 patients (45%) in the LCS group had previous abdominal or pelvic surgery. These differences were not statistically significant. Average operating room time was less when the LCS was used but did not reach statistical significance. Average blood loss was nearly the same whether the LCS was used or not ( Table 3 ) . The LOS was less for the LCS group , and this did reach statistical significance. One patient developed postoperative intra-abdominal bleeding in the no LCS group. No intra-abdominal bleeding complications occurred in the LCS group. Yet, three patients overall had postoperative bleeding from a stapled anastomosis, for an incidence of 3.5%. One patient had the bleeding controlled by colonoscopic cauterization at the circular staple line. Another patient after sigmoidectomy had an unsuccessful colonoscopic attempt to control the bleeding and required transanal suture of the staple line after 3 units of blood were transfused. A third patient bled from a stapled ileocolic anastomosis after heparin therapy was started to treat a postoperative pulmonary embolus. The bleeding stopped when the heparin was discontinued. However, when heparin was again started, the patient rebled, and, therefore, a vena caval filter was placed. Of note was that none of these patients who bled, bled from the area of dissection with the LCS, even when heparin therapy caused anastomotic bleeding. There were no patients who required readmission for delayed bleeding. Hence, there were no early or late bleeding complications in the areas of dissection with the LCS. Additionally, there were no other early or late complications which could be related to the use of the shears. This report is a single-institution, single-surgeon's experience with LAC, and dates from the first reported cases of LAC. 1 , 2 Although many new instruments and technologies have been introduced since then, the fundamental surgical techniques and principles described then have not changed. The most difficult steps in LAC are intracorporeal mobilization of the colon and division of the mesentery. The learning curve for these techniques is much longer than for the techniques required for laparoscopic cholecystectomy, and this has slowed the widespread use of LAC for patients needing colon resection. This study was done to assess the use of a new technology, the ultrasonically activated shears, for mobilization of the colon and division of the mesentery. The ultrasonically activated shears were developed to apply ultrasonic energy to unsupported tissue. 15 The jaws of the shears consist of an active blade and an opposing passive (not ultrasonically activated), movable tissue pad. This allows the surgeon to grasp tissue and vessels within the jaws of the shears, and coapt the endothelium of any vessels in the tissue. The ultrasonic energy is then transmitted to this tissue and can seal blood vessels and divide what has been grasped. The shears have been shown to facilitate completion of other advanced laparoscopic procedures such as division of the short gastric arteries during Nissen fundoplication 16 , 17 and division of the infundibulopelvic ligament during laparoscopic-assisted vaginal hysterectomy. 18 The active blade of the shears vibrates longitudinally at 55,500 Hz. Depending on the power setting of the generator, the active blade will move 50-100 microns with each oscillation. Touching the active blade to tissue transfers mechanical energy from the blade to the tissue. This mechanical energy breaks Hydrogen bonds in the protein of the tissue, resulting in a sticky coagulum which seals blood vessels. This will allow blood vessels up to 3 mm to be sealed with the shears, without the need for any other method to achieve hemostasis. 19 Relatively little heat is generated compared to other energy sources, since most of the energy delivered is mechanical energy. The relatively low level of heat generated increases the safety with which the instrument can be used adjacent to other viscera, such as the small intestine or great vessels. The largest or named arteries in the mesentery may need to be controlled by other means, such as clips, ligatures, or the endoscopie linear cutting stapler. We found that when the ultrasonically activated shears were utilized, the need for scissors, pre-tied loops, clips, and linear cutting staplers was markedly reduced. In situations in which blood vessels were less than 3 mm (as in colon resections for benign disease when high vascular pedicle division was not necessary) these were no longer needed, and the entire dissection could be completed with the shears. When we compared LAC done with and without the shears, the overall operative times and blood loss were similar. Although the operative times with the shears were a little bit shorter, this could have been due to an increase in our skills as we progressed along our learning curve. However, as our skills improved, and partly due to the availability of the shears, we attempted and completed many more difficult procedures than we would have tried without the shears, as documented by the 20% higher incidence of diverticulitis in the LCS group. These more difficult cases inevitably would have taken more time than most of the cases we tried initially if we had not had the shears. Therefore, the shorter length of time in the group in which the shears were used, although not great, is probably significant since we were often doing more difficult cases with the shears. It is our opinion that use of the shears greatly facilitated successful completion of these more complex cases. The literature documents a decreased length of stay following LAC. 3 – 11 In the present study, a similar LOS of 5-6 days is noted. Although the LOS for the group in whom we used the shears was less, this difference is probably due to changes in our postoperative management as we became more comfortable and familiar with the recovery of patients after LAC. Since the patients treated without the shears were all treated early in our experience (before the shears were available), the decrease in length of stay was probably related to our experience. With experience we learned that early advancement of the diet and earlier discharge are possible because the patient has less pain and a shorter ileus following LAC. We do not see a reason why the use of the ultrasonically activated shears would reduce pain and shorten ileus, nor do we see a reason why the use of the LCS would explain the shorter length of stay. The ultrasonically activated shears are a safe and effective device for mobilizing the colon and dividing the mesentery during LAC. For the experienced laparoscopic surgeon, use of the shears can reduce the time required for routine cases of LAC and can facilitate the completion of more difficult cases. For the inexperienced laparoscopic surgeon, there is no substitute for appropriate training, but the shears have the potential to shorten the learning curve for the inexperienced surgeon by facilitating the two most difficult technical parts of LAC. | Review | biomedical | en | 0.999997 |
10323167 | Laparoscopic-assisted colon resections were first reported in 1991. 1 – 4 Initial enthusiasm for these procedures was high, and it was hoped that the benefits of laparoscopic cholecystectomies would also apply to laparoscopic colon surgery. However, port-site recurrences in laparoscopic colon resections for malignant disease have created concern about laparoscopic surgery for colon cancer. Current prospective studies on laparoscopic surgery in colon cancer will hopefully determine the incidence of port-site recurrence and whether this can be prevented. Until the question is answered, we believe laparoscopic colon resections should be reserved for benign disease. In this setting, laparoscopic colon resection offers many advantages including decreased postoperative pain, decreased hospital stay, and an earlier return to normal activities. Our report is the result of a study of a series of 38 patients who underwent laparoscopic colon surgery; 33 patients had benign conditions including diverticulitis, villous adenomas, and large adenomatous polyps, while five patients had colostomy closures. This group of patients was compared to 39 patients undergoing open colon resections for both benign (15) and malignant (24) disease. From October 1992 to October 1997, 38 patients had laparoscopic-assisted colon resections for benign disease (Group A). During this same period, 39 patients had elective open colon resections: 15 for benign disease (Group B) and 24 patients for malignant disease (Group C). Patients who underwent laparoscopic colon resections for known malignant disease and all patients who had emergent colon resections were excluded. Patients who had resections for polyps that subsequently were shown to have invasive cancer were included. Group A included resections of the right colon (16), left colon (1), sigmoid (10), and transverse colon (2), as well as three subtotal colectomies, one low anterior resection (LAR), and five colostomy takedowns. Group B consisted of resections of the right colon (6), left colon (2), sigmoid colon (3), and four colostomy takedowns. Group C included resections of the right colon (10), left colon (3), sigmoid colon (6), transverse colon (1), as well as two subtotal colectomies and two LARs. All of the removed lesions were localized preoperatively by colonoscopy and/or barium enema. In five patients undergoing laparoscopic resection, intraoperative colonoscopy was also performed. All patients had a standard mechanical bowel preparation plus oral and intravenous antibiotics. The majority of operations were performed by surgical residents, and all laparoscopic cases were under the supervision of one surgeon (MEF). Patients undergoing laparoscopic-assisted right hemicolectomy were placed in the supine position. The initial port was placed by open technique and subsequent ports were placed under direct vision. Port sites varied with the location of the tumor but were generally placed 2-3 cm to the left of midline in a line between the xiphoid and the symphysis pubis. Three 12 mm ports (including the camera port) were used in the majority of cases. Additional ports were placed if needed. Most of the dissection was carried out by the harmonic scalpel (Ethicon Endo-Surgery), and atraumatic clamps were used to manipulate the bowel. Once adequate mobilization was achieved, a 10 cm transverse incision was made to the right of the umbilicus and the colon externalized. The resection, ligation of the blood supply, anastomosis, and closure of the mesenteric defect were performed extracorporeally. Pneumoperitoneum was recreated, and the abdomen was inspected for bleeding. Left colon resections were performed with the patient in a modified lithotomy position. The ports were placed in a mirror image of port placement for a right hemicolectomy and were all 12 mm ports. The dissection and division of the bowel distally was performed intracorporeally, as was the ligation of much of the blood supply. A small, left lower-quadrant transverse incision was made, the segment of bowel externalized, amputated, and the anvil of a circular stapler placed in the end of the proximal colon. The bowel was reintroduced into the abdomen, the incision was closed, and the pneumoperitoneum was recreated. The anastomosis was performed intracorporeally using a circular stapler placed transrectally. A similar technique was used for colostomy takedowns. Laparoscopic patients started a clear liquid diet as soon as they recovered from anesthesia. Patients were discharged when they were tolerating their diet and pain was adequately controlled with oral analgesics. Patients undergoing open procedures were fed when their ileus resolved and were discharged when tolerating their diet and pain was controlled with oral analgesics. Retrospective analysis was performed on the following information: operating room time (from time in to time out of room), operating time (from skin incision to skin closure), estimated blood loss, length of hospital stay (LOS), days until first bowel movement, intraoperative complications, postoperative complications and deaths (within 30 days), and readmissions. The three groups of patients had similar demographic characteristics with regard to age and gender ( Table 1 ) . Types of resections performed, as well as surgical indications, were well matched between Group A and Groups B and C ( Table 2 , 3 ) . Mean operating room time was 162 minutes for Group A, 132 minutes for Group B, and 130 minutes for Group C. Mean operating time was 122 minutes for Group A, 99 minutes for Group B, and 95 minutes for Group C. Mean estimated blood loss was 134 milliliters (ml) for Group A, 210 ml for Group B, and 203 ml for Group C. The average length of stay (LOS) was 3.4 days for Group A, 7.5 days for Group B, and 7.4 days for Group C ( Table 4 ) , and excluded preoperative days. The average time to the first postoperative bowel movement was 2.4 days for Group A, 5.2 days for Group B, and 5.3 days for Group C. Group A had three intraoperative and seven postoperative complications: two small bowel enterotomies and one colotomy (secondary to passing a circular stapler in the rectum), which were recognized intraoperatively and repaired without sequelae. Three wound infections, two postoperative bleeding episodes (one requiring 4 units packed red blood cells (PRBCs) and 2 units of fresh frozen plasma (FFP) for a previously undiagnosed coagulation defect), one intraabdominal abscess (requiring readmission) and one prolonged ileus (greater than 7 days) also occurred. Group B had no intraoperative complications and two postoperative complications: one prolonged ileus and a single instance of urinary incontinence (requiring two additional hospital days before resolution). Group C had two intraoperative complications and seven postoperative complications: two enterotomies, two anastomotic leaks, two wound infections (one leading to wound dehiscence), one intra-abdominal abscess, one prolonged ileus and one death. The death resulted from respiratory complications. Two patients required readmission: one for pneumonia and one for a pelvic abscess. These are summarized in Table 5 . The role of laparoscopy in colon surgery is currently being debated. While the initial enthusiasm for this procedure was high, numerous reports of port-site recurrences when done for malignant disease have discouraged many surgeons from performing laparoscopic colon resections for cancer. Our series demonstrates that laparoscopic colon resections for benign disease can be done safely and with many benefits to the patient. One of the advantages is a decrease in hospital stay. In our series, the average LOS for laparoscopic resections was four days fewer than the open group. This has been a consistent finding when others have looked at this variable. 5 – 8 Estimated blood loss averaged 70 cc less in the laparoscopic cases when compared to the open cases. We and other investigators have shown an earlier return to bowel function. Most studies comparing laparoscopic colon resections to open resections show that the laparoscopic patients tolerate their diet earlier. Some argue that patients undergoing laparoscopic colon resections tolerate their diet earlier because they are fed earlier, 9 but other data support earlier return of bowel function in laparoscopic cases. Bohm et al., 10 demonstrated that the normal myoelectric activity of the stomach, small bowel, and colon returned faster in dogs that underwent laparoscopic right colon resections than in those receiving a traditional open procedure. In addition, median time to the first postoperative bowel movement was 26 hours in the laparoscopic group versus 36 hours in the open group. Other factors that may contribute to a faster return of bowel function in laparoscopic patients are decreased narcotic analgesic usage and less intraoperative manipulation of the bowel. Theoretical advantages to laparoscopy include less intra-operative fluid loss and thus less postoperative fluid shifts, fewer adhesions leading to fewer postoperative bowel obstructions, and less immunosuppression, possibly resulting in improved survival. One disadvantage has been the increased duration of the operation. Operating time for laparoscopic cases was longer than for open cases (24 minutes). Others have noted a 30 to 40 minute increase in operating time for segmentai resections and even longer for total abdominal colectomies. 8 , 11 Patients undergoing laparoscopic resections had an equivalent number of localization studies preoperatively but were more likely to have an intraoperative colonoscopy due to the loss of tactile sensation in identifying lesions. Complication rates are comparable between laparoscopic and open procedures in this study and others. 7 – 9 , 12 – 14 This study compares favorably with others comparing laparoscopic colon resections to open procedures in terms of complication rates, operating times, and lengths of stay. However, studies that include more extensive resections will have higher complication rates and longer operating times. 11 These studies have also documented the safety of laparoscopic colon resection. There is a learning curve associated with performing laparoscopic colon resections. 11 , 13 – 15 In a study by Simons et al., 15 11 to 15 cases were needed to reach a consistent and predictable operating time that did not vary by more than 30 minutes. Others have felt the learning curve to require as many as 70 cases, and clearly there is a more pronounced learning curve than with other laparoscopic procedures. Since laparoscopic colon resections for malignant disease can be more difficult, the procedures necessary to gain these skills should be performed first on patients with benign disease. Our series demonstrates that laparoscopic colon resections for benign disease can be done safely and result in fewer days in the hospital with an earlier return of bowel function than open colon resections. In addition, we believe that surgeons should gain laparoscopic experience on benign disease while awaiting the results of ongoing trials to determine the safety of resections in malignant colon disease. | Review | biomedical | en | 0.999996 |
10323168 | Inguinal hernia repair has been a common procedure performed by general surgeons for the past 100 years. 1 The traditional treatment has been a conventional open repair including such methods as Bassini, Shouldice, or Liechtenstein. Of all of the methods, the Shouldice repair has been one of the most scientifically evaluated methods for hernia repair and is claimed to be the gold standard for comparison with the newer techniques. 2 Recently, a new approach was introduced to repair hernias, which was the laparoscopic method. An advantage of the laparoscopic approach is that it allows patients to recover faster, with less pain. 3 However, disadvantages are the more expensive charge and longer surgical time. 2 , 4 The dilemma thus becomes, Is it more cost-effective to perform a more expensive procedure for a quicker return to work? We conducted a retrospective study of inguinal hernia repairs performed by one surgeon at the same institution using the laparoscopic pre-peritoneal approach and the modified Shouldice technique, comparing surgical time, postoperative recovery time, charge, and time to return to work and to activities. A retrospective study was performed involving patients undergoing inguinal hernia repair from January 1996 to January 1998. All the patients had elective hernia surgery performed by the same surgeon in the same institution. A total of 85 patients were evaluated, with 45 undergoing the laparoscopic repair and 40 undergoing an open modified Shouldice repair. Operative records were examined, and telephone interviews were conducted on all 85 patients. Patient selection involved males from 20 to 75 years old. Patients who had both a laparoscopic repair and open repair were included in the study. Patients with more than two recurrences of inguinal hernia repair were excluded. Patients who had multiple hernia repairs by other surgeons were also excluded. The two methods of surgical repair performed were either a modified open Shouldice repair or a pre-peritoneal laparoscopic repair. The modified Shouldice repair involved the reduction of the hernia, ligation of hernia sac, and reconstruction of the hernia floor. The reconstruction involved a closure of the transversalis fascia with a 2-0 Proline suture in a running overlapping fashion. The conjoined tendon and free edge of Poupart's ligament were also approximated in a running fashion using O-Proline, as described by the Shouldice technique. The repair was performed with local lidocaine under monitored anesthesia. The laparoscopic method was a pre-peritoneal technique. The procedure was performed under general anesthesia. An Origin PB2 dilator using balloon dissection was used to form the pre-peritoneal space without entering the abdominal cavity. Isolation of the spermatic cord was done, and a polypropylene mesh was placed over the myoperitoneal orifice with tackers. The mesh was keyholed for the cord structure to pass through freely. Injection of 30 cc. of 0.5% marcaine was then placed within the pre-peritoneal space prior to closing. Data was collected in two parts. The first involved a telephone interview with the patients undergoing hernia surgery within the two-year period. Patients were asked the same questions using the same scale for evaluation. A 1 to 10 scale, with 1 being minimal and 10 being intense, was used to grade pain responses. The questions were as follows: 1) Severity of pain upon arrival home on the day of surgery. 2) Number of days until pain was gone. 3) Number of days until normal activities such as walking and climbing stairs resumed. 4) Number of days that the patient was out of work. 5) Number of days to start performing strenuous exercises including jogging, mowing lawns, or participating in athletics. Patients were excluded if they were unable to complete the entire interview. Patients who were retired or unemployed were asked when they would have returned to work had they been working. The second part of the study involved evaluation of the operating room record. The data collected included surgical time, recovery room time, and operation charge. The data was obtained for both laparoscopic and open hernia repairs. The surgical time was counted from time of skin incision to skin closure. The recovery room time was from exit from operating room to time of discharge. The charge included the cost for the room, anesthesia, and equipment. The data was evaluated by comparing the results of the laparoscopic technique to the open technique. Simple and bilateral hernia repairs were included. A T-test and Mann-Whitney test were used to statistically evaluate the comparisons. Statistical difference was achieved with a p value less than 0.05 regarding the Mann-Whitney and T-test comparisons. A total of 85 patients were evaluated, with 45 patients undergoing laparoscopic repair and 40 undergoing open repair. The laparoscopic groups included 14 patients with simple hernia repair and 31 with bilateral repair. The open technique included 28 patients with single repair and 12 undergoing bilateral hernia repair. The comparison of the laparoscopic group to the open technique group, regardless of single or bilateral repair, can be seen in Table 1 . The surgical time is 75.71 minutes vs 46.33 minutes, recovery room time is 137.96 minutes vs 68.88 minutes, and charge is $2223.47 vs $1004.88 for laparoscopic vs open procedure, respectively. The comparison of laparoscopic vs open regarding severity of pain is 4.78 to 5.65, days until pain resolution is 4.41 to 5.11, days to resume activities is 2.42 to 4.71, days out of work is 11.58 to 22.68, and days to resume athletic activities is 19.18 to 26.09. The comparison of pain severity and resolution of pain were not statistically different. The results comparing single laparoscopic and open single hernia repairs can be seen in Table 2 . The surgical time is 66.57 minutes vs 40.21 minutes, recovery room time is 138.64 minutes vs 67.57 minutes, and charge is $2190.07 vs $925.25 for laparoscopic vs open procedure. The comparison of laparoscopic vs open regarding severity of pain is 4.86 to 5.14, days until pain resolution is 3.96 to 4.57, days to resume activities is 2.43 to 4.23, days out of work is 10.43 to 20.82, and days to resume athletic activities is 18.07 to 25.20. Only the comparison of days to return to work was statistically significant. The results comparing bilateral laparoscopic and open bilateral hernia repairs can be seen in Table 3 The surgical time is 79.84 vs 60.58 minutes, recovery room time is 137.65 minutes vs 71.92 minutes, and charge is $2238.55 vs $1190.50 for laparoscopic vs open repair. The comparison of laparoscopic vs open regarding severity of pain is 4.74 to 6.83, days until pain resolution is 4.61 to 6.38, days to resume activities is 2.42 to 5.83, days out of work is 12.10 to 27.00, and days to resume athletic activities is 19.68 to 28.17. The comparison of pain severity and days to return to work were statistically significant. The use of laparoscopic surgery has revolutionized general surgery. The change can be easily demonstrated by evaluating gallbladder disease. 1 Patients are now able to have a laparoscopic cholecystectomy performed in less surgical time than an open cholecystectomy. Postoperatively, laparoscopic patients have a shorter recovery time with less pain and are able to return to work and activities sooner than patients undergoing the open techniques. The great success seen with gallbladder surgery can also be applied to hernia surgery. The laparoscopic hernia repair has now become a possible alternative to the traditional open technique. The dilemma is that the laparoscopic method allows patients to have less pain, 5 and return to work and activities sooner, 4 , 6 but this is achieved with a longer surgical time and a higher charge. Therefore, the cost-effectiveness of this procedure needs to be evaluated. 2 The results of our study comparing overall laparoscopic repair to the open technique indicated that the laparoscopic repair had a longer surgical time by 29.38 minutes, longer recovery room time by 69.08 minutes, and a higher charge by $1218.59, than the open technique. The explanation of these differences can be attributed to several factors. The laparoscopic pre-peritoneal approach requires general anesthesia, causing longer operating room time, whereas the open technique can be performed under local anesthesia. The longer surgical time for the laparoscopic procedure is caused by the additional time needed to enter and dissect the pre-peritoneal space with the balloon. This fact is supported when comparing the time for the laparoscopic single repair (66.57 minutes) with the laparoscopic bilateral repair (79.84 minutes). The average time to perform the second hernia repair in the bilateral patient is 13.27 minutes since the pre-peritoneal space is already dissected. This is compared to the single vs bilateral open technique when the additional time to complete the second repair is 20.37 minutes. The longer surgical time can be related to the novelty of the pre-peritoneal procedure. The length of time for our surgical laparoscopic procedures included early experience for the surgeon and staff. Our early operative experience included cases with associated indirect hernias, which required more extensive dissection and, thus, longer surgical times. The learning curve with this laparoscopic procedure resulted in longer times initially, which have noticeably decreased with experience. Patients undergoing laparoscopic procedures receive general anesthesia, and, therefore, recovery room time is considerably longer. These patients require more observation and have more side effects such as confusion and nausea. The open technique procedure patients receive local anesthesia and are, thus, able to be discharged sooner since they do not have the systemic effects of general anesthesia. Concern for the longer recovery room time has introduced the use of epidural anesthesia as a possible alternative to reduce this time factor. A significant difference is also seen when comparing charges for the two procedures. There are several explanations for the average $1000 to $1264 more expensive cost for the laparoscopic procedure. First, the charge for a laparoscopic operating room is $2-$5 per minute more expensive than a general surgery room. Second, the laparoscopic kit required ranges from $400 to $600. This kit includes the dissector and trocars. Third, the cost for general anesthesia is greater than for local anesthesia. The second part of the study involves the postoperative follow-up including the pain severity, pain duration, days to resume activities, and days to return to work. Comparing the overall results of pain severity and duration did not indicate a statistically significant difference. However, the results indicate a trend that the laparoscopic repair did appear to have less pain, which resolved quicker. This difference is supported when evaluating the pain severity in patients undergoing bilateral hernia repair. These patients had statistically significantly less pain than the open technique. This difference can possibly be attributed to less dissection and smaller incision in the laparoscopic repair. Additionally, the Shouldice procedure involves sutures placed under tension as well as requiring dissection through tissue planes along with tissue retraction. The laparoscopic procedure has minimal dissection with placement of a mesh support without tension. Patients undergoing laparoscopic repair were able to return to activities, resume athletic activities, and return to work sooner than the open repairs. This was statistically significant and may be attributed to several reasons. When laparoscopic surgery initially began with gallbladder disease, the main advantage was less pain, shorter hospital stays, and quicker return to work. The success of the laparoscopic cholecystectomy can be applied to laparoscopic hernia repair. Patients understand that the laparoscopic method is designed for less pain and are, thus, prepared to expect to return to activities and to work sooner. Psychologically, the laparoscopic patient expects to recover faster. Second, patients have surgical repair done without tension or a large surgical incision. Patients are also encouraged to walk sooner because there is no tension along the suture lines or tissue planes. Thus, the fear of breaking a suture is removed. Less pain keeps patients from being discouraged from walking or returning to work. An advantage of laparoscopic repair is the quicker return to work. However, this is achieved with a longer surgical procedure time, longer recovery room time, and greater charge. In the present health care crisis, where cost effectiveness is carefully evaluated, the more costly laparoscopic procedure can be considered cost-efficient. For example, in March 1997, the U.S. Department of Labor, Bureau of Labor Statistics reported that employer costs for employee compensation for civilian workers, in private industry and state and local government, in the United States averaged $19.22 per hour worked. Thus, if a person misses 11-15 days of work at 8 hours per day, the cost to the employer may be as much as $1760 to $2310. Since the laparoscopic procedure costs on average $1000 to $1200 more, the savings per person could range from $500 to $1400. Patients in this study undergoing laparoscopic hernia repairs were able to return to work sooner and resume activities and more strenuous athletic activities faster than patients undergoing the traditional open modified Shouldice technique. The results obtained in this study showed a higher charge for the laparoscopic procedure with longer surgical and recovery room time. The more rapid return to work and to activities may outweigh the higher charge and longer surgical and recovery room time. | Study | biomedical | en | 0.999997 |
10323169 | The majority of laparoscopic complications occur in the initial phases of the procedure at the time of Veress needle and trocar insertion. In a series of patients with major vascular injuries during gynecologic laparoscopies, 76.5% of the accidents took place during the initial phase of surgery. In this series, the majority of injuries were secondary to insertion of the umbilical trocar. 1 Abdominal entry injuries are not limited to vascular damage but include perforation of any intra-abdominal organ. A recent series of 26 complications caused by trocars includes 12 vascular injuries, 9 bowel injuries, 3 bladder perforations and 2 incisional hernias. 2 A number of patient-related factors are associated with an increased incidence of abdominal entry injuries; these include a history of prior surgeries, intra-abdominal adhesions and patient's physical habitus. Other factors associated with injury are procedure-related and include patient positioning, surgeon's level of experience, surgical equipment (trocars, needles) as well as technique of insertion. In brief, the standard procedure for abdominal entry involves blind insertion of the Veress needle with the patient positioned flat. Upon documentation of intraperitoneal positioning using the hanging drop, hiss and syringe aspiration test, insufflation is preformed using CO 2 up to an intra-abdominal pressure of 15 mm Hg. At this point, the trocar is blindly inserted. 3 Alternative techniques of trocar insertion have been described. 4 , 5 Although deemed safer, these approaches have not completely eliminated the risk of injury and are more time-consuming and laborious than the standard technique and involve the use of additional grasping instruments on the patient's skin, which may result in undesired scarring. In order to reduce these kinds of complications, we introduced some simple modifications to the standard abdominal entry technique that has been used in our procedures since April 1989. 6 Since April 1989, we performed 3041 procedures using the high-pressure trocar insertion technique as described below. With the surgeon standing on the patient's left side and the patient supine, the left thumb (with or without a sponge) is inserted into the umbilicus as deep as possible, after which the thumb and surrounding umbilicus are rolled over the lower left forefinger, stretching and widening the umbilical fossa, which is further enlarged with the blunt back end of the scalpel. A No. 15 blade is used to make a vertical midline incision on the inferior wall of the umbilical fossa, extending to and just beyond its lowest point. In thin patients, this incision frequently traverses the deep fascia, but intraperitoneal injury is avoided by the pulling of the umbilicus onto the surgeon's forefinger, a maneuver that controls the incision's depth. A disposable Veress needle is grasped near its tip, like a dart, between the thumb and forefinger. The lower anterior abdominal wall is stabilized, not elevated, by grasping its full thickness in the operator's fist and by pulling it downward to bring the umbilicus below the aortic bifurcation. The Veress tip is then inserted at a right angle to the anterior abdominal wall for a distance of 1 cm. Insertion of the Veress needle should be an anatomic exercise, with the surgeon cognizant of the anatomic structures traversed. Individual layers can be felt: deep fascia and peritoneum or, occasionally, peritoneum alone. If the Veress needle is inserted according to these principles, little need exists for testing to ensure proper position of the needle. After complete insertion, the needle is connected to the CO 2 insufflator flowing at 3 to 9 liters/min until a pressure of 25 to 30 mm Hg is obtained, usually after at least 5 liters ( Table 1 ) . The umbilical or first puncture trocar, with its surrounding trumpet-valve trocar sleeve, is placed within the umbilicus. It is not necessary to lift the anterior abdominal wall during insertion of the trocar after establishment of pneumoperitoneum at 25 to 30 mm Hg, as the parietal peritoneum and skin move as one unit with a greater distance between the abdominal wall and the aorta. The trocar should be palmed so that only 1 cm of the sharp tip protrudes beyond the operator's fingers. Following shallow penetration to seat the trocar at a 90° angle in the fascia-peritoneum anatomical funnel created where skin, deep fascia and peritoneum meet, the trocar is upturned to approximately 60°. This continuous motion is almost straight down at first and then becomes almost horizontal, with the wrist rotating nearly 45°. Pressure by pushing on the trocar is then increased until the fascia gives way. The trocar rarely penetrates more than 1 cm. Twisting of the trocar while under pressure is not done. The result is a parietal peritoneal puncture directly beneath the umbilicus. While holding the sleeve against the abdominal wall, the trocar is removed, and the operator hears a rush of gas out of the abdomen. The high pressure setting used during initial insertion of the trocar is lowered as soon as safe abdominal entry is documented to diminish the development of vena cava compression and subcutaneous emphysema. The total amount of time in which the intra-abdominal pressure is 25–30 mm Hg is less than 3 minutes. In cases with known or suspected extensive intra-abdominal adhesions, a special entry technique may be used. The Veress needle is inserted in the left ninth intercostal space, anterior axillary line and again pneumoperitoneum is established to 30 mm Hg. A 5 mm trocar is inserted at the left costal margin, giving a panoramic view of the entire peritoneal cavity. When it is available, a 2 mm scope can be used through a Veress needle. 7 The lower quadrant trocar sleeves are placed above the pubic hairline and lateral to the rectus abdominalis muscle found by direct inspection of the anterior abdominal wall. A relatively constant intra-abdominal pressure between 10 to 15 mm Hg is maintained during long laparoscopic procedures. There were no vascular injuries related to umbilical trocar insertion. Two bowel perforations occurred where bowel was directly adherent to undersurface of the umbilicus. Both cases were immediately repaired: one laparoscopically and the other by laparotomy without further complications. The 30 mm Hg intra-abdominal pressure generates a greater distance between the peritoneum and large abdominal vascular structures, allowing a safer umbilical trocar insertion. In addition, the straight-down initial thrust avoids bowel stuck immediately below the umbilicus. As the 30 mm Hg pressure is maintained for less than 3 minutes, the risk of deep venous thrombosis or CO 2 embolism is minimal, and none of the patients in this series experienced these complications. Nevertheless, the whole surgical and anesthesiological team must be aware of the intra-abdominal pressure at all times in order to remember to decrease the pressure after the abdomen is entered. Trocar entry has been a concern of many laparoscopic surgeons for a long time. In 1974, Hasson developed a technique called “open-laparoscopy” to minimize risk of large vessel injury during entry. 5 It is particularly appropriate for patients with suspected abdominal wall adhesions or for muscular males or children with strong abdominal walls. Although this method has recently been recommended for all laparoscopy by general surgeons in New York State, data concerning increased safety in trained and experienced hands are lacking. Furthermore, open laparoscopy does not necessarily eliminate complications in patients with previous abdominal surgery. 8 In fact, one of the authors (H.R.), who uses this technique only in cases with known extensive bowel adhesions, has entered bowel in each of six attempts at open laparoscopy. Using the standard technique of elevation of the skin of the lower anterior abdominal wall with the surgeon's hand often does not elevate the underlying peritoneum away from the viscera. The distance between the posterior peritoneum and the anterior abdominal wall increases as the abdomen is insufflated in proportion to the pressure obtained. With the 15 mm Hg pressure, the surgeon can almost always palpate aortic pulsation. At 30 mm Hg, it is not usually possible, and the anterior abdominal wall is elevated and fixed so that it will not be squashed or compressed towards the posterior abdominal wall upon downward pressure with a trocar. An additional advantage is that the increased intraperitoneal pressure acts as a counter pressure to the surgeon's thrust, which aids in controlling the depth of trocar penetration. Others authors advocate direct trocar insertion without pneumoperitoneum. This is a suitable method in selected patients without previous abdominal surgery and easily distendable abdominal wall. Strict attention must be paid to the standard surgical principles of good relaxation, adequate skin incision, sharp instruments and anatomy. The reported incidence of bowel injury has been similar to series using pneumoperitoneum , although prospective comparative studies have not been performed. 9 Towel clips have been used to elevate the skin around the umbilicus for Veress needle and trocar insertion. This requires the assistance of a third hand to hold one towel clip while the surgeon is inserting the Veress needle and the trocar and holding the other towel clip. 10 One of the critiques to this approach is the possibility of developing additional scars in some patients with a tendency to keloid due to the towel clips. We do not use shielded trocars. Manufacturers and distributors have recently been asked to voluntarily eliminate safety claims from the labeling of the shielded trocars by the Food and Drug Administration (FDA). The letter to the manufacturers says “FDA is unaware of any data, published or unpublished, showing that these shielded trocars provide any additional protection from injury to bowel, blood vessels, or other organs, when compared to conventional trocars. In fact, review of FDA's own MDR database, manufacturer's complaint files and others reports makes it clear that such injuries do occur with shielded trocars and that the incidence of these injuries is not uncommon.” 11 The high-pressure abdominal entry technique has the advantage of potentially reducing intra-abdominal trocar related injury without requiring additional instrumentation or additional training. Although the potential risks of prolonged exposure to elevated intra-abdominal CO 2 pressure need to be kept into account, the very short exposure time and the absence of complications in this large series point to the safety of the approach. Concerns that insufflation to 25 mm Hg can embarrass respiration, venous return, and cardiac output are unfounded. We never observed a decrease in blood pressure or difficulties with ventilation. The simplicity of the approach makes it acceptable for the surgeon in training, as well, reducing the general risk of complications. | Study | biomedical | en | 0.999997 |
10323170 | With the advent of minimally invasive surgery, there has been a growth of surgical procedures requiring the insufflation of a distending gas for surgical exposure. The physiologic effects of insufflating the intra-abdominal cavity (pneumoperitoneum) have been well described in the literature. 1 – 4 It is recognized that although laparoscopic surgery is associated with a low morbidity, there are significant cardiopulmonary and acid-base alterations that must be considered. 5 As more experience was gained, new procedures were developed, some of which included the use of a distending gas outside the confines of the peritoneal cavity. Such is the case in laparoscopic herniorraphy in which the preperitoneal space is insufflated with carbon dioxide. Additionally, other procedures, such as laparoscopic anti-reflux and colon surgery, that require a pneumoperitoneum involve violation of the peritoneal lining in order to perform the dissection. The opening of the peritoneum allows for the dissection of gas into the local tissue planes. Although this is helpful with the dissection, it also affords an opportunity for significant pneumodissection outside the operative field. The dissection of gas into the extraperitoneal space creates a more dynamic environment when compared to the relatively static space of the intra-abdominal cavity. This may become a significant problem as the complexity and length of laparoscopic surgical procedures increase. The preperitoneal approach to laparoscopic hernia repair provides a model to evaluate the physiology of extraperitoneal CO 2 (carbon dioxide) insufflation. The purpose of this study is to answer the following questions: 1) To what extent is preperitoneal CO 2 absorbed? 2) Does distention of the preperitoneal space result in cardiovascular changes? and 3) To what extent is there dissection of gas within these extraperitoneal tissue planes? In order to evaluate these questions, a porcine model was developed for the insufflation of the preperitoneal space utilizing insufflation pressures commonly employed for laparoscopic hernia repair. After a ten-day acclimation period, 11 adult male pigs weighing between 36 and 45 kg were anesthetized with an IM injection of ketamine hydrochloride (ketaset, 20 mg/kg, Fort Dodge Laboratories, Fort Dodge, Iowa) and Xylazine (2mg/kg, Butler Co., Columbus, OH). The animal was then placed on mechanical ventilation (Ohio V5A Modulus Anesthesia Gas Machine) with an initial tidal volume of approximately 15 cc/kg and maintained under general anesthesia with 1-2% isoflurane (Floran, Anaquest, Madison, WI) while receiving a continuous infusion of lactated ringers at 80 cc/hr. A common carotid arterial line was placed and monitored continuously via an ICU monitor (model HP66, Hewlett Packard, Waltham, MA). A pulmonary artery catheter was then floated into the pulmonary artery wedge position and monitored continuously. Initial arterial blood gas values were obtained prior to the start of the study period, and the animals minute ventilation adjusted to obtain levels of PCO 2 between 38 and 44 with pH between 7.40 and 7.45. Once the blood gas was normalized during this initial stabilization period, the minute ventilation was fixed for the remainder of the study. In addition to arterial blood gas values, hemodynamic parameters were also recorded during this initial stabilization period. Balloon dissection of the preperitoneal space was accomplished through a small infraumbilical incision. The anterior fascia of the rectus abdominus was incised and the rectus split in the direction of its fibers for exposure of the posterior sheath. A 10 mm Origin ™ balloon dissector was then passed along the posterior sheath into the preperitoneal space and insufflated with 15 pumps of the bulb inflator. A 10 mm Hasson trocar was placed and secured for insufflation of CO 2 gas, and a midline transabdominal port was placed in the subzyphoid position in order to monitor for rupture of the peritoneum. Once the blood gas was normalized to the target range, the 180-minute study period began. The preperitoneal space was first insufflated with 10 mm Hg CO 2 gas for 90 minutes. Arterial blood gas values (pH, PCO 2 , and PO 2 ) and hemodynamic parameters (CO - cardiac output, PCWP - pulmonary capillary wedge pressure, CVP -central venous pressure, and PAS/PAD - pulmonary artery systolic and diastolic pressures) were recorded every 15 minutes. End tidal CO 2 (EtCO 2 ) was also recorded using a Hewlett-Packard continuous in-line CO 2 monitor. Cardiac output was determined using the thermodilution technique with the mean of three injections per data point calculated by the hemodynamic module. CVP, PCWP, Pulmonary artery pressures, and arterial blood pressures were transduced directly. At the end of the initial 90 minutes at 10 mm Hg, the preperitoneal pressure was increased to 15 mm Hg for an additional 90 minutes. During the insufflation period, animals were monitored for the development of gross pneumodissection outside the confines of the pelvis, which was arbitrarily defined as palpable subcutaneous emphysema above the umbilical port. At the end of the 180-minute study period, each animal was sacrificed using a lethal dose of Beuthanasia and necropsy of the abdominal wall performed in order to determine the extent of auto-dissection of gas within the tissue planes of the abdominal wall. The data was analyzed using analysis of variance. Statistical significance was considered to be p value less than .05; error bars were calculated using standard error of the mean. The arterial blood gas and hemodynamic data collected during the stabilization period and 180-minute study period was analyzed with respect to 1) the insufflation time (180 minutes) and 2) the insufflation pressure (0, 10, 15 mm Hg). While the data was collected, the peritoneum was monitored for rupture via the transabdominal subzyphoid port. Two animals demonstrated peritoneal rupture and subsequent pneumoperitoneum shortly after increasing the pressure to 15 mm Hg. The data from these animals at 15 mm Hg was excluded from the analysis. The mean of each hemodynamic parameter was calculated at each 15-minute data point for all the animals. Table 1 represents the mean of each parameter at the beginning of the study, at the end of the first insufflation period with 10 mm Hg (after 90 minutes of insufflation), and, finally, the mean at the end of the study period after insufflation with 15 mm Hg pressure. During the 180-minute study period, there was no statistically significant change in hemodynamic parameters when analyzed with respect to time ( Table 1 ) . Analysis of arterial blood gas values with respect to time demonstrated statistically significant rises of PCO 2 and decreases in pH ( Table 2 ) . There was an increase in the slope of the PCO 2 curve and a corresponding decrease in the pH after increasing the pressure to 15 mm Hg . The mean value for each measured parameter at a pressure of zero (prior to insufflation) and at each pressure of 10 and 15 mm Hg was analyzed with respect to one another. The average CO, CVP, and PCWP at a pressure of 15 mm Hg was statistically changed when compared to pressures of 10 and 0. Other hemodynamic parameters did not differ significantly ( Table 3 ) . Similarly, we noticed that at a pressure of 10 mm Hg, there was no statistically significant difference in PCO 2 and pH when compared to blood gas values at a pressure of zero. However, when the pressure was elevated to 15 mm Hg, there was significant increase in the absorption of carbon dioxide with a corresponding acidemia ( Table 4 ) . Necropsy demonstrated that all animals had pneumodissection beyond the confines of the pelvis into the layers of the abdominal wall. However, six animals were noticed to have gross clinical evidence of pneumodissection outside the surgically dissected field during the study period. In all instances, this did not occur until increasing the insufflation pressure to 15 mm Hg and was manifested by palpable subcutaneous emphysema of the entire anterior abdominal wall. The animals with clinical evidence of pneumodissection demonstrated greater degrees of blood gas changes as evidenced by the pH and PCO 2 ( Table 5 ) . Significant pneumodissection in laparoscopic procedures is not uncommonly observed, particularly when the CO 2 is exposed to extraperitoneal tissue planes. Clinically this may present with extensive subcutaneous emphysema, pneumomediastinum and pneumothorax. These sequelae are usually self-limiting once insufflation ceases; however, during insufflation, rapid rises in end-tidal CO 2 can be observed in this setting. Carbon dioxide has traditionally been the insufflation gas of choice, although the use of other gases has been proposed. 6 Results from several studies indicate that hypercarbia, with its associated decrease in blood pH, is primarily due to transperitoneal absorption of the carbon dioxide. Seed et al observed that anesthetized patients undergoing laparoscopic surgery developed increases in EtCO 2 . 7 Ho et al, using a porcine model, were able to directly measure increased carbon dioxide production, with secondary acidemia, after insufflation of CO 2 gas during laparoscopic cholecystectomy. 8 They found that transperitoneal absorption of CO 2 , not increased dead-space and impaired ventilatory function, was responsible for the development of hypercapnia and acidemia. The current study showed that at 10 mm Hg pressure in the preperitoneal space, there was no significant absorption of carbon dioxide. However, after increasing the pressure to 15 mm Hg, there is a statistically significant increase in PCO 2 and decrease in pH with much greater degrees of change observed in the animals with clinical evidence of pneumodissection. It is possible that the development of hypercarbia and acidemia that occurred toward to end of the study period may still have developed if the pressure was left at 10 mm Hg for the entire 180 minutes. This is difficult to answer unless the animals were randomized to two separate pressure groups (one group at 10 mm Hg and a second at 15 mm Hg). However, when the pressure was increased to 15 mm Hg, there was an almost immediate trend in CO 2 retention with a corresponding acidemia, and in six of the eleven animals, a gross evidence of pneumodissection developed that was absent when the pressure was at 10 mm Hg. It is our contention that the process of pneumodissection markedly increases the effective surface area for gas diffusion. Anecdotally, we have observed similar phenomena in humans with significant hypercarbia during laparoscopic procedures in association with pneumodissection developing outside the confines of the surgical field. In our patients, we have only observed this situation at pressures of 15 mm Hg and have noted that the hypercarbia responds rapidly to decreasing the insufflation pressure to 10 mm Hg. These observations suggest that there is a threshold pressure that must be breached in order for pneumodissection to occur, and maintenance of the absorptive capacity of this dynamic space also requires a minimal pressure. Reports of acute cardiovascular compromise during abdominal insufflation is well documented. 9 During insufflation of the abdomen for laparoscopy, it is believed that at lower pressure levels there is augmentation of venous return with a corresponding increase in CVP and right heart filling. This results in an associated increase in cardiac performance as would be predicted by the Starling curve. However, higher levels of insufflation cause significant IVC compression that results in compromised venous return and, hence, a decrease in cardiac performance. 10 , 11 Kelman et al. demonstrated that progressive increases in abdominal pressure to 20 cm H 2 O was accompanied by increases of CVP and cardiac output. Greater increases to around 40 cm H 2 0 resulted in measurable decreases in CVP and cardiac output. 10 Kashtan confirmed these findings in a canine model with the additional caveat that the magnitude and direction of change not only depends on the level of abdominal pressure but also on the intravascular volume status. 11 The mechanical effect of increased abdominal pressure, however, alone may not explain the complex physiologic changes that occur during laparoscopic surgery. In addition to the direct mechanics that insufflation has on the cardiovascular system, there are also changes in acid-base homeostasis and reflex sympathetic activity that must also be considered. In a porcine model by Ho et al., they found that at standard insufflation pressures of 15 mm Hg, the CVP and cardiac output remained unchanged. However, there was a measurable depression in the stroke volume with a compensatory increase in heart rate, thereby maintaining CO. They go on to state that the reduction in stroke volume may be the result of a cardiac depressant effect that CO 2 has on myocardium and that this depressant effect may be partially masked by the sympathetic stimulation on the heart rate from high PCO 2 levels and the stress of abdominal insufflation. 12 In addition, the cardio-depressant effects of anesthesia are also confounding factors that can alter intraoperative cardiovascular physiology. For these reasons, it is impossible to explain a single mechanism for the cardiovascular changes noted during laparoscopic surgery. This study demonstrated no significant changes in hemodynamic parameters over the entire 180-minute study period when analyzed with respect to time. But when these values were compared with respect to the different insufflation pressures, there was an increase in CVP and PCWP, with a decrease in CO after increasing the insufflation pressure to 15 mm Hg. The elevated CVP and PCWP that was seen may possibly be explained by augmented venous return secondary to increased pressure and pneumodissection of gas that occurred at 15 mm Hg. Although, one would normally expect augmented cardiac performance with elevated venous return as predicted by the Starling Curve, it is possible that this increase was negated by the cardiac depressant of the anesthetic and hypercarbia, resulting in the decrease in CO that was observed. Although laparoscopic surgery with insufflation of CO 2 gas is safe and generally associated with minor physio-ogic consequences, there is the potential for significant CO 2 retention to develop in certain situations. In the case of extraperitoneal laparoscopic surgery, there is a potential plane for gas to dissect, thereby increasing the effective absorptive surface area for gas diffusion with a less predictable physiology than the fixed surface area of the intra-abdominal cavity. At the commonly used pressure of 10 mm Hg for preperitoneal hernia repairs, this pneumodissection is minimal. However, the current study suggests that when higher pressures are used, there is a threshold above which significant pneumodissection can occur, resulting in dynamic changes in CO 2 absorption. Insufflation of the preperitoneal space with CO 2 gas does not cause significant alterations in hemodynamics and blood gas changes at a pressure of 10 mm Hg. However, when a pressure of 15 mm Hg is used to insufflate this space, there is evidence of decreased pH and cardiac output, with elevated CVP and CO 2 retention. This correlates with greater pneumodissection of the gas within the layers of the abdominal wall when elevated pressures are used. | Review | biomedical | en | 0.999997 |
10323171 | Tube thoracostomy remains the standard of care for the treatment of pneumothorax and simple effusions in most hospitals. 1 The 8.3 French pigtail catheter (Cook Inc., Bloomington, IN) was designed for pericardial drainage but was first applied as an alternative to chest tube placement for the treatment of postoperative pleural effusions on the liver transplant service at our institution. A favorable initial experience stimulated broader application of this technique, and the pigtail catheter now enjoys acceptance for chest drainage in a variety of clinical settings. The objective of this study was to evaluate the efficacy of the pigtail catheter as an alternative to tube thoracostomy for pneumothoraces and simple effusions. We reviewed all consecutive inpatient pigtail catheter insertions performed between January and October 1996 at the University of Pittsburgh Medical Center. The 8.3 French pigtail catheter (C-PCS-830-LOCK) is constructed of radiopaque polyethylene and is 40 centimeters in length . There are six side holes at the distal end of the catheter. It is packaged with a needle, guidewire, and dilator for insertion using the modified Seldinger technique as well as a serrated, tapered catheter connector which allows easy attachment of the catheter to a standard thoracic drainage system. All procedures were done at the bedside under local anesthesia and without radiologie guidance. Salient technical aspects of pigtail catheter insertion include appropriate use of local anesthetic and needle insertion that barely “walks over” the top of the rib to avoid the intercostal bundle. We typically employ a small (22 gauge) “finder needle” before inserting the larger needle provided with the kit. Air or pleural fluid should be easily withdrawn with the needle, and passage of the guidewire into the pleural space should be virtually effortless. Development of an adequate tract with the dilator and insertion of the pigtail so that the sideholes are well within the pleural cavity are important for proper function. The pigtail catheter is attached to a standard thoracic drainage system and suction applied for pneumothoraces. Pre- and post-placement radiographs were reviewed by a thoracic radiologist (CRF) who determined effusion or pneumothorax volume before and after catheter placement. Pneumothorax and effusion volumes were calculated by determining the average intrapleural distance [AID; AID = (A + B + C) / 3, where A = maximum apical interpleural distance, B = interpleural distance at mid-point of upper half of lung, and C = interpleural distance at midpoint of lower half of lung] and applying a nomo-gram to calculate the percent pneumothorax/effusion volume.2 Therapeutic success was defined as freedom from a second intervention (repeat pigtail placement, tube thoracostomy, or operation) within 72 hours after removal of the pigtail catheter. One hundred and nine pigtail catheters were placed in 86 patients during the nine-month study period. Mean age was 56.3 years (range 16 to 80 years). There were 50 females and 36 males. Fifty-one of 109 insertions (47%) were performed on mechanically ventilated patients. Coagulopathies (defined as an international normalized ratio greater than 1.5 and/or a platelet count below 80,000/mm 3 ) were present at the time of 26/109 (24%) insertions. Seventy-seven effusions and 32 pneumothoraces were treated with pigtail catheters. Etiologies of effusions included: postoperative (3D, sympathetic (26), congestive heart failure (7), empyema (5), and other (8). The pneumothorax group included: central line-related (10), trauma (9), spontaneous (7), and postoperative (6). Treatment details are outlined in Table 1 . Therapeutic success rates in the pneumothorax and effusion groups were 81 and 86%, respectively. There were no major complications related to pigtail catheter insertion. The clinical history and description of failures in the effusion and pneumothorax groups are detailed in Table 2 . Among initial failures in the pneumothorax group, one was associated with malposition of the catheter four days after placement (and initial resolution of the pneumothorax). In three additional placements (in two patients), severe underlying parenchymal disease (severe emphysema in one case and diffuse pulmonary abscesses in another) likely predisposed the pig-tail catheter (and in one case multiple chest tubes) to failure. Exclusion of the two patients (three catheter placements) with severe underlying parenchymal disease and prevention of pigtail migration in the fourth would have yielded a success rate of 30/32, or 94% for pneumotho-races. Eleven of 77 catheter placements for pleural effusions were not successful. Four failures were associated with loculated (non-layering) fluid collections that required either operation or radiographically-guided drainage for resolution. In two cases, pigtail catheters were removed when they were draining in excess of 1000 ml of fluid per day, and the underlying effusions re-accumulated. Exclusion of patients with pre-placement evidence of loculated non-layering effusions and postponement of pigtail removal in the face of excess drainage would have yielded a success rate of 94% for effusions treated by pig-tail catheter drainage. Tube thoracostomy is the gold standard for drainage of pleural fluid and air at most medical centers in the United States. Experience on the liver transplant service at our institution suggested that pneumothoraces and simple effusions could be successfully treated with the 8.3 F pigtail catheter. The current study reviews our experience with 109 catheters inserted in 86 patients over a nine-month period. The high clinical success rate combined with the absence of insertion-related complications strongly supports broader use of the pigtail catheter for drainage of simple effusions and pneumothoraces. Although sporadic reports supporting the use of small-bore catheters for thoracic drainage have appeared in the literature, this technique has not enjoyed widespread application in clinical practice. Sargent reported using a 9 F catheter to treat pneumothoraces in 1970. 3 Lawless and colleagues from the University of Pittsburgh used an 8.5 F pigtail catheter for the treatment of pneumothorax in 16 neonates and small children with a high success rate. 4 Robinson applied Tenckhoff catheters for palliative drainage of malignant pleural effusion in nine patients. 5 Martin reported the use of a small-bore 13 F catheter with an integral one-way valve for the treatment of simple pneumothoraces in 84 patients. 6 All catheters were placed in the second intercostal space in the midclavicular line. No patients were mechanically ventilated. The success rate of 85% in this series was similar to our experience. Conces et al. treated small, asymptomatic, iatrogenic pneumothoraces with a 9 F catheter inserted in the second intercostal space under fluoroscopic guidance with a success rate of 87%. 7 A recent extensive review of techniques of thoracic drainage mentions small-bore catheters only in the context of the treatment of spontaneous or post-needle biopsy pneumothoraces in neonates. 1 A similar review does not include the small-bore catheter as a therapeutic option. 8 The 8.3 F pigtail catheter is now our method of choice for draining air and free-flowing simple effusions from the pleural space. The utility and advantages of the pig-tail catheter for thoracic drainage are supported by the results of this study, with no associated complications in over 100 consecutive insertions. These patients were quite ill: one-half were on mechanical ventilation, and one-quarter had a significant coagulopathy. In general, we do not attempt to correct coagulopathies before inserting the pigtail catheter, and this feature alone makes this technique an attractive alternative to tube thoracostomy for the critically ill, coagulopathic patient. Clinical success rates were high and comparable to previously reported rates for tube thoracostomy. 9 For effusions in the current series, 86 percent of initial pigtail placements were clinically successful. Similarly, 81 percent of pigtails inserted for pneumothoraces were successful. Review of failures in both groups ( Table 2 ) suggests that refinement of patient selection and management strategies would yield even higher success rates for the treatment of both pneumothoraces and effusions. Among physicians at our institution, there is wide acceptance of the pigtail catheter for thoracic drainage on a variety of clinical services. We strongly believe that the pigtail catheter causes substantially less pain than traditional tube thoracostomy, by virtue of its size in relation to the normal intercostal space. The average intercostal space in an adult (measured at the 5th intercostal space in the mix-axillary line) is 8.8 ±1.4 millimeters. A 24 F chest tube (the smallest size commonly used for the described indications) has an outer diameter of 8 mm, while a 32 F chest tube has an outer diameter of just under 11 mm. Chest tubes, with their excessive size, cause pain by compressing the neurovascular bundle at the top of the interspace, as well as by levering open the interspace. In contrast, the 8.3 F pigtail catheter has a diameter of only 2.8 mm and does not impinge on the neurovascular bundle or alter the geometry of the intercostal space . It has previously been shown that routine chest tube placement is frequently associated with an unacceptable pain level. 10 Success using the pigtail catheter demands adherence to proper patient selection and attention to details at the time of insertion. A lateral decubitus chest radiograph is a rapid and simple way of assuring that an effusion is free-flowing and, therefore, likely to respond to pigtail drainage. Nonlayering effusions should be drained with radiographie guidance. Classic teaching mandates placement of a thoracic drainage tube high in the chest for evacuation of air, and low for evacuation of fluid. In practice, however, with free-flowing effusions and pneumothoraces (in which there is free communication throughout the pleural space), we have not found the site of placement of the pigtail catheter to have any bearing on procedural success. Therefore, we place most pigtail catheters in the 4th-6th intercostal space in the mid-axillary line for maximum patient comfort and ease of insertion. Because the diaphragm can be elevated well into the pleural space (particularly in the presence of effusion and parenchymal collapse), we advise insertion of the catheter in a “safe zone” above the sixth intercostal space to avoid subdiaphragmatic catheter placement with its attendant complications. We restrict the use of the pigtail catheter for patients with pneumothoraces and for those with nonloculated simple effusions. We do not use the pigtail catheter on newly postoperative patients, trauma patients, or patients with suspected hemorrhagic effusions. The pigtail catheter may have utility for exudative (stage I, American Thoracic Society) empyemas, although our limited experience treating this entity with small-bore catheter drainage precludes a definitive conclusion; stage II (fibrinopurulent) and stage III (organized) empyemas require more invasive management. The pigtail catheter probably also has limited utility in the treatment of malignant pleural effusions, as the ability to perform talc pleurodesis through the catheter is restricted. In our hands, the pigtail catheter yielded rapid radiographie resolution of both pneumothoraces and pleural effusions. Failure to produce radiographie improvement should prompt insertion of either a second pigtail or a chest tube. One should have a low threshold for additional radiographie study of a pleural fluid collection that does not respond to initial drainage maneuvers. In conclusion, our enthusiasm for application of the pig-tail catheter for thoracic drainage is substantiated by the findings of this study. The pigtail catheter provides safe, reliable, and effective drainage of pneumothoraces and free-flowing simple effusions and is a reasonable alternative to tube thoracostomy when applied to appropriate patients. Adoption of this technique will provide the clinician with a valuable addition to his/her armamentarium that allows effective pleural drainage with minimal patient discomfort. | Study | biomedical | en | 0.999996 |
10323172 | There are currently over 200,000 patients on some form of dialysis in the United States, and the number continues to grow at a significant rate. 1 , 2 Since the introduction of a chronic indwelling catheter in 1976, peritoneal dialysis has been a viable option for patients with end stage renal disease. 3 Continuous Ambulatory Peritoneal Dialysis (CAPD) is increasing in popularity as an alternative to hemodialysis for several reasons. CAPD has many advantages over hemodialysis including cost, simplicity, patient independence and improved nutrition. 4 The use of peritoneal dialysis catheters also has some disadvantages, most of which are related to complications with the catheter. Peritonitis, catheter infection, and mechanical malfunction are the most common complications. 3 Catheter malfunction is usually secondary to migration of the catheter out of the pelvis or occlusion of the catheter by the omentum or adhesions. In the past, several different techniques have been used to salvage malfunctioning catheters. Open revision and fluoroscopic-guided manipulation were the most often used techniques until the late 1980's when the use of the laparoscope became more popular. 5 In our series, we found that laparoscopy offers an alternative approach for revision of these catheters and for primary placement of peritoneal dialysis catheters in patients with previous abdominal surgery. We performed a retrospective review of seven patients who, over an 18-month period, underwent placement or revision of a peritoneal dialysis catheter using laparoscopy. All patients had end-stage renal disease. In five patients, diabetes was the cause of their kidney failure; one was secondary to reflux nephropathy and one was due to nephrotic syndrome. All patients had general endotracheal anesthesia. A Hasson trocar was placed in the infra-umbilical position using an open technique in all patients. A pneumoperitoneum was then obtained, and the laparoscope was introduced. In the two patients who underwent primary placement of the catheters, a 5 mm trocar was placed to the left of the umbilicus under direct vision . With a camera through the periumbilical port and a blunt instrument through the second port, the abdomen was explored and any adhesions were lysed. A single-cuffed catheter was then placed into the abdomen through the 5 mm port and the distal end was then centered in the pelvis . The 5 mm port was then removed, bringing the proximal end of the catheter out the 5 mm cannula site and leaving the cuff within the rectus sheath. One of the patients who had the catheter placed primarily had chronically elevated liver function tests and also underwent a percutaneous liver biopsy. In the five patients who underwent revision of malfunctioning catheters, the umbilical trocar was placed using the technique described above. One or two other 5 mm trocars were also placed to allow for catheter manipulation and lysis of adhesions. Four patients were found to have omental adhesions surrounding the catheter . Three patients were found to have fibrin clot within the catheter, and in one patient the small bowel was adhered to the catheter. Infusing and draining dialysis fluid prior to closing tested all catheters. The fascia at the umbilical port site was closed, and the skin was closed with subcuticular sutures. All procedures were completed laparoscopically, none required conversion to an open procedure. In followup, there was one early complication at two weeks, which required removal of the catheter for an exit-site infection. This catheter was still functional despite the infection. A second catheter remained functional but was removed eight months later at the time of a combined kidney-pancreas transplant. The remaining five catheters are still functional with an average follow-up of ten months. All patients underwent successful peritoneal dialysis in the immediate postoperative period without evidence of leak or other postoperative complications. There continues to be an ever-increasing number of patients requiring treatment for end-stage renal disease. At present, approximately 15–20% of these patients are maintained on peritoneal dialysis. 3 The number of new patients who are beginning this form of treatment, either by choice or that of their primary physician, is only expected to increase. The laparoscopic approach to these patients offers several advantages. It provides a good view of the peritoneal cavity and allows the surgeon to directly visualize the cause of the malfunction. It allows for laparoscopic manipulation of catheter position, removal of fibrin plugs, and creates minimal bleeding which postoperatively can cause the catheter to plug with blood and fibrin products. In addition to these advantages, the fact that the laparoscopic ports can be quickly and securely closed insures the rapid reinstitution of peritoneal dialysis rather than interim hemodialysis requiring a temporary catheter. There is also the benefit of the initial laparoscopic placement of peritoneal dialysis catheters in patients who have had prior abdominal surgery. In this setting, laparoscopic lysis of adhesions can be performed and can allow for peritoneal dialysis in patients who would otherwise require hemodialysis. Failure of this technique is mainly associated with catheters that have recurrent infections and have been encased by massive adhesive process secondary to infection. We feel after one attempt at laparoscopic revision, these patients should be avoided and another technique used. We have found in a small series of patients that laparoscopy offers an alternative approach in the management of patients requiring peritoneal dialysis. Since the submission of this paper, there are six other patients who have undergone laparoscopic revision of their peritoneal dialysis catheter. The six patients were all found to have revisable problems at the time of their laparoscopic procedure. Five of these patients had omental adhesions, and one of the patients had encasement of the peritoneal dialysis catheter with bowel. | Review | biomedical | en | 0.999997 |
10323173 | There is a strong association between pregnancy and gallstones. When biliary colic or acute cholecystitis occurs during pregnancy, medical treatment is indicated. 1 – 3 Spontaneous abortion and congenital abnormalities are associated with cholecystectomy during the first trimester of pregnancy. Premature labor, on the other hand, can occur during the third trimester of pregnancy. 4 – 6 We present three cases of laparoscopic cholecystectomy during pregnancy: two during the second trimester and one at the beginning of the third trimester. Laparoscopic cholecystectomy has the advantage of faster recovery due to less pain than open cholecystectomy. 7 A 30-year-old white female, 22 weeks pregnant, was admitted with two episodes of right upper quadrant abdominal pain associated with nausea and vomiting. Ultrasonography of the gallbladder revealed gallstones with a normal size common bile duct. Because conservative treatment with diet failed, the patient was admitted for laparoscopic cholecystectomy. She received indocin to prevent premature contractions. A Hasson trocar was placed, and the abdominal cavity was insufflated with carbon dioxide, with the maximum insufflation pressure at 15 mm Hg. The patient was placed on the left lateral decubitus position. Laparoscopic cholecystectomy was performed without incident. In the recovery, room she had fetal heart monitoring, which did not reveal any bradycardic episodes. She was then admitted to the ante-partum unit and discharged the following day, tolerating oral diet. The final pathology revealed chronic calculous cholecystitis. She subsequently delivered a full-term baby boy weighing 7 lb 13 oz. A 25-year-old female, 26 weeks pregnant, was admitted to the hospital with two episodes of biliary colic, which resolved with conservative management. A third episode occurred, and she was again admitted, placed on antibiotics and taken to the operating room for laparoscopic cholecystectomy on the following day. A Hasson trocar was placed, and the abdominal cavity was insufflated with carbon dioxide with maximum insufflation pressure of 12 mm Hg. Intraoperative and postoperative fetal heart monitoring did not reveal any fetal heart compromise, although the patient had a few contractions. The patient was discharged on the second postoperative day, tolerating oral diet. The final pathology report was chronic calculous cholecystitis. She delivered a healthy, full-term 8 lb boy. A 24-week pregnant female had two previous admissions for cholecystitis, which were treated with analgesics and antibiotics. The ultrasound revealed gallstones with a thickened gallbladder wall and a normal size common bile duct. Because the pain recurred, the patient was admitted for laparoscopic cholecystectomy. The patient had a Hasson trocar placed, and the abdominal wall was insufflated with a maximum pressure of 15 mm Hg. No perioperative fetal monitoring was applied. She was dis-charged on the first postoperative day, tolerating oral diet and delivered a full-term baby with vacuum-assisted vaginal delivery. The final pathology again revealed chronic calculous cholecystitis. Biliary colic during pregnancy can most often be man-aged successfully with diet and analgesics. If acute cholecystitis is suspected, antibiotics can be added, and cholecystectomy can be postponed until after delivery. If these measures are not successful, cholecystectomy is indicated. 8 The second trimester is the safest time to perform the procedure. 9 – 12 During the first trimester, fetal malformation because of ongoing organogenesis is the major concern associated with anesthesia and abdominal surgery. During the third trimester, premature labor is the most important complication of cholecystectomy, 4 – 6 although cases of successful laparoscopic cholecystectomy during that trimester have been reported. 13 Recently, Graham et al. published six case reports of laparoscopic cholecystectomy during pregnancy and performed a literature research of 105 published similar cases. They concluded that, although the above procedure is technically feasible in all three pregnancy trimesters, the incidence of spontaneous abortion and premature delivery is lower during the second trimester. 9 There are 14 cases of laparoscopic cholecystectomy reported during the third pregnancy trimester. 9 Only one patient had pre-term delivery due to hypertension. The higher incidence of pre-term labor after open cholecystectomy (40%) 14 during the third trimester of pregnancy justifies the laparoscopic approach when cholecystectomy is necessary. Our only third trimester pregnant patient who underwent laparoscopic cholecystectomy had a full-term, uneventful delivery. The most common abdominal procedures performed during pregnancy are appendectomy, ovarian cystectomy, laparoscopy for ectopic pregnancy and laparoscopic cholecystectomy. 15 Prophylactic tocolysis with intravenous magnesium have an uncertain effect on the incidence of pre-term labor. 5 , 16 Our patients who underwent laparoscopic cholecystectomy received indocin with good results. The effect of carbon dioxide pneumoperitoneum is unknown. 4 Although the carbon dioxide can cause physiologic alterations in the fetus, the elimination of carbon dioxide from the placental circulation is rapid and should not cause serious problems. A case of gasless laparoscopic cholecystectomy has been reported by lafrati et al. 15 Intraperitoneal pressure of carbon dioxide should be kept at a minimum. In our cases, carbon dioxide pneumoperitoneum with a maximum insufflation pressure of 15 mm Hg did not cause any fetal compromise, as shown by the good perioperative course of the three patients, as well as the healthy babies that resulted. Concerns have been expressed about the effect of venous flow from the lower extremities with carbon dioxide pneumoperitoneum during pregnancy. Specifically, the application of intermittent pneumatic compressors cannot eliminate the phenomenon of venostasis during pregnancy. 17 None of our patients developed deep vein thrombosis during laparoscopic cholecystectomy. We found the use of a Hasson trocar to be extremely useful, because the open technique of port placement avoids injury to the uterus. Most other case reports of laparoscopic cholecystectomy during pregnancy report using the same technique. 18 , 19 We felt that intraoperative cholangiogram is risky for the fetus due to the radiation exposure and prolonged anesthesia. However, this topic is controversial. 13 We did not use intraoperative cholangiography during our cases because there was no evidence of choledocolithiasis on the ultrasonography or the laboratory evaluation. If cholangiography is necessary, a lead shield should be placed over the entire infraumbilical area to protect the fetus. 20 We conclude that laparoscopic cholecystectomy during the second and early third trimester of pregnancy with perioperative fetal monitoring is safe for the mother and the fetus. | Other | biomedical | en | 0.999995 |
10323174 | Mucocele of the appendix is a nonspecific term that is used to describe an appendix abnormally distended with mucus. This may be the result of either neoplastic or non-neoplastic causes and may present like most appendiceal pathology with either mild abdominal pain or life-threatening peritonitis. Urologie manifestations of mucocele of the appendix have rarely been reported. We report our experience with a woman with mucocele of the appendix and review the relevant literature. A 34-year-old female presented to our service with the complaint of lower abdominal and back pain increasing in severity over the last month. She stated she experienced a single episode of gross hematuria one month ago and that although she had not noted other occur-rences of hematuria, the abdominal pain consistently was worse with urination. The patient described the pain as sharp and constant. She denied nausea, vomiting or a change in bowel habits, but had been having occasional fever. She was seen twice in the Emergency department without diagnosis or resolution. The patient's past medical history included a tubal ligation, a cholecystectomy, and a pituitary adenoma diagnosed but not treated. She denied any history of kidney stones. The patient is married with two children, smokes one-half pack per day and drinks alcohol occasionally. Her family history is remarkable only for thyroid disease. Physical exam was significant only for a diffusely tender abdomen upon palpation without peritoneal signs. The pain was more pronounced on the right anterior abdomen and right flank. The remainder of the physical exam was unremarkable. Labs were noncontributory other than a white blood cell (WBC) count of 12,000/ml. Cystoscopy, upper GI endoscopy and colonoscopy were negative. KUB (kidney, ureter, bladder) plain film was significant for a pea-sized opacity seemingly outside the right lower border of the right kidney. Intravenous pyelography revealed a duplex ureter on the right and no filling defects in the urinary system and confirmed an opacity apparently outside the free edge of the right kidney . Computed tomography of the abdomen revealed a 7 cm × 1.5 cm tube-like structure extending from the cecum to the liver and was otherwise within normal limits . At laparoscopy, the patient was found to have a grossly dilated appendix that extended from the retroperitoneal cecum to the liver posteriorly. It was detached from the liver and successfully removed intact laparoscopically . The patient's postoperative course was uneventful. Pathologic examination revealed a benign mucinous cystadenoma with mucocele formation . Mucocele of the appendix has been described to occur more often in females and at an average age over 50. l It was originally characterized in 1973 with the term retention cyst to describe a sterile outflow obstruction in the appendix that was dilated and swollen with glairy mucus. The term mucocele has more recently been substituted. Patients almost always present with symptoms that are characteristic of any number of abdominal or pelvic conditions. The porcelain appendix and the so-called volcano sign are two nonspecific diagnostic clues, but CT remains the most suggestive tool. Mucoceles are occasionally diagnosed incidentally in the course of other surgery. On ultrasound, they have been known to mimic ovarian cyst torsion. Mucoceles can be classified histologically into three types: Mucinous neoplasms of the appendix may be benign, taking the form of hyperplasia or cystadenoma, or may more rarely occur as cystadenocarcinoma, which is malignant. Both benign and malignant mucoceles may spontaneously rupture, secondary presumably to hypersecretion of mucus. These patients may present with a peritoneal cavity filled with mucus, informally described as “jelly belly.” Mucocele characterized only by mucosal hyperplasia is an entity that macroscopically resembles a hyperplastic colorectal polyp. Cystadenomas are also benign and may be treated similarly. Simple appendectomy with free margins is curative for these nonmalignant mucoceles as long as they have not ruptured. Treatment for malignant mucoceles is distinct in that the histologie diagnosis following appendectomy warrants a return to the operating room for a right hemicolectomy. Because the neoplastic diagnosis is only determined by pathology, removal of the appendix requires caution as inadvertent rupture may lead to seeding of the malignancy causing pseudomyxoma peritonei. Although rupture of a mucinous cystadenocarcinoma does not result in systemic metastasis, excessive mucin in the peritoneum and pseudomyxoma peritonei may cause death due to infection or intestinal obstruction. Laparoscopy can be used as a diagnostic tool in those cases which are equivocal. Conversion to laparotomy may be indicated if there is special concern for the ability to remove the appendix intact or if more extensive resection is warranted, as in malignancy. Various etiologies have been reported. Mucoceles secondary to obstruction have been reported to occur in endometriosis, cystic fibrosis, and in cases of carcinoid. Additionally, mucocele has occasionally been reported to cause small bowel obstruction secondary to volvulus and to intussusception. There has been one report of a mucocele secondary to diverticulitis. 2 Mucocele of the appendix rarely presents with urologie manifestations. Local or mass effects of mucoceles have been reported to cause hydronephrosis, and classic symptoms of urinary tract infection (UTI), but have very seldom been reported to cause hematuria alone. 3 , 4 In one case report, mucocele was responsible for infertility, which resolved upon surgical removal of the diseased appendix. A variety of abnormalities of the appendix underscores the need for routine pathologic examination of the appendix. Histopathology often changes both the diagnosis and the treatment, as in occult parasitic infestation and malignancies. Although uncommonly reported in the literature, urologie symptoms may be a manifestation of appendiceal pathology. In this case, back pain with hematuria was the primary presentation of a benign cystadenomatous mucocele of the appendix. Laparoscopy allowed for definitive diagnosis and therapy. We recommend laparoscopy as the initial intervention and emphasize the need for the careful intact removal of a suspected mucocele of the appendix. | Clinical case | biomedical | en | 0.999997 |
10323175 | First described by Gilchrist in 1894, 1 blastomycosis was thought initially to exist in two distinct forms, cutaneous and systemic. Later investigations by Schwartz and Baum, 2 established lung as the primary portal of entry, with end organ (ie, skin, bone, soft tissue) involvement secondary to hematogenous dissemination. Endemic distribution of the organism occurs mainly along the Mississippi and Ohio River basins of the Midwestern and southeastern United States. While there is no overall age, race, sex, or occupational predilection for the disease, people exposed to the soil in endemic areas are at greatest risk. 3 , 4 After inhalation of the mycelia, conversion to the yeast form occurs at body temperature (37°C). Host defense mechanisms recruit neutrophils and form non-caseating granulomas with giant cells in an attempt to contain spread of the yeast. Only one-half of infected patients are symptomatic; presenting complaints include chills, fever, and transient pleuritic chest pain. Chest radiography demonstrates lobar or segmentai consolidation. 5 , 6 Serologie tests for diagnosis of blastomycosis include serum complement fixation assays or antibody A identification with immunodiffusion or radioimmunoassay/enzyme-linked immunosorbent assays. All are fraught, however, with low specificity and failure to reliably diagnose the disease in the acute setting. Definitive diagnosis relies on growth of the organism from body fluids or biopsy specimens. Treatment of localized pulmonary disease with oral azole derivatives has been successful. Intravenous amphotericin B treatment is reserved for critical pulmonary infection, central nervous system disease, and infection in patients with concomitant immunodeficiency syndromes. A 37-year-old woman presented to her primary care physician with a chief complaint of productive cough, fever to 39°C, and shaking chills. A presumptive diagnosis of pneumonia was made, sputum cultures demonstrated normal respiratory flora, and she was begun on a two-week course of oral antibiotics. Showing no improvement after this course of therapy, a chest radiograph demonstrated progression to a right pleural effusion. Cultures from a thoracentesis specimen were sterile. She continued to spike fevers, and computed tomography of the chest showed consolidation of the right lower lobe and re-accumulation of a loculated pleural effusion. She was transferred to our institution for further evaluation and treatment. The patient is a well-developed, well-nourished woman in mild distress, who complained of right-sided pleuritic chest pain with inspiration and reported occasional hemoptysis. Diminished breath sounds were noted at the right base. Heart examination showed a regular rhythm and no murmurs or rubs. No skin lesions were observed, and she was neurologically intact. The peripheral white blood cell count (WBC) was 16,100 cells/mm 3 . Thoracentesis revealed pleural fluid with a pH = 7.3, WBC = 2692 cells/mm 3 , glucose = 105 mg/dl, LDH = 433 IU/L, and total protein = 5.5 gm/dl. Chest radiography showed persistent atelectasis of the right lower lobe with an effusion. Fiberoptic bronchoscopy demonstrated no endobronchial lesions, and sputum cultures obtained were negative at two weeks. Chest radiographs showed a right lower lobe atelectasis and a large pleural effusion . After placement of a thoracic epidural infusion catheter, she underwent general endotracheal anaesthesia with a double lumen tube to facilitate single lung ventilation. A thick empyema with visceral and parietal pleural stud-ding was encountered on VATS exploration. Frozen section analysis of pleural biopsy specimens was performed. Histological examination showed non-caseating granulomatous inflammation, while pleural biopsies demonstrated scattered, predominantly suppurative granulomata surrounded by fibrous tissue and occasional foci of mature adipose tissue. Higher magnification showed round yeast forms of Blastomyces species with thick cell walls, and multiple nuclei , with occasional broad-based budding forms . After biopsy, an endoscopie decortication of the right thorax was performed. The patient was begun on oral itraconazole (400 milligrams) twice daily. Postoperative chest radiographs showed regression of the effusion but persistent mediastinal and hilar adenopathy. Computed tomography showed no intracranial lesions. The patient was begun on a six-week course of intravenous amphotericin B. After one week of therapy, her chest radiograph showed significant improvement. She was discharged to home on postoperative day nine. Follow-up at six months showed complete recovery and a normal chest radiograph. Clinical presentation of pulmonary Blastomyces includes fever, chills, and pleuritic chest pain. While most cases present radiographically with lobar or segmentai consolidation, this patient's recurrent effusion and pleural studding noted on video-assisted thoracoscopic examination of the thorax is uncommon. In 1964, the Blastomycosis Cooperative Study of the Veterans Administration 3 examined 198 patients with this disease and identified only four patients with effusions. Similarly, Sarosi et al. 7 found no effusions in their series of 18 patients, and Rabinowitz et al. 6 found no pleural disease in 51 patients with pulmonary blastomycosis. Other investigators have found varying degrees of pleural involvement with this disease. Kinasewitz 8 found pleural thickening in 88% of 26 patients, but only four cases with even small effusions. Thirteen of 63 patients had small effusions reported by Sheflin and colleagues 9 in a radiographie review series. Failla 10 examined seven cases of pulmonary blastomycosis at their institution and noted the uncommon findings of two patients with large pleural effusions and one with an endobronchial lesion. Diagnosis of blastomycosis is by culture of the organism from bronchial washings or lung biopsy specimens, or direct histopathologic identification. Granulomatous inflammation with characteristic fungal elements of large budding yeast cells with double refractile walls demonstrating Blastomyces dermatitidis are seen. Given the disease's low propensity for pleural involvement, it is not surprising that thoracentesis is generally non-diagnostic. This patient's operative finding of diffuse pleural blastomycotic studding might explain her development of a recurrent loculated effusion. It is not clear, however, why B. dermatitidis was not cultured from the two thoracentesis samples obtained, given the widespread pleural involvement, or why the patient developed hemoptysis. The postoperative progression of hilar adenopathy prompted evaluation of her central nervous system for evidence of systemic spread of infection. Although oral ketaconazole is the preferred treatment for pulmonary blastomycosis, this patient's thoracic adenopathy and low-grade fever resulted in a six-week course of intravenous amphotericin B (33 milligrams per day). Post-pneumonic effusion is best treated by thoracostomy tube drainage. Failure of this technique, coupled with negative sputum and effusion fluid cultures, resulted in VATS pleural debridement and biopsy. Blastomycosis pneumonia only rarely progresses to effusion and empyema. This patient's hemoptysis, loculated effusion, and hilar adenopathy are uncommonly associated with Blastomyces dermatitidis infection. Advantages of VATS over open debridement and biopsy include a limited incision with less potential for operative site infection and diminished postoperative pain while providing a thorough evaluation of the thorax and lung. We recom-mend VATS over the open thoracotomy with radiological localization approach for evaluation of patients with post-pneumonic effusions of unknown etiology. | Review | biomedical | en | 0.999998 |
10323176 | Bowel obstruction is a complication which is not uncommon after laparoscopic colectomy. Previous studies have reported incidences of 0.8-2.5%. 1 – 3 In most cases, the cause of the bowel obstruction is an incarcerated Richter's hernia in the trocar site. Here we report a case with a transmesenteric hernia after laparoscopic sigmoid colectomy in which a loop of small bowel was incarcerated in a surgery-related mesenteric defect. A 47-year-old woman was admitted to our hospital for surgical treatment of sigmoid colon cancer. Barium enema and colonoscopic study demonstrated a 1.5 cm × 1.8 cm polypoid lesion in the sigmoid colon . Biopsy revealed well-differentiated adenocarcinoma. Based on a diagnosis of submucosal cancer, laparoscopic-assisted sigmoid colectomy was selected for the optimal treatment. The operation was performed with curative intent using a gasless technique previously described. 4 Anastomosis was undertaken using the double stapling method. Lymph node dissection with low ligation of the inferior mesenteric artery was per-formed simultaneously. The mesenteric defect resulting from bowel resection was not completely closed because of technical difficulty. Figure 3 shows the resected specimen. Pathological examination revealed submucosally invasive cancer without lymph node involvement. On the 20th postoperative day, the patient developed abdominal distension, nausea and vomiting. Plain abdominal X-ray films demonstrated a small bowel obstruction. A decompression tube (long tube) was inserted, and, on the 26th postoperative day, the bowel obstruction showed improvement. However, after initiating liquid meals, the patient again developed bowel obstruction. Abdominal X-rays after ingestion of contrast medium showed a severely dilated small bowel . The anal edge of the dilated loop was located near the anastomosis, and laparotomy was performed. Abdominal exploration revealed a dilated loop of small bowel incarcerated within the mesenteric defect. Adhesiolysis was performed. The postoperative course was uneventful, and the patient was discharged on day 21 after the second procedure. Laparoscopic approaches to colonie malignancies have gained acceptance for selected patients as a new, minimally invasive therapeutic modalities. The incidence of bowel obstruction after laparoscopic colectomy is reported to be 0.8-2.5%. 1 – 3 The most frequent cause of the bowel obstruction is Richter's hernia at the trocar site. Such conditions have been reported after gastroentero-logical as well as urologie and gynecological laparoscopic operations. 5 – 12 To prevent Richter's hernia, fascial closure has been recommended, even for 5 mm trocar sites. 7 Vanclooster 13 and Tsang 14 reported cases developing hernia after laparoscopic herniorrhaphy in which a loop of small bowel became incarcerated in the incompletely closed peritoneum. In this case, which to our knowledge is the first case in the literature, a loop of small bowel became incarcerated in the incompletely closed mesentery. Closure of the mesentery after bowel resection is sometimes very difficult due to the limited operative field, especially at the most proximal portion of the mesentery. However, this case strongly suggests the need for complete closure of the mesentery to prevent bowel incarceration. Meticulous suturing or clipping should be performed to avoid transmesenteric hernia after laparoscopic colectomy. | Clinical case | clinical | en | 0.999995 |
10323177 | A 46-year-old female presented with vague gastrointestinal symptoms, chronic right upper quadrant abdominal pain and intolerance to fatty food and diarrhea for the last two years. She was given the diagnosis of irritable bowel syndrome and had been followed by her internist. She had undergone an abdominal hysterectomy with bilateral salpingo-oophorectomy for menometrorrhagia and fibroids 15 years ago but was otherwise healthy. During the work-up, an ultrasound (U/S) of the abdomen revealed several small stones in the fundus of the gallbladder and a common bile duct of normal diameter. The appendix was not visualized in the U/S. She was then scheduled to undergo an elective laparoscopic cholecystectomy. Pneumoperitoneum was established with CO 2 gas insufflation under general anesthesia in the usual fashion. Laparoscopic visualization of the abdominal cavity revealed a small brownish mass at the tip of the appendix resembling a lymph node. The gallbladder was removed via the standard American laparoscopic technique (4 ports). The camera was then moved from the supraumbilical port to the subxyphoid one. Using the same instruments, ie, two Endo grasps plus a Multifire Endo GIA 30 stapler, the appendix was removed by stapling across the base of the appendix and across the mesoappendix. Pathologic examination demonstrated a carcinoid tumor in the tip of the appendix, which measured 0.3 × 0.2 cm . The margins of the specimen were clear, no lymph nodes were seen, and there was no mesoappendix invasion. The patient was discharged 23 hours after the operation. She remains asymptomatic at six months follow-up visit. Merling, in 1838, described the gross pathology of carcinoid tumor of the appendix, and in 1907 Oberndorfer coined the term “Karzinoide.” 1 , 2 Carcinoid tumors are the most common gut endocrine tumors. 3 They arise from the foregut, midgut, and the hindgut. Carcinoid tumors of the midgut arise from the distal duodenum, jejunum, ileum, appendix, and ascending and right transverse colon, with the appendix and terminal ileum being the most common locations. Carcinoids share common histochemical characteristics with other gut cells; they originate as solid nests of small monotonous neuroendocrine cells with high rare mitoses and occasional acinar or rosette formation. They have a high amine content in the cytoplasm, are capable of amine precursor uptake, and are able to decarboxylase these products to synthesize amines and/or peptides, 4 such as tachykinins (substance P, neurokinin A, neuropeptide K) and others. Godwin reviewed 2,837 cases of carcinoid tumor and found that the majority originate from the gastrointestinal tract (85%), but they were also found in the lung, ovary, and biliary tracts. Most were in the appendix, rectum, and ileum. Age-adjusted incidence rates were higher for black males. Carcinoid tumors showed several differences from other kinds of tumor, including a low age for appendiceal and lung cases and low male/female and black/white ratios in the lung. Godwin's percentages of concurrent neoplasm and multiple carcinoids were low compared to other series. 5 Survival varies with anatomical location and histological characteristics. Five-year relative survival rates ranged from 99% (appendix) to 33% (sigmoid colon). However, survival for colon cases was not as low as expected on the basis of the high rate of metastases. 5 Histologically, there are five generally accepted carcinoid growth patterns, ie, insular (type A), trabecular (type B), glandular (type C), undifferentiated (type D), and mixed type. 6 The mixed tumors with acinar and glandular pattern have the best median survival time (4.4 years), whereas the undifferentiated has the poorest median survival (0.5 year). 6 In decreasing order of median survival time in years, the growth patterns ranked as follows: mixed insular plus glandular, 4.4; insular, 2.9; trabecular, 2.5; mixed insular plus trabecular, 2.3; three pooled low incidence rate mixed growth patterns, 1.4; glandular, 0.9; and undifferentiated, 0.5. 7 The site of origin and rate of metastases varies, with appendix being the most common site of carcinoid. The frequency of metastasis from carcinoid of the appendix varies between 1.4% and 8.8%. Carcinoids of the appendix and rectum smaller than 2 cm have a 5-year survival rate of approximately 100%, 8 – 12 declining to 40% when the tumor diameter is larger than 2 cm. However, metastases have been reported from carcinoid of the appendix less than 2 cm in size. 13 , 14 While the risk of metastasis of these tumors has been correlated with their size, invasion of the mesoappendix is predictive of an increased risk of metastasis for carcinoid tumors of the appendix less than 2 cm in size. 13 Tumors with liver metastases have a 5-year survival rate ranging from 21% to 42%. 15 , 16 The clinical presentation of carcinoid tumors depends on their location with a wide clinical spectrum varying from asymptomatic to the malignant carcinoid syndrome. Carcinoids of the appendix and small intestine are slowgrowing and silent. They are, in most instances, found incidentally at operation, as in our case. A third of patients with carcinoid present with years of intermittent abdominal pain often ascribed to other gastrointestinal or biliary disease, or to the irritable bowel syndrome. Malignant carcinoid tumors may present with symptoms of mechanical bowel obstruction due to fibrosis, adhesions and kinking of the intestine. Other constitutional symptoms include weight loss, diarrhea, upper gastrointestinal bleeding, intussusception, and abdominal mass. In patients with carcinoid of the foregut (atypical carcinoid), the urine contains only slightly elevated levels of 5-hydroxyindoleacetic acid (5-HIAA), but large quantities of 5-hydroxytryptophan (5-HTP) and 5-hydroxytrypta-mine (5-HT), because these tumors are deficient in dopadecarboxylase, which is responsible for the conversion of 5-HTP to 5-HT. The excess 5-HTP in the plasma is directly excreted into the urine. In carcinoid of the midgut (typical carcinoid) most of the 5-HTP is rapidly converted to 5-HT, which is taken up by platelets. The excess of 5-HT is converted by monoamino oxidase (MAO) and aldehyde dehydrogenase (DA) to 5-HIAA, which then is excreted in high concentrations in the urine. It is interesting to note that serotonin-rich foods, such as bananas, plantains, pineapples, kiwi fruits, wal-nuts, hickory nuts, pecans, avocados, and acetaminophen may artificially elevate 5-HIAA. 8 Numerous techniques can identify the primary site of the tumor. A chest radiograph or computed tomography (CT) may show a bronchial or mediastinal tumor 17 , 18 Double-contrast gastrointestinal studies still best define the primary neoplasm. Appendiceal tumors frequently escape radiological detection until large enough to be discovered by computed tomography (CT). The hypervascular nature of carcinoid tumors and their metastases allows superior mesenteric arteriography of the small bowel and cecum to be used when the scanning procedures are not revealing. The “spokewheel” configuration of the desmoplastic mesenteric masses and lymph node metastases are best seen by CT, whereas hepatic metastases can be demonstrated by CT, CT-angioportography (CTAP), ultrasonography (US), magnetic resonance imaging (MRI), and octreotide scintigraphy. Percutaneous needle biopsy with radiological guidance may confirm the diagnosis of carcinoid tumors and their metastases. Hepatic arteriography is frequently performed in preparation for hepatic embolization or chemoembolization. 19 , 20 Early resection offers the patient the best chance of cure. Carcinoid should be resected regardless of the presence of metastases. Appendiceal carcinoid tumors are rarely malignant, and lesions smaller than 1.5 cm can be safely resected by appendectomy only. If the tumor is at the base of the appendix, a cecectomy may prove necessary. Tumors larger than 1.5 cm require an ileocolectomy. 21 , 22 Laparoscopic resection of carcinoid tumors from the stomach, 23 gallbladder, 24 proximal 25 and distal 26 rectum have been described. Laparoscopic appendectomy has been compared to open procedures, and the results showed open procedures taking less time, but there were more wound infections than in the laparoscopic procedure. Patients with acute appendicitis recuperated more quickly from the laparoscopic procedure, as evidenced by the time until eating regular diet, period of hospitalization, incidence of nausea and pain medications on postoperative day one. 27 – 31 We report a case of incidental laparoscopic resection of a carcinoid tumor at the tip of the appendix during an elective laparoscopic cholecystectomy. Even though the risk of metastasis from carcinoid correlates with the size, ie, greater than 2 cm, there are several cases of metastatic disease from carcinoid tumors smaller than 1 cm in diameter. Based on review of the literature, we suggest that any suspicious lesion of the appendix found during laparoscopic exploration of the abdominal cavity should be removed. We believe that the magnification provided by the laparoscope, ie, up to 18 times, and the ability to inspect the abdominal cavity allowed us to identify the lesion and to perform laparoscopic appendectomy while adding minimal morbidity to the procedure. | Clinical case | clinical | en | 0.999997 |
10330396 | HeLa cells, stably transfected with GFP-NFAT , were grown in Dulbecco's modified Eagle medium ( GIBCO BRL ) with 10% fetal bovine serum on plastic dishes. Treatment of intact cells with trichostatin A (Wako BioProducts) to induce expression of GFP-NFAT, and with ionomycin ( Calbiochem Corp. ) to induce import of GFP-NFAT into the nucleus was done as described . As indicated, permeabilized cells were subjected to a preincubation step in the presence of ATP, either including or excluding RanQ69L and cytochrome c-NES (cc-NES) at final concentrations of 20–25 and 100 μg/ml, respectively. After preincubation, cells were washed twice with transport buffer (20 mM Hepes-KOH, pH 7.3, 110 mM KOAc, 2 mM Mg[OAc] 2 , 2 mM DTT, and 1 μg/ml each of leupeptin, pepstatin, and aprotinin). Nuclear export reactions and quantitation of nuclear export by flow cytometry were performed as described . The anti–CRM1 peptide antibody was described in Kehlenbach et al. . For immunoprecipitation, immunofluorescence, and immunogold-EM, the antibody was affinity purified on a column consisting of the COOH-terminal peptide against which it was raised, coupled to CNBr-Sepharose 4B ( Pharmacia LKB Biotechnology Inc. ). The anti–RanBP1 antibody was raised in rabbits and affinity purified using recombinant RanBP1 coupled to CNBr-Sepharose 4B. Anti–Can/Nup214 antibody was kindly provided by Dr. Gerard Grosveld. The monoclonal antibodies RL1 and mAb414 were from Affinity BioReagents , Inc. and BAbCO, respectively. The monoclonal anti-importin β-antibody (ascites fluid; Affinity BioReagents , Inc.) used for immunoprecipitations was diluted 1:1 with 5 mg/ml BSA in transport buffer. The polyclonal anti-importin β-antibody used for immunoblots was raised against recombinant glutathione- S -transferase (GST)–tagged protein and purified using recombinant 6× His-tagged protein. For immunoblotting, proteins were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose using standard methods. Blots were blocked with 5% milk powder in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) overnight. HRP-coupled goat anti– mouse or donkey anti–rabbit IgG ( Pierce Chemical Co. ; 1:10,000 in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) were used as secondary antibodies. The enhanced chemiluminescence system ( Pierce Chemical Co. ) was used for visualization of proteins. Recombinant wild-type Ran and RanQ69L were prepared as described . For preparation of GST-Ran, the cDNA was cloned into a pGEX-2T ( Pharmacia LKB Biotechnology Inc. )–derived expression vector. The protein was expressed in Escherichia coli and purified using glutathione-Sepharose beads ( Pharmacia LKB Biotechnology Inc. ). To load Ran with nucleotides, soluble protein (1 mg/ml) was treated with 10 mM EDTA, 2 mM DTT, and 2 mM GDP or GMP-PNP. Nucleotides were added from a 20-mM stock in 20 mM Hepes-KOH, pH 7.2, and 20 mM MgCl 2 . After 30 min at 30°C, MgCl 2 was added to a final concentration of 20 mM and the sample was diluted with an equal volume of transport buffer to 0.5 mg Ran/ml. For loading of GST-Ran, the protein was first bound to glutathione-Sepharose beads ( Pharmacia LKB Biotechnology Inc. ) at 3 mg/ml of packed beads. After incubation with 2 mM GMP-PNP, 10 mM EDTA, and 2 mM DTT for 30 min at 30°C, 20 mM MgCl 2 was added and the beads were washed with transport buffer. Recombinant RanBP1 (purified by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration) was kindly provided by Dr. R. Mahajan (The Scripps Research Institute). GST-tagged p62, p58, and p54 were expressed and purified as described in Hu et al. . A 6× His-tagged COOH-terminal fragment of Can/Nup214 was cloned into pTrcHisA (Invitrogen Corp.). 6× His-Can/ Nup214 and the 6× His-tagged RBD1 of RanBP2 were purified using Ni-NTA Superflow-beads (QIAGEN Inc.), according to the manufacturer's instructions. Native CRM1 was partially purified by ammonium sulfate precipitation and ion-exchange chromatography as described , dialyzed against transport buffer and frozen in liquid N 2 at ∼160 μg CRM1/ ml (as compared by SDS-PAGE to a BSA standard). Aliquots were stored at −80°C. When samples were analyzed by SDS-PAGE followed by Coomassie staining, CRM1 appeared as the most abundant protein with an estimated purity of ∼60–70%. To prepare cc-NES, 5 mg cytochrome c ( Sigma Chemical Co. ) in 1.4 ml PBS was activated with 7 mM sulfo-SMCC ( Pierce Chemical Co. ) for 1 h at room temperature. Free crosslinker was removed by chromatography over a PD10 column ( Pharmacia LKB Biotechnology Inc. ) equilibrated with PBS. 2 mg of NES-peptide (the Rev-sequence CLPPLERLTL, synthesized at the Scripps Core Facility) was added and the reaction was incubated overnight at 4°C. Free peptide was then removed by chromatography over a PD10 column equilibrated with transport buffer, and the protein (∼2 mg/ml) was stored at 4°C. Cytosol was prepared as described and dialyzed into triethanolamine buffer (20 mM triethanolamine, pH 7.4, 2 mM MgCl 2 ). To isolate the cytosolic factor that restores transport after preincubation of cells in the presence of RanQ69L, cytosol (10 mg of protein) was loaded on a FPLC Mono Q column (HR 5/5; Pharmacia LKB Biotechnology Inc. ) and 1-ml fractions were eluted with an 18-ml linear gradient of 0 to 500 mM NaCl in triethanolamine buffer at a flow rate of 0.4 ml/ min. Maximal nuclear export activity was recovered around 280 mM NaCl. 900 μl of the fraction containing peak activity (as determined by the nuclear export assay) was concentrated to 300 μl by vacuum dialysis and applied to a gel filtration column (Superdex 200 HR 10/30; Pharmacia LKB Biotechnology Inc. ) equilibrated in transport buffer. The sample was chromatographed at 0.4 ml/min and 0.5-ml fractions were collected. The nuclear export activity eluted with an apparent molecular mass of ∼30 kD. For immunofluorescence staining, digitonin-permeabilized cells were allowed to bind to poly lysine–coated coverslips for 15 min at 4°C, fixed for 6 min with 4% formaldehyde in PBS at 20°C, and then treated with 0.2% Triton X-100 in PBS for 5 min at 20°C. After washing, cells were incubated with purified anti–CRM1 antibody at 1–2 μg/ml for 1 h at 20°C in 2% BSA in PBS. Texas red donkey anti–rabbit IgG (Pierce) was used as secondary antibody. Confocal microscopy was performed using an MRC1024 (Bio-Rad Laboratories) attached to an Axiovert S100TV ( Carl Zeiss, Inc. ). Microscopic parameters were identical for pairs of pictures. Digital data were processed identically, using Adobe Photoshop (Adobe Systems Inc.). To carry out immunogold-localization, cells were permeabilized by one cycle of freeze-thawing and incubated in transport buffer with anti–CRM1 antibody (2 μg/ml) for 2–3 h. Samples were washed and incubated with anti–rabbit IgG coupled to 5 nm gold ( Sigma Chemical Co. ) as secondary antibody. After washing with transport buffer, cells were fixed with 2% glutaraldehyde for 15 min, postfixed with 1% osmium tetroxide, and stained en block with 1% uranyl acetate. Cells were then dehydrated in serial ethanol concentrations and embedded in Eponate 812 (Ted Pella, Inc.). Thin sections were analyzed using a Phillips EM 208, without further staining. All steps were performed at room temperature. For immunoprecipitation, 4 × 10 6 digitonin-permeabilized cells were solubilized for 20 min on ice in 1 ml NP40 buffer (1% NP40, 50 mM Tris-HCl, pH8, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl 2 , 60 mM β-glycerophosphate, 100 μM KF, 100 μM NaVO 4 , 2 mM DTT, 1 μg/ml each of leupeptin, pepstatin, aprotinin, and 300 mM NaCl). After centrifugation at 100,000 g for 30 min at 4°C, purified anti–CRM1 antibody (10 μg/ml) or anti–importin β antibody (ascites fluid, 1:100) was added. Samples were incubated at 4°C for 2 h and immunoreactive proteins were collected with protein A–Sepharose ( Pharmacia LKB Biotechnology Inc. ). The beads were washed four times with NP40 buffer and precipitated proteins were analyzed by SDS-PAGE, followed by immunoblotting or silver staining. To analyze the binding of NE-proteins to immobilized CRM1, 30 μg of purified anti–CRM1 antibody was first bound to 60 μl protein A–Sepharose beads. 2 × 10 7 digitonin-permeabilized HeLa cells were solubilized in NP40 buffer containing 300 mM NaCl and centrifuged at 100,000 g for 30 min. To load the protein A-anti–CRM1 beads with CRM1, beads were incubated with the permeabilized cell supernatant for 90 min at 20°C, and then washed four times with NP40 buffer. Only bands corresponding to CRM1 and IgG could be detected when a sample of these beads was analyzed by SDS-PAGE, followed by blotting and Ponceau staining. Nuclear envelopes were prepared as described and stored at −80°C. 200 OD 260 U of NE (derived from ∼6 × 10 8 nuclei) were solubilized on ice for 20 min in 1 ml NP40 buffer containing 450 mM NaCl. After centrifugation at 100,000 g for 30 min, the supernatant was diluted with NP40 buffer without NaCl to 150 mM NaCl. 60 μl of this extract was added to 6 μl protein A-CRM1 beads in the absence or presence of 25 μg/ml RanGDP or RanGMP-PNP and 100 μg/ml cc-NES. NP40 buffer containing 150 mM NaCl, and BSA (final concentration of 10 mg/ml) was added to give a final volume of 250 μl. After incubation at 20°C for 1 h, beads were washed four times with NP40 buffer containing 150 mM NaCl and bound proteins were analyzed by SDS-PAGE, followed by immunoblotting. For binding experiments using purified CRM1 and GST-p62, GST-p58, GST-p54, or 6×His-Can/Nup214, the recombinant proteins were bound to glutathione-Sepharose beads or Ni-NTA-superflow beads (QIAGEN Inc.) at 50–100 μg/ml. The beads were then treated with 30 mg/ml BSA in transport buffer for 20 min to block nonspecific binding sites. 5 μl CRM1 (∼160 μg/ml) was added to 6 μl of beads in the absence or presence of 25 μg/ml RanGDP or RanGMP-PNP and 1 mg/ml NES-peptide (CLPPLERLTL; from a 2-mg/ml stock in transport buffer) in a total volume of 100 μl containing 10 mg/ml BSA. After 90 min at 20°C, the beads were washed with transport buffer three times. All buffers involving Ni-beads contained 20 mM imidazole. The amount of bound CRM1 was analyzed by SDS-PAGE and immunoblotting. To analyze the release of CRM1 from RanGMP-PNP by RanBP1, ∼1 μg of purified CRM1 was bound to 6 μl GST-RanGMP-PNP beads in the presence of 1 mg/ml NES peptide and 10 mg/ml BSA in a total volume of 100 μl transport buffer. After extensive washing with transport buffer, the beads were incubated at 0° or 30°C with 5 mg/ml BSA and 1 mg/ml NES-peptide in the absence or presence of 120 μg/ml RanBP1 for 20 min. These high concentrations of RanBP1 were used because the matrix contains a high concentration of RanGTP, to which RanBP1 and CRM1 can bind. Released proteins were precipitated with 10% TCA for 5 min on ice and pelleted by centrifugation at 14,000 g for 15 min at 4°C. Samples were analyzed by SDS-PAGE, followed by immunoblotting. To analyze the release of CRM1 from permeabilized cells after the RanQ69L preincubation, they were subjected to a second incubation for 15 min in the absence or presence of RanBP1 or RBD1 of RanBP2. The cells were collected by centrifugation and equivalent amounts of pellet and supernatant were analyzed by SDS-PAGE, followed by immunoblotting. We previously found that the NES receptor CRM1 and the small GTPase Ran reconstitute efficient nuclear export of GFP-NFAT in digitonin-permeabilized HeLa cells that have been preincubated with ATP at 30°C in order to deplete shuttling export factors . To identify additional cytosolic export factors besides CRM1 and Ran, we had to find conditions to make them rate limiting in our assay. We therefore compared the ability of CRM1 and cytosol to promote NFAT export in HeLa cells after various preincubation steps that might more completely deplete export factors from the permeabilized cells. After our standard preincubation with ATP alone, we found that either CRM1 alone or cytosol alone stimulated nuclear export of GFP-NFAT to equivalent levels in the presence of an excess of wild-type Ran , consistent with our previous work . We then examined the effects of preincubation with RanQ69L, a mutant form of Ran that is insensitive to RanGAP and therefore is predominantly in the GTP-bound form . RanQ69L has been shown to promote nuclear export of substrates carrying a leucine-rich NES and to strongly inhibit NLS-mediated nuclear protein import . After preincubation of permeabilized cells with ATP and RanQ69L, the ability of CRM1 plus wild-type Ran to promote nuclear export was clearly reduced . In contrast, cytosol still retained its strong stimulatory effect on nuclear export. We next examined the effect of including an excess of NES substrate in the preincubation, which would be expected to more fully mobilize the endogenous export factors. For the NES substrate, we coupled the Rev NES to cytochrome c, a 12-kD protein that is expected to enter the nucleus by passive diffusion. Consistent with this possibility, the Rev-NES conjugate (cc-NES), but not a conjugate containing an export-defective NES, was able to compete for nuclear export of GFP-NFAT in vitro (data not shown). After preincubation of permeabilized cells with ATP, RanQ69L, and cc-NES, CRM1 and Ran were no longer sufficient to support any nuclear export of GFP-NFAT . Under these conditions, the GFP-NFAT remained in the nucleoplasm . Again, the addition of cytosol after this preincubation resulted in a strong stimulation of nuclear export. However, preincubation of cells with ATP and cc-NES alone did not affect the ability of CRM1 and Ran to support nuclear export (data not shown). These results clearly demonstrate that cytosol contains an activity distinct from CRM1 and Ran that is required for nuclear export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L. The export substrate cc-NES potentiates this requirement for an additional factor when added with RanQ69L in the preincubation. The cytosolic activity that stimulates export under these conditions could be a nucleocytoplasmic shuttling factor that is lost from the nuclei during the preincubation. Alternatively, RanQ69L and cc-NES could impose a block on nuclear export that is efficiently relieved by a cytosolic factor. In an attempt to distinguish between these possibilities, we decided to characterize the localization of GFP-NFAT and of CRM1, the NES receptor for NFAT, after incubation of permeabilized cells with RanQ69L. An export substrate containing a leucine-rich NES has been shown to bind cooperatively with RanGTP to CRM1 in vitro , forming a putative nuclear export complex. Preincubation of permeabilized cells with an excess of RanQ69L and cc-NES substrate should efficiently mobilize CRM1 into export complexes and perhaps trigger the transport of these complexes out of the nucleus. As we observed previously , incubation of permeabilized cells at 30°C with ATP alone led to a depletion of CRM1 from the cells, as compared with the 0°C control incubation . In contrast, incubation with RanQ69L , but not with wild-type Ran (data not shown), largely prevented the loss of CRM1 from the permeabilized cells. The high level of CRM1 retained by preincubation with RanQ69L could result either from inhibition of export of CRM1 out of the nucleus or from facilitated recycling/reimport back into the nucleus. Since incubation of the same number of cells in a 30-fold larger volume at the same concentration of RanQ69L did not change the level of CRM1 compared with the standard RanQ69L incubation as detected by immunoblotting (data not shown), RanQ69L appears to act by inhibiting export of CRM1, rather than by promoting its reimport. This notion is further validated by the data shown below. Thus, although RanQ69L promotes efficient nuclear export of NES-containing substrates in vivo , and also a moderate level of nuclear export in vitro , the release of the nuclear export receptor CRM1 from the permeabilized cells is impaired by RanQ69L. We next used immunocytochemical approaches to examine the localization of CRM1 in the nucleus with and without preincubation of the cells with RanQ69L. In previous work, CRM1 was found to be localized throughout the nucleoplasm as well as at the cytoplasmic and nucleoplasmic sides of the NPC in cultured cells . Consistent with this, by indirect immunofluorescence microscopy, we detected CRM1 predominantly in the nucleoplasm of digitonin-permeabilized cells . Incubation of cells with RanQ69L, but not with wild-type Ran (data not shown), led to a strong increase in staining at the NE together with a decrease in intranuclear staining . The localization of the export substrate GFP-NFAT did not substantially change upon incubation with RanQ69L . Very similar results were obtained when the cells were incubated with a combination of RanQ69L and cc-NES . When the Triton permeabilization was omitted after the fixation step before antibody labeling, a similar level of CRM1 was detected at the NE, even though the NE remained intact, as demonstrated by the inaccessibility of nuclear lamins to anti-lamin antibodies (data not shown). This suggests that CRM1 resides at the cytoplasmic side of the NPC after the RanQ69L cc-NES incubation. To further examine the localization of CRM1 under these conditions, we performed immunogold labeling of digitonin-permeabilized cells that had been incubated either at 0°C in buffer, or at 30°C with RanQ69L and cc-NES . The nuclei were permeabilized by freeze-thawing before immunolabeling. Under these conditions, the nucleoplasmic side of the NPC was freely accessible to gold probes, as demonstrated by strong labeling of the nucleoplasmic side of the NPC with antibodies to Tpr (data not shown), which is found on the nuclear side of the NPC . In the control cells, some anti-CRM1 gold labeling was seen throughout the nucleoplasm (not shown) and gold particles were occasionally found at the cytoplasmic side of the NPC . Strikingly, however, after cells were incubated with RanQ69L, the cytoplasmic periphery of every NPC was strongly decorated with gold, while essentially no labeling of the nucleoplasmic side of the NPC was evident. Labeling was specific, because it was completely abolished by preincubating the anti-CRM1 antibody with the peptide against which it had been raised. These results indicate that CRM1 crosses the NE and accumulates at the cytoplasmic periphery of the NPC when RanQ69L is present. Hence, RanQ69L appears to inhibit export by inducing the stable trapping of CRM1 at the cytoplasmic periphery of the NPC. Since this accumulation is observed only in the presence of RanQ69L but not wild-type Ran, the release of CRM1 from this binding site is likely to require GTP hydrolysis on Ran and/or a cytosolic release factor that is absent from the permeabilized cells. We next carried out biochemical analyses to identify the NPC proteins with which CRM1 associates in the presence of RanQ69L. Previous work showed that immunoprecipitation of an epitope-tagged version of the nucleoporin Can/Nup214 from an extract of cultured HeLa cells resulted in the coprecipitation of some CRM1 . Can/Nup214 is one of the FG-repeat–containing nucleoporins that is detected by the monoclonal antibodies RL1 and mAb414 . In addition to Can/Nup214, RL1 detects the nucleoporins RanBP2, Nup153, Pom121, Nup98, p62, p58, and p54 . As shown in Fig. 3 a, on silver-stained gels, we detected two proteins with apparent molecular weights of ∼70 and ∼220 kD (marked by asterisks) that specifically coprecipitated with CRM1 from a cell lysate after preincubation with RanQ69L at 30°C. These bands were not seen after preincubation at 30°C without RanQ69L . Addition of the peptide that had been used to prepare the anti–CRM1-antibody abolished the precipitation of CRM1 and also led to the loss of the coprecipitated proteins, indicating that the latter are in a complex with CRM1. In a similar experiment we used immunoblotting with the RL1 monoclonal antibody to analyze the NPC proteins that coprecipitated with CRM1 . As a control, we examined the proteins that coprecipitated with the import receptor importin β. No RL1-reactive proteins were detectable in the anti–CRM1 immunoprecipitate from cells that had been incubated in buffer at 0°C. In contrast, after incubation with ATP, RanQ69L, and cc-NES at 30°C, two RL1-positive proteins with the same apparent molecular weights as the coprecipitated proteins detected on silver-stained gels were coprecipitated with CRM1. The latter was identified as Can/Nup214, using a specific antibody against this protein (data not shown). The 70-kD protein is p62, as judged by its mobility on SDS-PAGE and by the specificity of RL1. The monoclonal antibody mAb414 detected the same two proteins as RL1 in these immunoprecipitates (data not shown). The p58, p54, and p45 subunits of the p62 complex were not detected in these immunoprecipitation experiments by either RL1 or mAb414 with our immunoblotting conditions, probably because larger amounts of these proteins are required for detection compared with p62 . However, since these proteins remain tightly associated with p62 under similar extraction conditions , they most likely are present in the immunoprecipitates with p62. The level of immunoprecipitated CRM1 itself in this experiment was the same with or without the RanQ69L preincubation , consistent with the finding that CRM1 is retained in the permeabilized cells during the RanQ69L treatment. As a control, we examined whether RL1 antigens coimmunoprecipitated with the import receptor importin β under these conditions. In contrast to the results obtained with CRM1, a distinct set of nucleoporins coprecipitated with importin β in this experiment. RanBP2 and Nup153 were the only detectable nucleoporins that appeared in anti–importin β immunoprecipitates from extracts of cells that had been incubated at 0°C . When cells were preincubated with ATP, RanQ69L, and cc-NES at 30°C, Nup153 no longer coprecipitated with importin β, although the amount of coimmunoprecipitated RanBP2 did not change significantly. The amount of importin β in the immunoprecipitate was clearly reduced after the RanQ69L incubation, indicating that importin β, unlike CRM1, is depleted from the nucleus under these conditions. Thus, the export receptor CRM1 and the import receptor importin β behave differently during preincubation with RanQ69L: RanQ69L promotes the association of CRM1 with a subset of cytoplasmic nucleoporins, but does not promote its release from nuclei, whereas RanQ69L induces the dissociation of importin β from a nucleoplasmic nucleoporin (Nup153) and stimulates release of importin β from the permeabilized cells. To investigate the binding of CRM1 to proteins of the NPC under defined in vitro conditions, we immobilized CRM1 on anti–CRM1 antibodies coupled to Protein A beads. The beads were then incubated with an NP40 lysate derived from purified rat liver NEs in the presence of RanGDP or RanGMP-PNP, a nonhydrolyzable GTP analogue. In some reactions, we added cc-NES, which enhances the binding of RanGMP-PNP to CRM1 (data not shown). Proteins bound to the CRM1 beads were detected on immunoblots using the RL1 antibody. In the absence of added cc-NES, the nucleoporins p62 and Nup153 and a faint band migrating below Nup153 bound to the beads in the presence of RanGDP . Very similar levels of these proteins bound when no exogenous Ran was added (data not shown). When the incubation contained Ran that had been loaded with GMP-PNP, Can/Nup214 and an increased level of p62 bound to the beads. Neither the level of Nup153 nor of the minor band below Nup153 changed upon addition of RanGMP-PNP. Although addition of cc-NES to the incubations had no effect when the samples contained RanGDP, cc-NES increased the amount of Can/Nup214 and p62 bound to the beads in the presence of RanGMP-PNP. As a control for the specificity of binding, we used Protein A beads that had been coupled to rabbit IgG instead of anti–CRM1 antibody, but were otherwise treated identically. Only a very faint band corresponding to Nup153 could be detected (data not shown), indicating that the binding of nucleoporins to the beads was specific for CRM1. The differential binding of Nup153 to CRM1 in Fig. 3 , b and c, probably results from the different experimental approaches: in the immunoprecipitation experiment , we examined CRM1 that is associated with proteins of intact NPCs at the end point of a transport reaction in a permeabilized cell assay. This reaction leads to accumulation of CRM1 on the cytoplasmic side of the NPC, whereas Nup153 is localized to the nucleoplasmic side. In contrast, in the in vitro binding experiment , solubilized proteins of the NPC are allowed to bind to immobilized CRM1. We next investigated the binding of partially purified CRM1 to recombinant proteins of the p62 complex (p54, p58, and p62) and to a COOH-terminal fragment of Can/ Nup214 that has previously been shown to bind to CRM1 in vivo . In this experiment, the nucleoporins (instead of CRM1) were immobilized on affinity matrices for the binding reactions. As shown in Fig. 3 d, RanGMP-PNP, but not RanGDP or GMP-PNP alone, strongly promoted binding of CRM1 to all three proteins of the p62 complex. The binding was further increased if an NES peptide was included in the reaction. We detected substantial binding of CRM1 to the COOH-terminal fragment of Can/Nup214 in the presence of RanGDP or free GMP-PNP (i.e., in the absence of Ran), and only a modest increase in binding when RanGMP-PNP was added. No binding was detected when we used Ni beads that had not been coupled with the Can/Nup214 fragment (data not shown). In the two different binding reconstitution experiments , RanGMP-PNP stimulated the nucleoporin/CRM1 interactions to different levels, depending on the source of nucleoporins and the nature of the affinity matrix. It strongly enhanced the interaction between Can/Nup214 and CRM1 when solubilized NEs were used as a source of nucleoporins , but only modestly enhanced the interaction when a recombinant fragment of Can/Nup214 was used . Conversely, RanGMP-PNP only modestly enhanced the association of p62 with CRM1 when p62 came from solubilized NEs, but strongly enhanced the association when recombinant p62, p58, and p54 were used . Aside from the different nature of the affinity matrices in the two cases , these differences may be due to the presence of components in the solubilized NEs that differentially affect the binding of Can/Nup214 versus p62 to CRM1. In addition, the short recombinant fragment from the COOH terminus of Can/Nup214 might be folded in a way that results in a different binding activity of the fragment compared with the full-length protein from NEs. Despite these quantitative differences, the results of these in vitro binding reconstitution experiments are in agreement with the results of the immunoprecipitation experiments involving extracts of permeabilized cells , which showed that RanQ69L induces the stable binding of CRM1 to Can/Nup214 and p62 in permeabilized cells. The stimulation of binding of CRM1 to these nucleoporins by RanQ69L/RanGMP-PNP suggests that these interactions reflect the association of an export complex separately with Can/Nup214 and p62. It remains possible that a ternary complex of CRM1, p62, and Can/ Nup214 can be formed. However, in light of our EM data, we think it unlikely that the end point of the transport reaction in the presence of RanQ69L, which is analyzed in the immunoprecipitation experiments , involves such a complex. This is because much of the CRM1 was found at the periphery of the NPC where Can/Nup214 is localized , which is distinct from the central location where p62 is found . Significantly, the above results imply that RanGTP not only promotes the formation of an export complex, but also targets it to specific sites in the NPC during transport. Since Can/Nup214 is located in a more peripheral cytoplasmic location in the NPC than the p62 complex, it is likely that the association of the export complex with Can/ Nup214 represents a late step in nuclear export. Release from this site is mediated by a cytosolic factor, at least in cells that have been preincubated with RanQ69L, thereby allowing efficient nuclear export in a subsequent reaction . To identify the cytosolic factor that restores export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L and cc-NES, we fractionated cytosol by column chromatography and tested individual fractions for their ability to promote export in the presence of exogenous Ran and partially purified CRM1. With analysis by ion exchange chromatography using a Mono Q column, we obtained a major peak of export activity in fraction 20, which eluted from the column at ∼280 mM NaCl . This peak fraction was further analyzed by gel filtration on an S200 column. A small peak of export activity was observed in fraction 19, at the position of a globular protein of ∼30 kD . A much larger peak eluting at the same position was obtained when total cytosol instead of a Mono Q fraction was used for gel filtration (data not shown). The apparent size of the active protein eluting from the gel filtration column prompted us to probe fractions of both purification steps for the presence of RanBP1, a previously characterized cytosolic RanGTP-binding protein of 28 kD . As shown in Fig. 4 , a and b (insets), the peak of RanBP1 detectable by immunoblotting coincides with the peak of export activity on both the Mono Q and the gel filtration columns. When the S200 samples were analyzed by silver staining, a protein with the same mobility as RanBP1 was the only species that peaked in fraction 19 (data not shown). This protein accounted for ∼10% of the total protein in that fraction. We next tested the ability of recombinant RanBP1 to stimulate nuclear export of GFP-NFAT after the RanQ69L cc-NES preincubation. We found that a combination of wild-type Ran and RanBP1 strongly stimulated nuclear export . Neither Ran nor RanBP1 alone had a significant effect. No further stimulation of export was obtained by adding purified CRM1 to Ran and RanBP1 in the reconstitution (data not shown). In a titration experiment (data not shown), we found that 50% of the maximal stimulation was obtained with ∼2 μg/ml RanBP1. RanBP1 was maximally active at a concentration of ∼15 μg/ml, whereas a higher concentration resulted in inhibition of nuclear export. These results demonstrate that RanBP1 stimulates nuclear export in cells that have been preincubated with RanQ69L and cc-NES, indicating that it relieves the block imposed by these reagents. This raises the possibility that RanBP1 or related proteins have a role in nuclear export under normal conditions. As discussed above , Ran and CRM1 support efficient nuclear export in permeabilized cells that have been preincubated with ATP alone. Nevertheless, RanBP1 does have a modest stimulatory effect on nuclear export in nonpreincubated cells, as demonstrated by the time course experiment shown in Fig. 4 d . Here, incubation of the permeabilized cells for 30 min reduced the nuclear fluorescence from 100 to 37 U in the presence of RanBP1, but only to 51 U in its absence. This RanBP1-mediated stimulation of nuclear export by ∼30% was obtained in four independent experiments. The observation that RanBP1 is not absolutely required for nuclear export under these conditions raised the possibility that another factor might provide RanBP1-like functions when the cells have not been preincubated with RanQ69L. RanBP2 is a giant nucleoporin that has four RBDs very similar to that of RanBP1 . RanBP2 is localized in close proximity to Can/ Nup214 on the cytoplasmic side of the NPC and is well situated to interact with an export complex bound to Can/ Nup214. In RanQ69L preincubated cells, the RBDs of RanBP2 are likely to be stably occupied by RanQ69L, which cannot be readily released because the RanQ69L mutant is insensitive to RanGAP. This situation would necessitate the addition of exogenous RanBP1 to promote export, even though the RBDs of RanBP2 would be available under normal conditions. To test whether the RBDs of RanBP2 can function like RanBP1 in our assay, we expressed them as 6× His-tagged fusion proteins and tested their ability to stimulate nuclear export of GFP-NFAT after preincubation of permeabilized cells with RanQ69L and cc-NES. As shown in Fig. 4 e, the combination of RBD1 of RanBP2 and Ran stimulates nuclear export to a similar extent as RanBP1 and Ran . RBDs 2–4 of RanBP2 were equally effective (data not shown). These results show that either RanBP1 or the RBDs of RanBP2 can release the block of nuclear export imposed by preincubating cells with RanQ69L and cc-NES. RanBP1 has been shown to promote the dissociation of RanGTP from the importin β-related proteins transportin and CAS, as well as from importin β itself . To test whether RanBP1 has a similar effect on the RanGTP-CRM1 complex, we bound purified CRM1 to GST-RanGMP-PNP and examined whether RanBP1 was able to release the CRM1 in a subsequent incubation. As shown in Fig. 5 a, RanBP1 released the majority of CRM1 into the supernatant (compare the levels of released and bound CRM1), whereas very little CRM1 was released in the absence of RanBP1. We next tested whether RanBP1 might have a similar effect on CRM1 that is trapped in a complex with Can/ Nup214 at the cytoplasmic side of the NPC as a consequence of incubation of cells with RanQ69L and cc-NES. Since the binding of CRM1 to Can/Nup214 is strongly promoted by RanGTP, the dissociation of RanGTP from CRM1 would be expected to promote the release of the latter from the NPC. Indeed, after a first incubation of permeabilized cells with RanQ69L, a second incubation with RanBP1 strongly reduced the amount of CRM1 at the NE as seen by immunofluorescence microscopy . Without RanBP1, cells exhibited a strong nuclear rim staining for CRM1, very similar to the one obtained without a second incubation . The release of CRM1 from NE of RanQ69L-preincubated cells was analyzed also by immunoblotting . Whereas a major proportion of CRM1 remained associated with the cells (pellet, P) in the absence of RanBP1, the addition of RanBP1 resulted in the recovery of most CRM1 in the released fraction (supernatant, S). A similar effect was obtained with RBD1 of RanBP2 . Taken together, these results suggest that RanBP1, and probably the RBDs of RanBP2, are involved in the disassembly of the export complex at a terminal nucleoporin site (Can/Nup214). RanBP1 and RanBP2 apparently act by releasing RanGTP from CRM1, thereby promoting release of the export complex from the NPC and dissociation of the cargo-receptor complex. If the RBDs of RanBP2 are occupied by RanQ69L, exogenously added RanBP1 (or cytosolic RanBP1 in intact cells) or soluble RBDs of RanBP2 are required to fulfill this function. In this study, we used biochemical and functional approaches to search for new components involved in CRM1-mediated nuclear export. Our studies were based on the initial observation that incubation of permeabilized cells with RanQ69L and cc-NES results in strong inhibition of CRM1-mediated nuclear export. During this treatment, the nuclear export receptor CRM1 becomes highly concentrated at the cytoplasmic side of the NPC in association with Can/Nup214 and the p62 complex, concomitant with CRM1 depletion from the nuclear interior. We found that the cytosolic protein RanBP1 and the RBDs of the nucleoporin RanBP2 can relieve the transport inhibition imposed by RanQ69L by releasing CRM1 from the cytoplasmic nucleoporins. As discussed below, this indicates that RanBP1 and probably RanBP2 play a key role in a terminal step of nuclear export involving export complex dissociation from the NPC. Previous studies have indicated that CRM1 binds cooperatively to RanGTP and export cargo in the nucleus to form a putative export complex that is transported through the NPC. Several criteria indicate that the accumulation of CRM1 at the cytoplasmic side of the NPC during treatment of permeabilized cells with RanQ69L and cc-NES reflects terminal stages of nuclear export, as opposed to initial stages of reimport. First, the in vitro binding of CRM1 to the cytoplasmic nucleporins Can/Nup214 and to the p62 complex is promoted by components known to be involved in the formation of an export complex (RanGTP, or analogues thereof, and export substrate). Similarly, the accumulation of CRM1 at the cytoplasmic nucleoporins in permeabilized cells is dependent on RanQ69L, which is effectively locked in the GTP-bound form, and the block of export by RanQ69L is potentiated by the export substrate cc-NES. Furthermore, CRM1 is released from these cytoplasmic sites by RanBP1 and RBDs, a reaction that is compatible with an export intermediate but not with an import intermediate. RanBP1 has previously been proposed to have a role in disassembly of nuclear export complexes in the cytoplasm . Whereas Can/Nup214 is localized in the fibrils at the cytoplasmic periphery of the NPC, the p62 complex is situated on both the cytoplasmic and nucleoplasmic sides of the NPC near the central gated channel . Our data are consistent with the possibility that Can/Nup214 is the terminal binding site for the CRM1 export complex at the NPC, to which it is transferred after binding to the cytoplasmically oriented p62 complex. The apparent flexibility of the cytoplasmic fibrils , might allow them to interact with the NPC region containing the p62 complex, enabling direct transfer of the export complex to Can/Nup214. The fact that CRM1 is associated with the p62 complex as well as with Can/Nup214 in permeabilized cells preincubated with RanQ69L may be due to the saturation of all available CRM1 binding sites at Can/Nup214, resulting in the accumulation of CRM1 at a more proximal site (the p62 complex) in the export pathway. The movement of the CRM1 export complex through the NPC first must involve its association with nucleoplasmic nucleoporins. CRM1 from yeast (Crm1p) has been shown to interact by a two-hybrid assay with a number of nucleoporins, including mammalian Nup98 , which is localized to the nucleoplasmic side of the NPC . Nup153, a protein localized to the nucleoplasmic fibrils , may be another one of the nucleoplasmic binding sites for CRM1 in mammals, since we found that it associates with CRM1 in vitro (albeit not in a RanGTP-stimulated fashion). An additional site probably is the population of p62 complexes found on the nucleoplasmic side of the gated channel. It is striking that CRM1 was not detected on the nucleoplasmic side of the NPC after preincubation with RanQ69L. This result may be explained, at least in part, by the possibility that the affinity of the CRM1 export complex for cytoplasmic nucleoporins is higher than that for nucleoplasmic nucleoporins. In this respect, the p62 complex found on the cytoplasmic side of the NPC may not be biochemically identical to the nucleoplasmic p62 complex. It also is possible that RanGTP in export complexes acts at the central gated channel of the NPC to promote vectorial movement to the cytoplasm. Whatever the mechanism, the finding that RanGTP (RanQ69L) strongly promotes the binding/accumulation of the CRM1 export complex at the cytoplasmic side of the NPC indicates that RanGTP is important for the vectorial transfer of the export complex through the NPC. Thus, these results point to a second important function for RanGTP in nuclear export, in addition to its role in promoting the association of export cargo with CRM1 inside the nucleus. The behavior of the import receptor importin β was very different from CRM1 in our studies, arguing that the nucleoporin associations seen for CRM1 are specific and functionally relevant. Importin β appears to be more highly associated with the NPC than CRM1 in cultured cells at steady state, since substantial association of importin β with RanBP2 and Nup153 was seen in immunoprecipitates of permeabilized cell extracts, whereas no significant association of CRM1 with Can/Nup214 or the p62 complex was detected unless cells were pretreated with RanQ69L. High levels of importin β remained associated with RanBP2 after treatment with RanQ69L, but the association with Nup153 was no longer seen. These findings are consistent with previous work showing that adding GMP-PNP to Xenopus egg lysates results in loss of coprecipitation of importin β with Nup153 , and that RanGTP strongly promotes the binding of importin β to RanBP2 at the cytoplasmic side of the NPC in vitro . Incubation of cells with RanQ69L appears to promote the export (recycling) of importin β, since substantial amounts of the latter are released from the cells under these conditions. Thus, RanBP2 may be a terminal NPC binding site for the export of importin β, much like Can/Nup214 appears to be a terminal site for CRM1 export. This suggests that the recycling (export) of at least one import receptor uses nucleoporin binding sites that are at least partially distinct from those used by an export complex. Teleologically, it makes sense that these two pathways would be distinct, so that protein export would not compete with recycling of import factors. We have identified RanBP1 as the major cytosolic activity that relieves the block of nuclear export imposed by RanQ69L and cc-NES, and showed that it also stimulates export in cells that have not been preincubated with RanQ69L. RanBP1 has previously been suggested to have a role in nuclear import and nuclear assembly in higher eukaryotes. The yeast homologue of RanBP1, Yrb1p, also has been suggested to play a role in nuclear protein import and RNA export . However, the molecular basis for these previously observed functional effects, and whether they are direct or indirect, is not entirely clear. Recent biochemical studies have provided new insight on a potential role for RanBP1 in nuclear export. In addition to its role in activating RanGAP , RanBP1 has been found to reduce the inhibitory effect of certain importin β–related transport receptors (including CAS, transportin, and importin β itself) on RanGAP . This is a consequence of the ability of RanBP1 to release RanGTP from these transport receptors . This release reaction could allow the cytosolic RanBP1 both to promote RanGTP hydrolysis and to stimulate the dissociation of cargo from export complexes, thereby irreversibly dissociating the export complex. In this study, we have found that RanBP1 releases RanGTP from the export receptor CRM1 in a manner similar to other importin β–like receptors that have been examined. This not only is likely to promote cargo release from CRM1, but probably also is responsible for the release of CRM1 from the NPC, since RanGTP strongly enhances the association of CRM1 with cytoplasmic nucleoporins. We also observed some release of CRM1 from immobilized GST-RanGTP using very high concentrations of RanGAP in vitro (our unpublished observations). Thus, we cannot rule out that RanGAP also contributes to the release of CRM1 from the NPC. However, exogenously added RanGAP, even at high concentrations, gave no stimulation of nuclear export in nonpreincubated cells (our unpublished observations). Similarly, in a recent study, Englmeier et al. have described a small stimulatory effect of RanBP1, but not of RanGAP, on nuclear export in vitro. The isolation of a native complex consisting of a nucleoporin, CRM1, export cargo, and RanGTP will be required for further studies on the function of RanBP1 and RanGAP. RanBP1 stimulates nuclear export to a modest level (∼30%) in our in vitro assay when the permeabilized cells have not been preincubated with RanQ69L and cc-NES. Under these conditions, RanBP2 most likely fulfills similar functions as RanBP1, since RBDs of RanBP2 are active both in releasing CRM1 from NEs and in stimulating nuclear export after preincubation with RanQ69L. It is interesting to note that there is no RanBP2 homologue in yeast. Yeast cells therefore rely on the activity of RanBP1-like proteins for nuclear import and export . In fact, Yrb1p appears to be the only essential RanBP1-like protein in yeast. Richards et al. detected efficient export of an NES-containing reporter protein after injecting RanG19V into the nuclei of cultured cells. RanG19V, like RanQ69L, is insensitive to RanGAP and is therefore predominantly in the GTP-bound form. Unlike our in vitro studies involving incubation of permeabilized cells (depleted of most of the endogenous Ran) with RanQ69L, the microinjection studies were done in cells containing a high concentration of wild-type Ran. Thus, the RBDs of endogenous RanBP1 and RanBP2 may not have been completely blocked by RanG19V in this study, and this could explain how efficient export could occur in this situation, in contrast to the results we have obtained in our studies. We were unable to detect any accumulation of the export cargo GFP-NFAT at the NE under conditions of RanQ69L preincubation. This could be due to the vast excess of cc-NES and/or other endogenous export substrates in the reaction, which would compete with NFAT for binding to CRM1. However, even when export complexes are arrested at the cytoplasmic side of the NPC by RanQ69L, cargo may slowly dissociate from CRM1 even though CRM1 remains bound to the NPC. This could explain results we obtained in our previous study , in which we found a low but significant level of nuclear export of GFP-NFAT in the presence of RanQ69L, allowing a single round of nuclear export but no recycling of the export receptor. Our data suggest a working model for CRM1-mediated nuclear export that is shown in Fig. 6 . We propose that an export complex of cargo, CRM1, and RanGTP that forms in the nucleus first binds to the nucleoplasmic fibrils of the NPC. It subsequently is translocated through the central gated channel via the p62 complex on the nucleoplasmic and cytoplasmic sides of the NPC, and then associates with the peripheral nucleoporin Can/Nup214 at a terminal NPC site. The binding of RanBP2 and/or cytosolic RanBP1 to the RanGTP in the export complex at this site then dissociates RanGTP from CRM1, leading to the release of the cargo from CRM1 and, concomitantly, the dissociation of CRM1 from Can/Nup214. GTP hydrolysis on Ran by cytosolic or RanBP2-associated RanGAP would make export essentially irreversible. Our data suggest a new function for RanGTP in promoting the vectoriality of nuclear export: in addition to stimulating the binding of cargo to CRM1 in the nucleus, RanGTP in an export complex promotes targeting to the cytoplasmic side of the NPC. It will be interesting to examine whether RanGDP, which might be part of a nuclear import complex , could have an analogous role in targeting the import complex to the nucleoplasmic side of the NPC. | Study | biomedical | en | 0.999998 |
10330397 | The Saccharomyces cerevisiae strains used in this study are listed in Table I . Rich medium (YPD) and supplemented minimal medium (SMM) were prepared according to Kaiser et al. . To evaluate growth at low pH, YPD was adjusted to pH 3.8 with HCl (this medium remained at pH 3.8 throughout the growth of a yeast culture). For some experiments, SMM was buffered to pH 6.5 using 50 mM MOPS and 50 mM MES. Genetic manipulations were performed according to standard protocols . DNA manipulations were carried out as described in S ambrook et al. . pAF70 carries the SEC24 gene in the centromere vector pCT3 . pKR34 and pKR41 carry the 3.8-kb KpnI/SalI fragment containing the SEC24 gene from pAF70 in the 2μ vectors pRS426 ( URA3 ) and pRS425 ( LEU2 ), respectively. pKR17 carries the LST1 gene on a 3.5-kb fragment in the centromere vector pRS316 ( URA3 ). A subclone of the LST1 gene from pKR17 into the 2μ vector pRS426 gave yeast transformants at a very low efficiency because of the toxicity of LST1 sequences when present at high copy. To study the toxic effects of LST1 , pKR35 was constructed which contains the entire LST1 coding sequence expressed from pGAL1 on pCD43 ( URA3 ). pKR35 will prevent growth under conditions of full induction on galactose medium, establishing that overexpression of Lst1p is toxic to yeast cells. Under conditions of partial induction of pGAL1–LST1 in cells grown on raffinose, pKR35 will complement lst1 Δ:: LEU2 for growth on acidic medium. This shows that the LST1 open reading frame carried on pKR35 still posses LST1 function. Epitope-tagged LST1 was constructed as follows. First, the NotI site in the polylinker of pKR17 was deleted with a 350-bp SmaI/NaeI fragment (pKR17Δ), and then a 12-bp linker carrying a NotI site was inserted at the Eco47III site (at codon 13 of LST1 ) of pKR17Δ to make pKR17N. pKR17HA carries the 100-bp NotI fragment from pGTEPI , which encodes three tandem copies of the hemagglutinin (HA1) epitope, inserted into the NotI site of pKR17N. Restriction analysis using sites flanking the point of insertion revealed that two 100-bp inserts (six HA epitopes) were present in pKR17HA. pKR17HA was transformed into CKY536 to make CKY535 ( MAT a lst1 Δ:: LEU2 leu2-3 , 112 ura3-52 [pKR17HA]). The following plasmids and strains were constructed for use in the sec13-1 synthetic-lethal screen. The plasmid pKR1 carries SEC13 on a 1.8-kb SalI/ BamHI fragment excised from pCK1313 , inserted into pRS316 . pKR4 carries the same 1.8-kb SalI/ BamHI fragment and a 3.8-kb NheI/BamHI fragment containing ADE3 from pDK255, both inserted into the vector pRS315 . CUY563 and CKY45 were crossed to produce a MAT a ade2 ade3 leu2 ura3 sec13-1 segregant, which was transformed with pKR4 to give CKY423. The mating type of CKY423 was switched by ectopic expression of the HO gene to give CKY424. Cultures of CKY423 and CKY424 were mutagenized by irradiation with a germicidal UV lamp at a dose resulting in 10% cell survival. Mutagenized cells were plated on YPD at a density of 150 colonies per plate. After 5 d of growth at 24°C, colonies with a solid red color and no white sectors were selected for further analysis. The dependence of the nonsectoring phenotype on the sec13-1 mutation was tested by transforming candidate mutants with either pKR1 or pRS316. Strains that sectored after transformation with pKR1, but not after transformation with pRS316, were scored as sec13-1 –dependent. Complementation tests were performed by mating mutants isolated from CKY423 with those isolated from CKY424. Zygotes isolated by micromanipulation were scored for their ability to form sectored colonies on YPD plates. The genes defined by these complementation groups were designated LST . All lst mutant strains were backcrossed to a parental strain twice. The lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus, using the integrating plasmid p1312 . The integrants were grown on YPD and cells from white sectors (indicating loss of pKR4) were isolated. The integration of a wild-type copy of SEC13 was confirmed by the ability of the cells from white sectors to grow at 36°C, a temperature that is restrictive for the sec13-1 mutation. Owing to the poor growth of lst9 strains, we were not able to construct a lst9 single mutant by this method. To test for synthetic-lethal interactions between lst mutations and mutations in sec genes, lst mutants CKY435 ( lst1-1 ), CKY436 ( lst2-1 ), CKY437 ( lst3-1 ), CKY438 ( lst4-1 ), CKY439 ( lst5-1 ), CKY440 ( lst6-1 ), CKY441 ( lst7-1 ), and CKY442 ( lst8-1 ) were crossed to the sec mutants CKY45 ( sec13-1 ), CKY50 ( sec16-2 ), CKY78 ( sec23-1 ), and CKY450 ( sec31-2 ). Inviability of a given lst sec double mutant was inferred from crosses where lethality segregated as a two-gene trait (most tetrads giving a segregation pattern of 1:3 for lethality), an outcome that was easily detectable since crosses to wild-type typically gave >95% spore viability. The segregation pattern of the sec mutation in the surviving sister spores was used as an additional test to establish that the inviable spores always carried the sec mutation, and therefore were not the result of random spore death. Replacement of the chromosomal LST1 gene with an allele disrupted with the LEU2 gene was constructed as follows. pKR18 carries the 5′ half of LST1 on a 2.0-kb Xho1/HindIII fragment inserted into pRS316. A 2.0-kb HindIII/BamHI fragment containing the LEU2 gene from plasmid pJJ252 and a 250-bp BclI/SacI fragment from the 3′ noncoding region of LST1 were inserted into pKR18 to construct pKR28. The NH 2 -terminal coding region of LST1 (except for codons 1–13) was removed by deleting a 1.7-kb Eco47III/MscI fragment from pKR28 to generate pKR28Δ. The lst1 Δ:: LEU2 construct, liberated from pKR28Δ by digestion with XhoI, was transformed into the wild-type diploid strain CKY348 ( MAT a / α leu2-3,112/leu2-3,112 ura3-52/ura3-52 ). On sporulation and dissection, this diploid gave four viable spore clones, and haploid segregants carrying lst1 Δ:: LEU2 were confirmed by Southern blotting. One such segregant was further backcrossed to our S288C genetic background to give strains CKY536 and CKY542. Pma1p activity was assayed by proton efflux from intact cells into the external medium. Cells were grown to exponential phase in YPD at 37°C, washed, and then stored in deionized water at 4°C overnight. Cell number was measured by light scattering, and a total of 25 A 600 units (∼5 × 10 8 cells) was suspended in 5 ml of 100 mM KCl, 10 mM glycine, pH 4.0. The pH of the cell suspension was measured using a combination electrode at 25°C with constant stirring. Once the pH had stabilized (∼10 min), glucose was added to a final concentration of 40 mM and the ensuing drop in pH was recorded at 30-s intervals over 15 min. In comparison of wild-type (CKY443) and lst1 Δ (CKY536) strains, both suspensions had identical cell concentration as measured by light scattering, and showed the same response to calibration pulses with HCl. The intracellular location of Pma1p in wild-type (CKY443) and lst1 Δ (CKY536) cells was examined by indirect immunofluorescence microscopy using techniques described previously . Strains were grown exponentially in SMM medium, pH 7.2, at 30°C. Cells were fixed in 3.7% formaldehyde and then converted to spheroplasts by digestion with lyticase. Both primary and secondary antibody incubations were for 1 h at 25°C. Affinity-purified anti-Pma1p antibody was prepared as follows. A crude preparation of yeast membranes was resolved by preparative SDS-PAGE, and after transfer of proteins to a nitrocellulose membrane by electrophoresis, the strip of membrane that contained Pma1p was excised. Rabbit antiserum to Pma1p was applied to the nitrocellulose strip, and after the strip was washed with 20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween 20, the bound antibody was eluted with 100 mM glycine, pH 2.8, 500 mM NaCl, 0.5% Tween 20. Affinity-purified Pma1p was used at a 1:100 dilution and FITC-conjugated anti-rabbit IgG was used at 1:200 dilution. Mounting medium was supplemented with 4′,6-diamidino-2-phenylindole (DAPI). Micrographs were taken with a Nikon Eclipse TE300 microscope with a Hamamatsu Orca C4742-95 CCD camera. For the localization of Lst1p-HA, CKY535 was grown on SMM to exponential phase and prepared as described above. For visualization of Lst1p-HA, the 12CA5 antibody (Berkeley Antibody Co., Inc.) was used at a 1:5,000 dilution and FITC-conjugated goat anti–mouse IgG was used at a 1:50 dilution. Rabbit anti–Kar2p polyclonal serum (a gift of M. Rose, Princeton University, Princeton, NJ) was used at a 1:1,000 dilution and rhodamine-conjugated goat anti–rabbit IgG was used at a 1:200 dilution. Samples were viewed and imaged using a Nikon Optiphot 2 microscope and a Photometric ImagePoint CCD camera. Images were recorded using IP-Lab software (Molecular Dynamics, Inc.). Cell organelles were fractionated on equilibrium density gradients as previously described . Cultures were grown exponentially at 24°C and then shifted to 37°C for 3 h. 1.6 × 10 9 cells were collected by centrifugation and suspended in 0.5 ml STE10 (10% [wt/wt] sucrose, 10 mM Tris-HCl, pH 7.6, 10 mM EDTA) with a protease inhibitor cocktail (1 mM PMSF, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 2 μg/ml aprotinin) and lysed by vortexing with glass beads. An additional 1 ml of STE10 was added, and the lysate was cleared of unbroken cells and large cell debris by centrifugation at 300 g for 2 min. The cleared extract (300 μl) was layered on top of a 5-ml, 20–60% linear sucrose gradient in TE (10 mM Tris-HCl, pH 7.6, 10 mM EDTA) prepared for an SW50.1 rotor ( Beckman Instruments, Inc. ). Samples were centrifuged 100,000 g for 18 h at 4°C and fractions of 300 μl were collected from the top of the gradient. Protein was precipitated from each fraction by the addition of 100 μl of 0.15% deoxycholate and 100 μl of 72% trichloroacetic acid. Protein pellets were collected by centrifugation at 13,000 g , washed with cold acetone, and then solubilized in ESB (60 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.02% bromophenol blue). Pma1p, Gas1p, and Sec61p were resolved by SDS-PAGE and were detected by immunoblotting. The relative amount of each protein in cell fractions was determined by densitometry using an Ultroscan 2202 (LKB Instruments, Inc.). The Golgi GDPase activity was assayed in gradient fractions before protein precipitation using standard methods . The subcellular distribution of Lst1p-HA was examined using techniques described previously . CKY535 carrying pKR17HA, which expresses Lst1p-HA, was grown to exponential phase in SMM without uracil. 2 × 10 9 cells were harvested, converted to spheroplasts, and then gently lysed by glass beads in 500 μl of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl 2 ) including the protease inhibitor cocktail. The cell extract was sequentially centrifuged at 500 g for 20 min, 10,000 g for 20 min, and 150,000 g for 60 min, to give one soluble and three particulate fractions. Release of Lst1p-HA from the particulate fraction was examined by treating cell extracts with 500 mM NaCl, 100 mM sodium carbonate, pH 11.5, 2.5 M urea, or 1% Triton X-100. After 1 h of incubation at 4°C, samples were centrifuged at 50,000 g for 30 min to separate soluble and particulate fractions. Fractions from both experiments were solubilized in sample buffer and analyzed by immunoblotting. Samples of 10–30 μl in ESB were resolved by SDS-PAGE and immunoblotting was conducted according to standard protocols . For transfer of Lst1p to nitrocellulose membranes, 0.1% SDS was included in the transfer buffer. The following antibodies were used: mouse monoclonal 12CA5 anti-HA at 1:1,000 dilution; rabbit anti-Pma1p (a gift of A. Chang, Albert Einstein College of Medicine, Bronx, NY) at 1:500 dilution; rabbit anti-Gas1p (a gift of H. Riezman, University of Basel, Basel, Switzerland) at 1:10,000 dilution; rabbit anti-Sec61p (a gift of R. Schekman, University of California, Berkeley, CA) at 1:3,000 dilution; rabbit anti-Gdh2p (a gift of B. Magasanik, Massachusetts Institute of Technology, Cambridge, MA) at 1:1,000 dilution; HRP-coupled sheep anti–mouse Ig and HRP-coupled sheep anti–rabbit Ig (Nycomed Amersham Corp. ) at 1:10,000 dilution. Blots were developed using chemiluminescence detection system (Nycomed Amersham Corp. ). The strains used for radiolabeling all carried the plasmid pNV31, which carries the SUC2 gene under the constitutive TPI1 promoter (a gift of M. Lewis, Medical Research Council Laboratories of Molecular Biology, Cambridge, UK). Wild-type (CKY540) and lst1 Δ (CKY542) strains were grown in SMM without methionine (buffered with 50 mM MES and 50 mM MOPS to pH 6.5) at 24°C to exponential phase, and then shifted to 37°C for 3 h before labeling. A sec12-4 strain (CKY541) was similarly grown to exponential phase at 24°C, but was shifted to 37°C 5 min before the addition of label. Radiolabeling and immunoprecipitation of invertase was performed as previously described . The yeast two-hybrid assay was used to test potential protein–protein interactions as previously described . Interactions were tested between either Lst1p or Sec24p fused to the LexA DNA-binding domain and Sec23p fused to an acidic transcriptional activation domain. The following plasmids were used: pPE81 carries SEC23 fused to the acidic activation domain of pJG4-5 ; pRH286 carries SEC24 (codons 34–926) fused to the lexA DNA-binding domain in pEG202 ; pKR37 carries LST1 fused to the lexA DNA-binding domain in pGilda (a derivative of pEG202 with pGAL1 ; provided by D. Shaywitz). Combinations of control and fusion protein plasmids, along with the reporter plasmid pSH18-34, were transformed into the strain EGY40 . Strains were grown exponentially in SMM with 2% raffinose as the carbon source. Galactose was added to a concentration of 2%, and incubation was continued for 10 h to induce fusion proteins expressed from pGAL1 . Assays for β-galactosidase activity were performed on cells lysed by disruption with glass beads . Activity was normalized to total protein determined by the Bradford assay (Bio-Rad Laboratories). A gene fusion expressing Lst1p fused to glutathione S-transferase (GST) was constructed by inserting the 3.0-kb BamHI/XhoI fragment of pKR17HA into pRD56 (a gift of R. Deshaies, California Institute of Technology, Pasadena, CA) to construct pRH254, which gives GST–Lst1p-HA (amino acids 14–927 of Lst1p) fusion expressed from pGAL1 . pPE123 is the SEC23 gene expressed from pGAL1 in pRS315 . Binding interactions were tested from extracts of CKY473 transformed with pRH254 (GST–Lst1p-HA) and either pCD43 (vector) or pPE123 (Sec23p). Cells were grown to exponential phase in SMM with 2% raffinose, galactose was added to 2%, and incubation was continued for 2 h at 30°C to induce pGAL1 expression. 5 × 10 8 cells were converted to spheroplasts as previously described and then gently lysed using glass beads in IP buffer (20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100) containing the protease inhibitor cocktail. The extract was diluted to 1 ml with IP buffer, and membranes were collected by centrifugation at 500 g for 20 min. This pellet was extracted with 1 ml of IP buffer and 600 mM NaCl for 10 min at 0°C to release membrane-bound protein complexes. After clarification by centrifugation at 90,000 g for 10 min, the extract was diluted threefold with IP buffer, and a 1-ml aliquot was removed and incubated at room temperature for 1 h with glutathione Sepharose 4B beads ( Pharmacia Biotech, Inc. ). The beads were washed twice with 200 mM NaCl, 20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100, and once in IP buffer without Triton X-100. Proteins were released from glutathione Sepharose 4B beads by solubilization in ESB. Samples of total lysate were prepared by adding 2× ESB to an equal amount of the diluted extract from the salt washed membranes. Samples were analyzed by immunoblots probed with anti-Sec23p antibody. For analysis of the membrane association of GST–Lst1p-HA and Sec23p, cells expressing GST–Lst1p-HA, Sec23p, or both GST–Lst1p-HA and Sec23p from pGAL1 , were grown in 2% raffinose and then induced by the addition of 2% galactose as described above. 2 h after induction, 2 × 10 7 cells were collected by centrifugation and resuspended in 20 μl of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl 2 ) with protease inhibitor cocktail. Cells were lysed by vigorous agitation with glass beads and an additional 500 μl of lysis buffer was added. The lysate was cleared of unlysed cells and large cell debris by centrifugation at 300 g for 3 min. 50 μl of the supernatant was reserved for a total extract sample and the remainder was centrifuged to pellet ER membranes at 10,000 g for 30 min at 4°C in a microcentrifuge. An equal number of cell equivalents of total extract, membrane-pellet, and supernatant fractions was solubilized in ESB and analyzed by immunoblotting. The cytosolic protein Gdh2p was found only in the soluble fractions, demonstrating cell lysis was complete (data not shown). To find new genes required for the budding of COPII vesicles, we screened for mutations that displayed synthetic lethality with the COPII mutation sec13-1 using a plasmid sectoring assay . Strain CKY423 has the chromosomal mutations ade2 ade3 sec13-1 and harbors the plasmid pKR4, which carries wild-type copies of SEC13 and ADE3 . This strain accumulates a red pigment because of the ade2 mutation, but the spontaneous loss of pKR4 during the growth of a colony gives white sectors of ade2 ade3 segregants. In this strain, a mutation that is lethal with sec13-1 will produce a nonsectoring colony. Mutagenesis of CKY423 and the isogenic strain of opposite mating type, CKY424, yielded 139 nonsectoring mutants . These strains were then tested for restored ability to sector after transformation with pKR1, which carries wild-type SEC13 , but lacks the ADE3 gene. By this test, 57 of the mutants had synthetic-lethal mutations that could be rescued by wild-type SEC13 . In backcrosses, 52 mutants gave a segregation pattern indicating that the trait was due to a single nuclear mutation . Matings between mutants identified 11 complementation groups using colony sectoring of the diploid as the criterion for allelic complementation. These complementation groups were designated LST (Table II ). One of the complementation groups was shown to comprise recessive lethal mutations in the SEC13 gene itself . Tests for rescue of the nonsectoring phenotype by plasmids carrying known sec genes showed that LST10 was SEC16 . To perform further genetic tests on the lst mutations, the lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus (Materials and Methods). Representative lst single mutants were then crossed to sec16 , sec23 , and sec31 mutants. For mutations in LST2 , LST3 , LST4 , LST5 , LST7 , and LST8 , only crosses to sec13-1 gave a segregation pattern indicative of a synthetic-lethal interaction (Table III ). We have subsequently shown that these LST genes relate to a function of SEC13 in the sorting of amino acid permeases in the late secretory pathway, and analysis of these genes is described elsewhere . Mutations in LST1 were inviable when combined with sec16 , sec23 , and sec31 mutations, and mutations in LST6 were inviable with sec16 and sec31 (Table III ). Importantly, mutations in LST1 and LST6 did not show synthetic lethality in parallel crosses to mutations in SEC17 or SEC18 , genes required for fusion of COPII vesicles. Given that synthetic-lethal interactions usually occur between mutations in genes involved in the same step of the secretory pathway, the tests for genetic interactions indicated that LST1 , and probably also LST6 , participate in vesicle budding from the ER. The LST1 gene was isolated by its ability to restore sectoring to CKY426 ( MAT a sec13-1 ade2 ade3 leu2 ura3 [pKR4]), a strain that forms solid red, nonsectoring colonies because of the presence of the lst1-1 mutation. CKY426 was transformed with yeast genomic libraries. 34 colonies that regained the ability to form white sectors were identified among 97,000 Ura + transformants. We expected this screen to yield plasmids carrying either SEC13 or LST1 . About half of the complementing plasmids were shown to carry SEC13 by restriction site mapping and by the ability to complement the temperature sensitivity of sec13-1 . The restriction maps of the remaining rescuing plasmids show that they represent two unrelated chromosomal regions. The clones p21-31 and p77-2 were selected as representatives of each region. The genomic sequence from p77-2 was inserted as an XhoI fragment into the integrating vector pRS306 to produce pKR20. For chromosomal integration, pKR20 was linearized by digestion with HpaI and transformed into CUY564 ( MAT α ade2 ade3 leu2 ura3 ). The resulting strain was crossed to the lst1-1 mutant CKY426 ( MAT a lst1-1 sec13-1 ade2 ade3 leu2 ura3 [pKR4]). After sporulation and dissection, the integrated pKR20 was found to be completely linked to the LST1 locus: sectoring segregated 2:2 and all sectored colonies were Ura + , whereas all nonsectored colonies were Ura − . Thus, p77-2 carries the LST1 gene. In parallel, the genomic sequence from p21-31 was inserted as an EcoRI/HindIII fragment into pRS306 to produce pKR7. pKR7 was integrated at its chromosomal locus after linearization with MscI and was then checked for linkage to lst1-1 . Tetrad analysis showed that pKR7 was not linked to LST1 and we concluded that pKR7 carries an unlinked suppressor gene. The 3.5-kb insert of p77-2 was inserted into the XhoI site of the centromeric vector pRS316 to construct pKR17. The base sequence of this insert was determined and found to contain a single open reading frame encoding a protein of 929 amino acids. This sequence corresponds to the open reading frame YHR098c located on chromosome VIII . The predicted amino acid sequence of LST1 shows significant similarity to SEC24 (YIL109C). The two proteins share 23% sequence identity that extends over most of their length , suggesting that Lst1p may have a function similar to that of Sec24p as a subunit of the COPII vesicle coat. One copy of the LST1 gene in the wild-type diploid strain CKY348 was disrupted to generate a lst1 Δ:: LEU2/LST1 heterozygote. Sporulation and dissection of this diploid gave >95% spore viability on YPD medium and the LEU2 marker segregated 2:2, showing that LST1 is not essential for growth. A lst1 Δ:: LEU2 mutant spore clone was crossed to sec mutants to test for synthetic lethality. In these crosses, both the temperature sensitivity of the sec mutation and the lst1 Δ allele marked by LEU2 could be followed independently. In crosses of lst1 Δ to sec12 , sec13 , sec16 , sec23 , sec24 , or sec31 mutants, inviability segregated as a two-gene trait (segregation patterns for dead:viable spore clones were 2:2, 1:3, and 0:4). Tests of the genotype of the surviving sister spore clones showed that the inviable spores in these crosses were always lst1 Δ sec double mutants. Crosses between lst1 Δ and sec17 or sec18 produced viable double mutants. These findings confirmed and extended our earlier tests for synthetic lethality with lst1-1 , and demonstrated that lst1 Δ was synthetically lethal with all the known genes required for COPII vesicle formation, but not with genes required for vesicle fusion. We evaluated the growth of lst1 Δ:: LEU2 mutants under a variety of conditions. On YPD, the lst1 Δ:: LEU2 strain grew, as well as an isogenic wild-type strain at temperatures ranging from 14 to 37°C. However, on SMM the lst1 Δ:: LEU2 strain grew poorly at temperatures above 30°C. Since YPD (pH 6.5) and SMM (pH 3.8) differed markedly in pH, we suspected that lst1 Δ mutants may be particularly sensitive to an acidic environment, and we tested the effect of pH on the growth of lst1 Δ mutants. Although lst1 Δ mutants grew as well as wild-type on YPD at all temperatures, when YPD was brought to pH 3.8, lst1 Δ mutants grew much more slowly than wild-type at 37°C . Conversely, on SMM buffered to pH 6.5, lst1 Δ and wild-type grew even at 37°C (data not shown). These results demonstrated that at high temperature, growth of the lst1 Δ mutant was sensitive to acidic conditions. Having identified conditions where LST1 was needed for growth, we investigated whether overexpression of SEC24 could supply the function lost in lst1 Δ. Some restoration of function was indicated by the ability of an lst1 Δ mutant to grow on acidic medium when provided with extra copies of SEC24 on either centromeric or 2μ plasmids . These findings imply some functional overlap between LST1 and SEC24 . In parallel tests for suppression, we found that the genes SEC12 , SEC13 , SEC31 , or SEC23 , when expressed from 2μ plasmids, could not restore the ability of an lst1 Δ mutant to grow on acidic medium. We found that the lst1 Δ mutation caused a selective defect in the trafficking of Pma1p from the ER, and we also examined the ability of overexpressed SEC24 to suppress this phenotype caused by the lst1 Δ mutation. By immunofluorescence microscopy, the proper localization of Pma1p to the cell surface was restored in an lst1 Δ strain that also carried SEC24 on a 2μ plasmid . In an attempt to test the effect of overexpression of LST1 , we found that LST1 on a 2μ plasmid severely impaired growth of wild-type yeast cells. To examine the response of cells to different doses of Lst1p, we designed a way to express different levels of Lst1p according to the amount of galactose in the growth medium. A wild-type strain (CKY473) carrying a plasmid that expressed LST1 from pGAL1 (pKR35) was spread on an SMM plate with 2% raffinose, a carbon source that allows yeast growth without repression of the GAL1 promoter. When these cells are exposed to a gradient of galactose concentrations, from 3 mg of galactose in a filter disk on top of the lawn, growth was inhibited in a halo 1.5 cm beyond the edge of the filter . A strain that did not contain pKR35 grew uniformly up to the edge of the filter, showing that the galactose itself was not inhibitory. Given the similarity of Lst1p to Sec24p, we asked whether the overexpression of SEC24 could compensate for overexpression of LST1 . Cells carrying both the pGAL1–LST1 plasmid (pKR35) and the SEC24 gene on a 2μ plasmid (pKR41) were tested in an identical halo assay, and were found to be resistant to the effect of galactose . Suppression by SEC24 appeared to be specific, since parallel tests of 2μ plasmids carrying SEC12 , SEC13 , SEC31 , or SEC23 failed to show suppression. It is worth noting that SEC23 expressed from a 2μ plasmid significantly slows the growth of our yeast strains. Any suppression afforded by overexpression of SEC23 might be counteracted by this inherent toxicity of SEC23 . A simple conclusion that can be drawn from these overexpression studies is that too great of a stoichiometric excess of Lst1p over Sec24p is lethal. This observation can be explained if Lst1p and Sec24p compete with one another in the assembly of vesicle coat complexes and that excess Lst1p causes sequestration of vesicle components into complexes that fail to satisfy some essential function of COPII. The sensitivity of lst1 Δ mutants to low pH suggested the involvement of Pma1p, which has been shown to be the limiting cell component for growth on acidic medium . The dependence of Pma1p activity on LST1 was supported by the observation that lst1 Δ mutants exhibited an unusual morphology characteristic of pma1 mutants. When lst1 Δ mutants were grown in low pH (SMM or YPD brought to pH 3.8) at 37°C, ∼10% of the cells formed multibudded rosettes; in some cases, as many as 15 daughters radiated from a single large mother cell . The unseparated daughter cells contained nuclei that could be stained with DAPI and the daughter cells could be separated from their mothers by micromanipulation, indicating they had completed cytokinesis. Cells depleted of Pma1p produce similar multibudded cells with attached daughters that had completed cytokinesis. In this case, multibudded rosettes are thought to form because a mother cell formed with sufficient Pma1p in the plasma membrane will continue to bud, whereas daughter cells formed after Pma1p transport is compromised will have insufficient Pma1p to form buds themselves . The morphology of lst1 Δ cells grown at relatively high pH (YPD or SMM buffered to pH 6.5) at 37°C appeared normal, with few cells having more than one attached daughter. As a more direct test of the effect of lst1 Δ on the activity of Pma1p, we measured the capacity of mutant cells to pump protons into the external medium. Wild-type and lst1 Δ strains were cultured in YPD at 37°C, conditions under which both strains grow equally well. After starvation by prolonged incubation in water, the cells were placed in a weakly buffered medium and proton efflux on addition of glucose was measured as a drop in extracellular pH. For both wild-type and lst1 Δ strains, addition of glucose produced a sharp decline in pH (after a 30-s lag), which began to level off after ∼5 min . Although the responses of wild-type and lst1 Δ cells were qualitatively similar, proton efflux from lst1 Δ cells was compromised: in the first 5 min after addition of glucose the rate of change in pH produced by the lst1 Δ mutant was 65% of that of wild-type. These findings indicate that the lst1 Δ mutant grown at 37°C has about half of the Pma1p activity as wild-type cells. To determine whether the reduced Pma1p activity in lst1 Δ mutants was due to a defect in the transport of Pma1p to the cell surface, we compared the localization of Pma1p in wild-type and lst1 Δ mutant cells by immunofluorescence microscopy. Cells were grown at 30°C in YPD medium to avoid possible secondary effects due to the pH sensitivity of lst1 Δ mutants. In lst1 Δ cells, Pma1p was located primarily at the nuclear periphery and at the cellular rim, indicating that a large proportion of Pma1p remains in the ER . This pattern of localization differed markedly from the surface localization of Pma1p in wild-type cells incubated at 30°C or in lst1 Δ cells incubated at 24°C (data not shown). We also examined the subcellular distribution of Pma1p in lst1 Δ cells by cell fractionation. Lysates from cells grown at 37°C for 3 h were fractionated on sucrose density gradients under conditions where the ER and plasma membrane are well separated on the basis of their buoyant density. Pma1p from wild-type cells was located in dense fractions of the gradient in a peak that was coincident with that of Gas1p, a GPI-linked plasma membrane protein . In contrast, <35% of the total Pma1p from lst1 Δ cells coincided with the plasma membrane marked by Gas1p protein and the majority of Pma1p was located in fractions containing the ER . Interestingly, the ER from lst1 Δ mutants (marked by Sec61p) reproducibly resolved into two peaks of different density, suggesting that accumulation of Pma1p segregates ER membranes into subdomains of relatively high and low density. Given that most of the Pma1p was located in the ER peak of higher density, it is possible that the density of the ER had been increased because of the accumulation of Pma1p. A similar increase in density of a portion of the ER is caused when folding mutants of PMA1 are retained within the ER . The fact that transport of Pma1p, but not of Gas1p, was affected by deletion of LST1 suggested that LST1 may be specifically required for the export of Pma1p from the ER. The absence of a general protein secretion defect in lst1 Δ mutants was implied by the normal growth of lst1 Δ mutants at 37°C in medium of pH 6.5 (the doubling time of both lst1 Δ and wild-type was 1.75 h in YPD), indicating a normal rate of expansion of the plasma membrane. As a specific test for the rate of ER to Golgi transport, pulse– chase experiments were performed to follow the rate of maturation of invertase from its core glycosylated ER form to the Golgi and secreted forms. No delay in invertase transport was observed in lst1 Δ mutants that had been grown at 37°C for 3 h, conditions that caused the accumulation of Pma1p . Similarly, no defect in the maturation of carboxypeptidase Y from the ER form to the Golgi and vacuolar forms of the enzyme could be detected (data not shown). We also considered the possibility that transport of Pma1p may be particularly sensitive to any subtle defect in vesicle formation. We addressed this possibility by examining the localization of Pma1p in sec24-1 and sec31-2 mutant cells at the semipermissive temperature of 28°C. Although the growth rate of both mutants was compromised at this temperature (doubling time on YPD: 2.9 h for sec24 and 2.4 h for sec31 , as compared with 1.7 h for wild-type), no accumulation of Pma1p was detected in the perinuclear region of either mutant by immunofluorescence microscopy (data not shown). Thus, partial defects in COPII functions did not lead to the extensive accumulation of Pma1p in the ER that was observed for lst1 Δ mutants. Taken together, comparisons between the lst1 Δ mutation and COPII gene mutations indicate that the lst1 Δ mutation is unusual in its ability to inhibit Pma1p exit from the ER without interfering with the transport of other cargo proteins. To examine the intracellular distribution of Lst1p, an epitope-tagged derivative was constructed by inserting six copies of the 10–amino acid HA near the NH 2 terminus of Lst1p. The HA-tagged LST1 was functional, as demonstrated by its ability to complement lst1-1 in a sectoring assay, and to restore the ability of a lst1 Δ mutant to grow on acidic medium at 37°C (not shown). In cells expressing Lst1p-HA that were fixed for immunofluorescence microscopy, staining was found primarily at the nuclear periphery . No signal was seen in cells expressing untagged Lst1p, verifying that the origin of the staining pattern was due to Lst1p-HA. Although Lst1p-HA staining largely coincided with the ER marker Kar2p, there were subtle differences in their patterns of localization: Kar2p appeared uniformly, distributed around the nuclear periphery, whereas Lst1p-HA staining had a more punctate appearance indicating that Lst1p might be concentrated in particular regions of the ER. In addition, weak punctate staining was observed throughout the cell body, some of which may correspond to ER membranes near the cell periphery. The intracellular distribution of Lst1p was also examined by subcellular fractionation. Cells expressing Lst1p-HA were converted to spheroplasts and then gently lysed. This cell lysate was subjected to differential centrifugation and most of Lst1p-HA was found to pellet at either 500 g or 10,000 g . All of the soluble marker protein Gdh2p was found in the 150,000 g supernatant fraction, indicating complete cell lysis (data not shown). The association of the Lst1p protein with the sedimenting fraction was analyzed by chemical treatment of cell lysates before centrifugation at 50,000 g . Incubation of cell extracts in 1% Triton X-100, 2.5 M urea, 100 mM sodium carbonate, pH 11.5, or 500 mM NaCl resulted in the release of a portion of the Lst1p-HA into the soluble fraction . The partial dissociation of Lst1p-HA from the sedimenting fraction by these agents suggested that Lst1p is a peripheral membrane protein that adheres tightly to the membrane. Sec24p was first identified as a protein that formed a 400-kD complex with Sec23p . Because of the similarity of Lst1p to Sec24p, we investigated whether Lst1p could also bind to Sec23p. To assay potential interactions by the yeast two-hybrid assay, LST1 was fused to the lexA DNA-binding domain (pKR37) and SEC23 was fused to an acidic activation domain (pPE81). Interaction between the two fusion proteins was tested by assaying for activation of a lacZ reporter gene. Induction of β-galactosidase was observed when the LST1 and SEC23 fusions were coexpressed, but not when expressed alone (Table IV ). The level of induction caused by interaction of LST1 and SEC23 was similar to that seen for interaction of SEC24 and SEC23 . To confirm the interaction between Lst1p and Sec23p, association of these proteins was examined in yeast cell extracts. The coding sequence of LST-1-HA (codons 14–927) was fused to GST and expressed in yeast from the pGAL1 promoter. SEC23 was also expressed from pGAL1 . Since both proteins are largely associated with intracellular membranes , membranes prepared from cells overexpressing both Sec23p and GST–Lst1p-HA were first extracted with 600 mM NaCl to release protein complexes from the membrane, the salt extracts were clarified by centrifugation at 90,000 g , and diluted to give a final concentration of 200 mM NaCl. GST–Lst1p-HA was isolated from the extracts by affinity to glutathione Sepharose beads. Sec23p was found in association with GST–Lst1p-Ha, but not in control extracts prepared from cells expressing Sec23p and GST alone . Together, these experiments show that Lst1p, like Sec24p, can form a complex with Sec23p. Sec23p and Sec24p have been shown to assemble onto the ER membrane as a complex . While working out conditions to optimize recovery of Sec23p bound to GST–Lst1p-HA, we discovered that assembly of an Lst1p/Sec23p complex appears to enhance the association of both proteins with the ER membrane. When both GST–Lst1p-HA and Sec23p were overexpressed in the same cell, >60% of the Sec23p, and 70% of the GST–Lst1p-HA were found in a fraction that pelleted at 10,000 g . This pellet contains most of the ER, as marked by the ER membrane protein Sec61p (data not shown). When material that pelleted at 10,000 g was suspended in 60% sucrose and applied to the bottom of a sucrose density gradient, >90% of the GST– Lst1p-HA and Sec23p cofractionated with the ER resident membrane protein, Sec61p, at a density corresponding to 45% sucrose, showing that GST–Lst1p-HA and Sec23p were associated with membranes (data not shown). In contrast to the case when Sec23p and GST–Lst1p-HA were expressed together, <10% of the Sec23p pelleted at 10,000 g in lysates from a strain overexpressing Sec23p alone. Similarly, <20% of the GST–Lst1p-HA pelleted at 10,000 g in lysates from a strain expressing GST–Lst1p-HA alone . Thus, when either Sec23p or GST–Lst1p-HA was overexpressed alone, most of the overexpressed protein was soluble, but when the proteins were expressed together, most of the proteins were associated with the ER membranes. These data support the observation that Lst1p can form a complex with Sec23p, and that the Lst1p/ Sec23p complex has affinity for ER membranes. By screening for mutants that exhibited synthetic-lethal genetic interactions with the COPII mutation sec13-1 , we identified the LST1 gene. Subsequent genetic tests showed that lst1 Δ is lethal when combined with mutations in genes required for COPII vesicle budding from the ER ( SEC12 , SEC13 , SEC16 , SEC23 , SEC24 , and SEC31 ), but lst1 Δ is not lethal when combined with mutations in genes that are required for vesicle fusion with the Golgi compartment ( SEC17 and SEC18 ). This pattern of genetic interactions indicated that LST1 participates in the process of vesicle budding from the ER, an expectation that was born out by the examination of the LST1 gene and its product. The following observations indicate a role for Lst1p as part of a COPII-like vesicle coat: (I) LST1 encodes a 90-kD protein that is homologous to the COPII-coat subunit Sec24p. The two proteins share 23% amino acid identity over their entire lengths. (II) Lst1p is a peripheral ER membrane protein as shown by immunofluorescence microscopy and cell fractionation. (III) Lst1p, like Sec24p, can bind to Sec23p as shown by tests for two-hybrid interaction and affinity purification of a complex of GST–Lst1p and Sec23p. (IV) Assembly of the Sec23p–Lst1p complex appears to enhance the membrane association of both Lst1p and Sec23p: when both proteins are overexpressed together, most associate with membranes, whereas either protein overexpressed alone is mostly cytosolic. (V) Although strains with chromosomal deletion of LST1 are viable and appear normal for secretion of marker proteins, these mutants show a pronounced accumulation of Pma1p in the ER, indicating a selective defect in ER to Golgi traffic. Based on these findings, we propose that Lst1p takes the place of Sec24p in a specialized COPII coat complex that is used for the recruitment of Pma1p into vesicles. Strains carrying lst1 Δ have the phenotypic hallmarks of a deficiency in Pma1p activity, including sensitivity to growth in an acidic environment, the formation of multibudded cells, and a decreased rate of proton efflux from intact cells. All three traits are expressed only at temperatures of 30°C and above, indicating that LST1 is only required for Pma1p activity at high temperature. Localization of Pma1p in lst1 Δ cells by immunofluorescence and sucrose density cell fractionation demonstrate that the transport of Pma1p from the ER is compromised in lst1 Δ at 37°C. Export of Pma1p from the ER cannot be completely dependent on Lst1p, since Pma1p transport appears normal in lst1 Δ mutants at 24°C. Even at 37°C, the block in Pma1p transport may not be complete since ∼35% of the total Pma1p fractionates with the plasma membrane, although some of the Pma1p detected in the plasma membrane in this experiment was probably synthesized before the shift to restrictive temperature. Therefore, it seems likely that Lst1p and Sec24p share the burden of transporting Pma1p from the ER. At 24°C, it appears that Sec24p (or some other protein) can compensate for the absence of Lst1p, but at temperatures of 30°C or higher, compensation is no longer possible unless extra copies of Sec24p are provided by expression from a multicopy plasmid. The transport defect caused by deletion of LST1 appears to be specific for Pma1p. Under conditions where a defect in Pma1p transport was observed in lst1 Δ mutants, transport of Gas1p, carboxypeptidase Y, and invertase was unaffected. Using growth as a more general assay for trafficking defects, we found that lst1 Δ mutants grew at an identical rate to wild-type at 37°C when we compensated for the defect in Pma1p transport by using media at pH 6.5. This indicates that rate of expansion of the plasma membrane, including the transport of all essential plasma membrane proteins, is not significantly affected by the absence of LST1 . We also considered the possibility that there may be differences among cargo molecules in their response to general defects in the protein transport machinery. Of particular concern was the possibility that Pma1p transport might be particularly sensitive to slowed ER to Golgi transport, such that a defect in transport too subtle to have an effect on our standard marker proteins might have a significant effect on the rate of transport of Pma1p. If this were the case, partial defects in other COPII components should also interfere with Pma1p transport. Therefore, we examined sec24 and sec31 mutants, but could find no evidence for a defect in Pma1p transport, even at semipermissive temperatures where the rate of growth was inhibited. Although Pma1p was the only essential protein for which we could detect a transport defect in lst1 mutants, a defect in the transport of any nonessential protein could have been overlooked by our analysis. Factors required for the transport of specific membrane proteins have been documented in a number of other cases. The SHR3 gene encodes an ER resident protein that is required for the transport of amino acid permeases out of the ER, but is not required for the transport of a variety of other proteins . A set of ER proteins, Vma12p, Vma21p, and Vma22p, are required for transport from the ER of the integral membrane subunit of the vacuolar ATPase . Similarly, mutational studies have shown that the small ER membrane protein Erv14p is specifically required for transport of the plasma membrane protein Axl2p out of the ER . Finally, Ast1p has been suggested to be a factor specifically needed for the transport of Pma1p from the Golgi compartment to the plasma membrane . In all of these cases, the question remains whether Shr3p, the Vma proteins, Erv14p, or Ast1p act directly in vesicular transport of their respective cargo molecules, or whether they are primarily involved in protein folding and influence protein sorting indirectly through quality control mechanisms. Because Lst1p appears to be a component of a vesicle coat, Lst1p seems more likely to have a direct role in the sorting of Pma1p rather than in its folding. Expression of a variety of dominant PMA1 mutations can cause accumulation of both mutant and wild-type Pma1p in proliferated ER . Similarly, the transport of wild-type Pma1p from the ER is blocked when PMA2 (an isoform of PMA1 ) or plant plasma membrane proton-ATPases are overexpressed in yeast . One proposal was that a special factor may be required for the transport of Pma1p from the ER in a manner analogous to the requirement for Shr3p in the transport of amino acid permeases . The specific role of Lst1p in the transport of Pma1p suggests that it may be the factor depleted by the expression of dominant forms of Pma1p. In the future, it may be possible to test this idea by evaluating the ability of Lst1p overexpression to reverse the effects of dominant PMA1 mutations. The mechanism by which Lst1p acts in the transport of Pma1p may be inferred from recent studies examining the recruitment of cargo molecules into COPII vesicles. Using ER-derived microsomes and purified COPII components, Kuehn et al. have shown that the Sec23p/Sec24p complex, along with Sar1p, associate with amino acid permeases and other integral membrane protein that are destined for the plasma membrane. In parallel experiments using mammalian microsomes, mammalian Sec23p/Sec24p and Sar1p were found to bind to microsomal membranes and form a complex that contains the cargo protein VSV-G . The conclusion from both experimental systems is that the Sec23p/Sec24p complex contains specific binding sites for the capture of membrane cargo proteins within the plane of the ER membrane. Based on the data presented here, Lst1p appears to be an isoform of Sec24p that is adapted for selection of Pma1p. This provides the first evidence that Sec24 family members carry information specifying the type of cargo molecules that are accepted by ER-derived vesicles. We have looked for association of Lst1p with ER-derived vesicles, but under the conditions of an in vitro budding reaction, a large quantity of Lst1p-HA is released from the membrane in soluble form. Soluble Lst1p-HA gives a high background in vesicle fractions preventing us from reliably determining whether there is a specific association of Lst1p with vesicles. In future experiments, it may be possible to isolate vesicles coated with Lst1p by performing an in vitro budding reaction using purified cytosolic components, including a purified complex of Lst1p and Sec23p. It may also be possible to determine whether vesicles that are formed using a Sec23p/Lst1p complex more efficiently incorporate Pma1p than vesicles formed using the Sec23p/Sec24p complex. Finally, it will be of interest to determine if there is direct binding of Lst1p to Pma1p. The identification of a Sec24p homologue that also acts in transport from the ER raises the possibility that the coats of ER-derived vesicles may be heterogeneous. It is possible that Sec23p/Lst1p complexes act to form a class of vesicle that is distinct from those formed by Sec23p/ Sec24p complexes. Alternatively, it is possible that the two complexes assemble together forming vesicles with coats of mixed composition. The identification of additional homologues of Sec23p and Sec24p suggest the existence of coats with even greater combinatorial complexity. We have identified a third Sec24p family member, which we call Iss1p, as a protein that binds to Sec16p. Iss1p (YNL049c) also binds Sec23p and appears to be associated with the ER membrane . In addition, the Saccharomyces genome contains an uncharacterized open reading frame (YHR035w) that is 21% identical to Sec23p . If each of the Sec23p and Sec24p homologues carry different determinants for cargo selection, and if mixed coats can form, the possible combinations of Sec23p and Sec24p homologues should allow the formation of a wide variety of COPII-like vesicles with different capacities to carry different cargo molecules. | Study | biomedical | en | 0.999997 |
10330398 | The Gi fraction was prepared from rat liver homogenates using a motorized Potter-Elvehjem homogenizer (rotating Teflon ® pestle at 2,300 rpm) in ice cold 0.25 M sucrose, 4 mM imidazole, pH 7.4, following protocols described previously . The GE fraction was isolated from liver homogenates in ice cold 0.25 M sucrose, 5 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl 2 , 4.5 mM CaCl 2 (STKCM) by a variation of the method of Bergeron et al. and Smith et al. in which discontinuous, rather than continuous, sucrose gradients were used to obtain Golgi fractions of high protein concentrations without having to resort to pelleting. After filtration through two layers of cheese cloth and centrifugation at 400 g max for 10 min, supernatants were adjusted to 1.15 M sucrose in STKCM and underlaid beneath a discontinuous gradient of 0.95 and 0.4 M sucrose in STKCM. After centrifugation at 200,000 g av for 90 min, Golgi fractions were collected at the 0.4/0.95 M sucrose interface. The WNG fraction 2 was isolated from 20% liver homogenates in 0.25 M sucrose containing 5 mM Tris-HCl, pH 7.4, and 5 mM MgCl 2 (STM). Unless otherwise indicated, all buffers also contained the proteinase inhibitors PMSF (1 mM) and aprotinin (200 U/ml). Instead of the motor driven Teflon ® pestle Potter-Elvehjem homogenizer (Thomas Scientific) used to prepare Gi and GE liver homogenates, the homogenates used for isolation of the WNG fraction were prepared after gentle homogenization of finely minced liver with the type B loose pestle of a Dounce homogenizer (12 strokes; Thomas Scientific). After filtration through two layers of cheese cloth, homogenates were routinely incubated at 4°C for 2 h to ensure that microtubules were depolymerized . After low-speed centrifugation at 400 g max for 5 min, the supernatant (S1) was saved and the pellet (P1) rehomogenized in half the original volume with five strokes of the type B pestle. After centrifugation at 400 g max for 5 min, the supernatant (S2) was combined with S1 and centrifuged at 1,475 g max for 10 min. The resulting pellet (P2) was combined with P1 and resuspended in 1.22 M sucrose in the above buffer (20% wt/vol original liver wet weight). A continuous gradient in STM of 0.25–1.10 M sucrose was generated above the load zone and centrifuged at 1,200 g av for 30 min, followed by 83,000 g av in the rotor (SW27; Beckman Instruments, Inc. ) for 1 h. The band at ∼1 cm above the load zone was collected, adjusted to 0.4 M sucrose in STM without proteinase inhibitors, and centrifuged at 1,475 g max for 10 min (P3). Final enrichment in large membranes was achieved by two successive rounds of resuspension in 0.25 M sucrose in STM followed by pelleting at 1,475 g max for 10 min. The final pellet was recovered in 0.25 M STM and represented 0.085 ± 0.02% ( n = 7) of the homogenate protein. The disrupted WNGdis fraction was prepared using methods identical to those used to obtain WNG fractions with the following modifications. After collection of the initial low-speed pellets at 1,475 g max for 10 min (P3, above), these pellets were rehomogenized in 0.25 M sucrose in 4 mM imidazole, pH 7.4, with proteinase inhibitors, and homogenized with 10 strokes of the motor driven Teflon ® pestle (2,300 rpm) of a Potter-Elvehjem homogenizer. After pelleting at 1,475 g max for 10 min and the supernatant saved, the pellet was resuspended, rehomogenized, and repelleted once more using the same protocol. The three resulting supernatants were combined and gently pelleted onto a 2 M sucrose cushion by centrifugation at 144,000 g av for 40 min. The pellicle was resuspended in 1.15 M sucrose in 4 mM imidazole buffer, pH 7.4, and underlaid in a sucrose step gradient of 0.95 and 0.4 M buffered sucrose. After centrifugation for 90 min at 200,000 g av , the interface at 0.4/0.95 M sucrose was collected as the WNGdis fraction. The isolated WNG fraction in 0.25 M STM was adjusted to 300 mM KCl and incubated at 150 μg/ml cell fraction protein for 1 h at 4°C. Membranes were recovered by centrifugation through 0.4 M buffered sucrose layered onto a 2-M buffered sucrose cushion at 201,000 g av for 1 h in an SW40 rotor ( Beckman Instruments, Inc. ). The band at the 0.4/2 M interface was then collected and mixed by gentle resuspension. For cisternal dissociation of WNG fraction (WNGI), the isolated WNG fraction was incubated at 150 μg cell fraction protein/ml in 4 mM imidazole, pH 7.4, 0.25 sucrose for 1 h at 4°C, and then collected as described above for KCl-treated WNG membranes. To determine the sedimentation characteristics of Golgi fractions, the different Golgi fractions (Gi, GE, WNG) were adjusted to 0.25 M sucrose in the respective buffers used to isolate each fraction, with 0.5 ml loaded onto a 0.4 M sucrose cushion (buffered as above) (2.0 ml in the SW60 rotor and centrifuged at the indicated g · min). The pelleted fractions were evaluated for their GalT content. For identification of the fusogenic component of the WNGdis fraction, the fraction was centrifuged at 200,000 g max for 40 min (Ti60 angle rotor; Beckman Instruments, Inc. ). The resultant pellet was resuspended in 0.25 M sucrose (4 mM) imidazole buffer, pH 7.4, and 0.6 ml of fraction (1.2–2.3 mg protein/ml) loaded onto a step sucrose gradient (10 [0.5 ml], 15 [0.5 ml], 20 [0.5 ml], 25 [0.5 ml], 30 [0.5 ml], 35 [0.5 ml], and 40% [0.4 ml] [wt:wt] preincubated 2 h at room temperature and 1 h at 4°C) and centrifuged 10 min at 20,000 rpm using the SW60 rotor. Fractions were collected (0.5 ml) and evaluated for their content of total protein, GalT, NAGT, and transport activities. The incorporation of UDP-[ 3 H]-GlcNAc into endogenous acceptors was measured as described . GalT assays were performed as described previously and, where applicable, the GalT-specific activity expressed in milliunits (nmol gal transferred/min · mg cell fraction protein). NAGT assays were performed as described by Vischer and Hughes using ovalbumin as acceptor and UDP- 3 H GlcNAc as sugar donor. 125 I-insulin was prepared as described by Frank et al. . To evaluate endosomal contamination, rats were anesthetized with sodium pentobarbital and injected with 5 μCi of [ 125 I]-insulin into the portal vein. 5 min after injection, the animals were killed and the livers were processed for homogenization and subcellular fractionation as described above. CHO Golgi-enriched membrane fractions were isolated from cells grown in suspension and homogenized in 0.25 M sucrose containing 10 mM Tris-HCl, pH 7.4, and proteinase inhibitors, by 12 passages through a steel ball homogenizer . CHO acceptor membranes were obtained from wild-type CHO cells (pro-5, American Type Culture Collection [ATCC]), while donor membranes were obtained from lec1 CHO cells (ATCC) 3.5 h after infection with vesicular stomatitis virus, as described by Weidman et al. . CHO cytosol was prepared as described . Protein concentrations were determined by the BCA method ( Pierce Chemical Co. ) using immunoglobulins as standard or by the Bradford method (Bio-Rad Laboratories) using bovine serum albumin as standard . Intra-Golgi transport between VSV-G–containing donor and the various NAGT I–containing acceptor membrane fractions was measured as incorporation of UDP-[ 3 H]-GlcNAc into immunoprecipitated VSV-G essentially as described in Taylor et al. , with the exception that all 25-μl assays contained 2 μl donor membranes (0.07–0.1 μg protein) and 2 μl CHO cytosol (1.4 μg protein), and were performed for 60 min at 37°C. For analysis of transport activity in fractions observed after rate-zonal centrifugation, the assay volume was increased to 50 μl while keeping buffer, sucrose, cytosol, and UDP-[ 3 H]-GlcNAc concentration constant; this change accommodated the larger volumes (10 μl) of liver Golgi fraction to be tested. The amount of acceptor membrane present in a given assay varied between experiments and is stated in the figure legends. Control experiments confirmed that all such assays were performed under linear conditions of either substrate addition by donor Golgi fractions and enzyme addition by acceptor Golgi fractions. Protein samples were first separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (Xymotech). The blots were incubated in 5% skim milk in TNT buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). Antibodies to β-COP (affinity-purified rabbit antipeptide antibody EAGE, supplied by Drs. T.E. Kreis and J. Lippincott-Schwartz or monoclonal antibody M3A5; Sigma Chemical Co. ) and ARF (mouse monoclonal antibody ID9, a gift of Drs. P. Randazzo and R.A. Kahn, NIH, Bethesda, MD) were used at dilutions of 1/1,000. Secondary antibodies conjugated to alkaline phosphatase were used to visualize immunoblots as described by Smith and Fisher . Polyclonal antibodies to GS15 were visualized using chemiluminescence according to the manufacturer's instructions (NEN Life Science Products). Antibodies to GS28 (monoclonal) and Vti-rp2 (polyclonal) were visualized by radioautography after incubation with 125 I rabbit anti–mouse antibodies and 125 I-labeled goat anti–rabbit, respectively. Quantitation of 125 I signals were by Phosphorimager analysis (Fuji Bioimaging). All SNARE antibodies were a kind gift from Dr. Wanjin Hong (Institute of Molecular and Cell Biology, University of Singapore). Membrane samples for electron microscopy were prepared using the random sampling filtration apparatus of Baudhuin et al. as described previously . Before filtration on HA 0.45-μm filters ( Millipore Corp. ), membrane fractions were fixed at 4°C overnight with an equal volume of 5% glutaraldehyde in 100 mM sodium cacodylate buffer at pH 7.4. Post-fixation was effected either with reduced OsO 4 (equal volumes of 4% OsO 4 , 4% potassium ferrocyanide) or for visualization of coats with tannic acid followed by uranyl acetate staining . Stereology was performed as described by Rabouille et al. for the estimation of both cisternal stacking, as well as cisternal lengths. For this analysis, a cisterna was defined as flattened if its length was at least twice its width. Furthermore, flattened cisternae were considered as stacked if the intercisternal space was less than the width of the cisternal lumen over at least 50% of the cisternal length. Flattened cisternal lengths were determined by tracing their length along the central axis of the sectioned lumen. Several Golgi fractions with varying degrees of structural integrity were prepared using selected buffer conditions, as well as homogenization and centrifugation procedures. The Gi fraction was floated from the high-speed microsomal pellet of hepatic extracts prepared by vigorous homogenization in imidazole buffer . Electron microscopy of membrane samples recovered by a filtration method that ensures random sampling verified that this fraction consisted largely of single Golgi cisternae . Direct floatation from total membrane extracts prepared by vigorous homogenization but in a less disruptive buffer (Tris-HCl containing Mg 2+ , K + , Ca 2+ ) yielded a fraction that contained a mixture of stacked and unstacked Golgi cisternae . Finally, a fraction consisting of long Golgi cisternae in a mostly stacked configuration, termed WNG, was isolated after gentler homogenization of liver and floatation from extracts enriched in large structures after their selection by low-speed centrifugation . Tannic acid staining of samples of this WNG fraction further revealed a large number of coated structures . Coated structures were not evident in the other fractions (data not shown). The buds in the WNG fraction were generally larger than expected and the intercisternal space considerably narrowed, presumably as a consequence of the staining protocol. Similar results were reported by Orci et al. in their initial description of COP I–coated vesicles (vesicle diameter/intralumenal space of 3:1). Biochemical characterization of the three preparations established that, despite similar yields, the WNG protocol resulted in significantly greater enrichment of the Golgi markers galactosyl transferase (GalT) and N -acetylglucosaminyl transferase (NAGT) (Table I ). Furthermore, endosomes, a frequent contaminant of hepatic Golgi fractions, were markedly reduced in the WNG fraction. The highly stacked membranes recovered in the WNG preparation should be representative of the cellular organelle since the yield of the homogenate galactosyl transferase activity (14.5 ± 1.6% (mean ± SD; n = 7)) was similar to that of the Gi and GE fractions (data not shown). The difference in size of the Golgi components containing the enzyme marker was confirmed by sequential differential centrifugation . This sedimentation analysis demonstrated that the WNG fraction was the largest with all GalT activity sedimenting completely even under centrifugation conditions as low as 15,700 g · min. By contrast, the GalT marker in the Gi fraction remained completely in the supernatant at 33,680 g · min, and 1.68 × 10 6 g · min were required to pellet 90% of the GalT activity. Previous studies had established that rat liver Golgi preparations can be substituted for the standard Golgi fraction isolated from wild-type CHO cells as a source of NAGT I in the Golgi transport assay . The three morphologically distinct hepatic Golgi fractions could therefore be compared as a source of NAGT1 for transfer to VSV-G–containing Golgi fractions isolated from a mutant CHO line lacking that activity . The three hepatic fractions displayed marked differences in the Golgi cell-free transport assay , which appeared inversely related to their size as determined by the sedimentation analyses of Fig. 3 . The WNG fraction, despite its abundant budding machinery, displayed very low ability to provide NAGT I in the transport-coupled glycosylation assay. In contrast, the unstacked Gi fraction had very high transport activity while the GE fraction displayed an activity similar to the standard acceptor Golgi fraction isolated from wild-type CHO cells . Note that all assays were performed under linear conditions of transport where neither substrate availability nor enzyme activity were rate limiting. The differences in transport activity did not result from differential enrichment in Golgi membranes since the inactive WNG fraction showed the highest enrichment in the Golgi marker GalT and NAGT I (Table I ). Similarly, the lack of transport activity by the WNG fraction did not result from lack of endogenous substrate accessibility; endogenous glycosylation assays , recently renamed “freeze-frame” glycosylation , established that all three Golgi fractions had a similar ability to transport UDP-[ 3 H]-GlcNAc intraluminally and transfer the sugar to endogenous glycoprotein acceptors using endogenous NAGT I . The lower activity of the WNG fractions could have been caused by a diffusible inhibitor absent in the other fractions or by the lack and/or consumption of an essential diffusible transport factor. This clearly was not the case, however, since addition of an eight-fold excess of WNG Golgi fraction on a protein basis (or ∼15-fold excess by GalT activity) had little effect on the extent of transport, measured with constant amounts (0.25 μg protein) of the most active acceptor Gi fraction . The results of this mixing experiment also eliminated endosomal membranes in the Gi and GE fractions (greatly diminished in the purer WNG fractions, see Table I ) as responsible for the higher activity of these preparations since endosomes provided by the Gi fraction did not stimulate the activity of WNG membranes. This was a relevant consideration since COP I coatomer proteins also associate with endosomes , thereby potentially complicating in vitro fusion assays. Finally, if the cis-Golgi network (CGN) contributed to the transport reaction, lower activity could have resulted from selective loss of this compartment during WNG preparation. The Western blot analysis presented in Fig. 4 D demonstrated that this was not the case since p58, a marker of the CGN and the intermediate compartment , was observed in both Gi and WNG fractions. Furthermore, the cis-Golgi network integral membrane proteins of the p24 family are highly abundant in the WNG fraction . We concluded from these experiments that the explanation for lower activity of the WNG membranes lay elsewhere, possibly in its structural integrity and/or associated proteins. Previous work has established that addition of GTPγS causes acceptor inactivation, possibly as a consequence of the recruitment of excess coatomer . The abundance of coated structures on WNG membranes described above suggested an examination of the relative ARF and coatomer content of our Golgi fractions. Western blot analysis revealed that WNG membranes contained a high amount of associated β-COP and ARF1 relative to the other hepatic Golgi fractions or to Golgi-enriched membranes obtained from CHO cells . Several experiments were performed to test the relationship, if any, between the high coatomer content of WNG membranes and their low transport activity. Brefeldin A (BFA) is a small fungal metabolite that interferes with recruitment of COP I coatomer on Golgi membranes and can thus be used to assess the putative role of COP I coatomer in the lower activity of WNG fractions. Fig. 5 C shows that incubation of CHO Golgi membranes in cytosol with or without additional GTPγS dramatically increased β-COP association, but that the WNG fraction appeared nearly saturated with β-COP and showed limited increase after incubations with cytosol. Under these conditions, BFA eliminated β-COP binding to the CHO Golgi fractions and reduced β-COP association with the WNG fraction by nearly 60% . Despite these clear effects of BFA on β-COP association, the drug had a negligible impact on transport reactions containing the inactive WNG fraction . Similarly, BFA did not affect the cell-free transport when NAGT I was provided by Gi or GE fractions (not shown); each Golgi fraction appeared fixed at its relative ability to serve as enzyme source, independently of BFA addition. We conclude that BFA cannot reactivate the inactive WNG fraction. KCl treatment has also been found to remove quantitatively β-COP from membrane fractions and was therefore chosen as an alternative to BFA to pursue this question. Incubation with KCl led to the complete removal of β-COP from the WNG fraction . However, complete removal of coatomer did not rescue transport activity since KCl-treated membranes (WNGKCl) displayed no enhanced acceptor activity . Experiments carried out in parallel established that KCl treatment of Gi membranes had no effect on their transport activity (data not shown). These experiments clearly established that the lower activity of the WNG fraction did not result from its elevated COP I coatomer content. To test whether simply increasing the amount of accessible membrane by Golgi cisternal unstacking or fragmentation would influence the cell-free transport assay, we developed methods to unstack and disrupt the WNG Golgi membrane fraction. The extent of disruption achieved after various treatments was assessed by differential centrifugation (1,570 g max ) under conditions where intact stacks sedimented, and disrupted ones did not . The distribution of Golgi membranes between pellets and supernatants was measured using GalT as a marker. Harsh homogenization of the parent low-speed pellet of the WNG fraction revealed a buffer-dependent effect on the release of membrane-bound GalT activity to the supernatant. When the buffer used for rehomogenization was the same sucrose–Tris-MgCl 2 buffer used in the initial liver homogenization protocol to prepare the WNG fraction, GalT activity sedimented at low speed . However, changing the homogenization buffer to sucrose-imidazole, the buffer used to isolate the Gi fraction, led to liberation of most of the GalT activity in the low-speed supernatant . Homogenization was only effective when the crude low-speed pellet was used (data not shown). However, organelle fragmentation within the low speed pellet was likely selective for Golgi components since the marked differences in distribution of GalT activity under these conditions were not reflected in the proportion of total protein sedimenting at low speed . Comparison of Golgi fractions isolated by floatation from parent low-speed pellets rehomogenized in the two different buffers revealed marked differences in their activity as Golgi acceptors. The WNGdis fraction obtained after disruption in imidazole displayed a 3.2-fold higher transport activity than measured with the control WNG fraction . Quantitative evaluation of cisternal unstacking by electron microscopy now revealed a majority of single short cisternae in the WNGdis fraction . These experiments demonstrated that disruption of stacked Golgi membranes leads to their activation for transport. Further experiments aimed at separating the effects of cisternal unstacking and fragmentation revealed that unstacking was not sufficient for enhanced transport. As shown in Fig. 7 , treatment of a WNG fraction in sucrose-imidazole without rehomogenization led to partial cisternal unstacking . This did not result in enhanced transport . We therefore concluded that the buffer, but not homogenization, was responsible for cisternal partial unstacking, and that increasing the surface area of cisternal membrane by partial unstacking was not sufficient to enhance transport activity. This result is noteworthy since it suggests an additional approach to characterize some of the molecular interactions leading to the characteristic stacking of Golgi membranes. To estimate the extent of Golgi fragmentation, we determined the average cisternal lengths in our various Golgi fractions using methods developed by Rabouille et al. . This analysis confirmed that Golgi fractions obtained after more disruptive homogenization protocols resulted in shorter cisternae . Using the fraction of cisternae with lengths <0.5 μm as a comparative measure, we found a statistically significant correlation ( r = 0.91) with acceptor activity . This suggests that activation may have resulted from either the release of tethered transport intermediates and/or the fragmentation of cisternae and anastomosing fenestrated elements on the rims of Golgi stacks or intercisternal continuities. Electron microscopy of filtered VSV-G containing CHO Golgi fractions revealed short cisternae that were also unstacked . As shown in Fig. 8 A, these CHO Golgi fractions had cisternal lengths comparable in size to those of the rat liver GE fraction, but shorter than the most intact WNG fraction. This observation is in general agreement with the relatively high activity of the CHO membrane preparation when used as source of NAGT I in the Golgi transport assay . The previous analysis established a correlation between appearance of cisternal fragments after harsh homogenization and transport activity, but did not identify the active membranes. To characterize the fusogenically active component in disrupted Golgi fractions and gain insight into the mechanism of activation, we designed sucrose gradients and centrifugation conditions based on data in Fig. 3 that could readily resolve by size small Golgi fragments from partially stacked membranes remaining in the WNGdis fraction (see Materials and Methods for details). By rate-zonal centrifugation , the bulk of proteins present in the WNGdis fraction cosedimented with GalT and NAGT I activity as a major peak in fraction 6. In contrast, transport (fusogenic) activity was partially resolved from the peak of GalT and NAGT I activities. Note again that all transport assays were carried out under nonsaturating conditions where signal was proportional to the amount of membrane protein added. Although some transport activity was detected throughout the gradient of Fig. 9 , even into fraction 8, the bulk of fusogenic activity was recovered in fractions 1–5. The relatively higher transport activity of structures sedimenting in early fractions is particularly evident when expressed as specific activity relative to protein content . Electron microscopy of filtered samples from each fraction confirmed the progressive increase in size from fractions 1 to 8 . Recognizable stacked Golgi cisternae were restricted to fractions 6 and higher, coinciding with the distribution of the bulk of NAGT I and GalT activity . Vesicular profiles were found throughout the gradient , but were only a minority of the pleomorphic structures evident in fractions 1–5 (transport active). Of note were the apparent continuities observed between Golgi cisternae, as well as between cisternal and vacuolar distensions in transport-attenuated regions of the gradient. Since the greater activity of membranes in early fractions could result from their specific SNARE content , we measured the distribution of three SNAREs known to localize to the Golgi complex of mammalian cells: GS15 , GS28 , and Vti-rp2 . Western blotting of fractions from a typical gradient identified all three v-SNARES in the transport active zone of the sucrose gradient . GS15 was particularly enriched in active fractions, but could not be reliably quantified because it was detectable only by chemiluminescence. Quantitation of 125 I-labeled secondary antibodies with a Phosphorimager confirmed the high concentration of the other two SNAREs, especially GS28, in transport active fractions . In conclusion, a GS15-enriched subcompartment of fragmented Golgi apparatus contained a minority of Golgi resident enzyme. This compartment, only releasable by physical fragmentation, appears responsible for the ability of this Golgi fraction to provide NAGT I in the cell-free transport assay. A combination of biochemical subcellular fractionation and morphological studies revealed an unexpected relationship between the extent of structural integrity of Golgi membranes and their ability to participate in fusion reactions leading to protein transport. These studies led to the identification of an active fusogenic fraction of defined SNARE content that can be released from inactive stacked preparations by vigorous homogenization in imidazole buffers. This work took advantage of a novel procedure for the isolation of structurally intact hepatic Golgi fractions with greatly reduced endosomal contamination. This method exploited the significant size differences between these compartments by first using very gentle homogenization procedures to maintain the structural integrity of large Golgi complexes, followed by a low-speed centrifugation step to eliminate smaller endosomes. Golgi complexes were then resolved from ER, mitochondria, nuclei, and large plasmalemmal sheets (major contaminants of the parent pellet as identified by electron microscopy; Fazel, A., and J.J.M. Bergeron, unpublished observations) by density gradient floatation; the low density of hepatic Golgi complexes resulting from their enrichment in lipoprotein particles facilitated this step. Morphological analysis of the resultant Golgi fraction revealed lipoprotein-filled stacked cisternae enriched in coated structures. Remarkably, cisternal lengths were much larger than those usually obtained, with 50% of the cisternae exceeding 0.5 μm and 20% exceeding 1.0 μm in length. Furthermore, in contrast to other Golgi fractions, >90% of the Golgi complexes were represented by stacked cisternae. Biochemical characterization revealed a marked reduction in endosomal contamination and a high enrichment in galactosyltransferase, ARF, and β-COP relative to hepatic or CHO Golgi fractions isolated by standard protocols. Because the yields achieved with this novel method are very similar to those obtained with standard methods, we conclude that WNG fractions do not represent an unusual subpopulation, but rather are highly representative of Golgi membranes in vivo. Love et al. previously established that the intra-Golgi cell-free transport assay measures transfer of NAGT I into VSV-G–containing cisternae (as opposed to vesicular transfer of VSV-G into NAGT I–containing cisternae), and proposed that this transfer involved small COP I vesicles. We therefore expected that highly stacked WT Golgi fractions with their elevated ARF and coatomer content would have the highest activity in the transport assay because they have abundant machineries for vesicle budding. Unexpectedly, the WNG fraction was largely inactive as a source of NAGT I in this assay. This was in contrast to the greater transport activity of the more fragmented but demonstrably less pure fractions derived by standard procedures from wild-type CHO cells, or rat liver homogenates (Gi, GE fractions). Several experiments ruled out trivial explanations for this lack of activity. Mixing experiments established that the low activity of the WNG fraction did not result from the presence of diffusible inhibitory factors, or the lack and/or consumption of diffusible limiting transport factors. The WNG fraction was not transport defective because it lacked cis-Golgi elements since p58, a marker of the intermediate compartment and cis-Golgi network , was present at similar levels in Gi and WNG fractions. Moreover, lack of activity was not caused by overcoating of membrane because treatments that reduced (BFA) or eliminated (KCl wash) nearly all traces of COP I stably associated with WNG fractions did not increase the activity of stacked membranes in the assay. Finally, a budding assay confirmed that the WNG fraction was not biochemically inert since it could sort cargo and generate secretory vesicles (D. Shields and J.J.M. Bergeron, unpublished observations). We therefore conclude that the low activity of the WNG fraction in the Golgi transport assay is not an experimental artifact and instead reflects a defining feature of the cell-free assay relevant to the mechanism of cargo transport in vivo. Further analysis revealed that, whereas unstacking or removal of coatomer from WNG fractions had little effect on transport activity, homogenization in imidazole-containing buffers readily led to their activation for transport. This activation could have resulted either from the release of tethered transport intermediates and/or fragmentation of fusogenic elements of cisternae, fenestrae, or intercisternal continuities. In the first case, the low activity of the WNG fraction would have resulted from a cytoskeletal matrix that restricted diffusion of transport intermediates but could be dislodged by homogenization. Recent tethering models for postulated intra-Golgi transport intermediates , as well as the demonstration of spectrin as part of a Golgi-associated cytoskeleton that includes microtubules and myosin , provide likely candidates for this matrix. However, several observations suggested that such an explanation is unlikely. For example, neither washing in 0.3 M KCl (to remove matrices) or prior incubation at 4°C to depolymerize microtubules had any influence on the activity of WNG fractions in the cell-free transport assay. Furthermore, incubations in sucrose imidazole that did lead to cisternal unstacking and therefore liberation of a matrix did not enhance the transport activity of the WNG fraction. More likely, our observations indicate that small Golgi-derived fragments play an important role as source of NAGT I in the assay, a possibility first suggested by the clear inverse correlation between average cisternal length and transport activity of various Golgi fractions . The demonstration, using rate-zonal centrifugation, that transport activity of a WNG fraction activated by homogenization in imidazole could be well resolved from the bulk of protein and sedimented less rapidly than more intact Golgi stacks and the peak of GalT or NAGT activities confirmed the importance of these fragments. . Our results are in agreement with those of Love et al. , who reported similar separation of a transport-active Golgi subfraction by velocity centrifugation from the bulk of larger (and less active) Golgi elements. Interestingly, the additional characterization of the transport active zone of our velocity gradients revealed a unique pattern of v-SNARES, especially GS15 and GS28, that could be relevant to their function in vivo. Antibodies to one of these, GS28, inhibited the same Golgi cell-free transport assay as used here , although a role in ER-to-Golgi transport has also been proposed . The role of GS15 and Vti-rp2 in the cell-free transport assay remain to be elucidated. The exact nature of the fusogenic components in our fragmented Golgi fractions or the budded fraction of Love et al. remains unknown, but ongoing purification to morphological homogeneity should settle the morphological identity of the structures. Previous work by Happe and Weidman established that the Golgi transport assay used here reconstitutes primarily heterotypic transfer between medial NAGT I–containing Golgi compartments and an earlier (cis/ERGIC-located) VSV-G–containing one. Our studies are therefore unlikely to be explained by a homotypic fusion model akin to the reassembly of Golgi fragments after mitotic disassembly or pharmacological disassembly via ilimaquinone . Our homogenization protocol to release active fragments produced membranes of the same size and type as those normally recovered by the standard isolation procedures used by Happe and Weidman and others. In contrast, generation of mitotic kinases and IQ-induced fragments likely involves changes in membrane composition and/or properties that would not be reproduced by the simple physical forces used here. Several recent studies that established that the Golgi apparatus is a dynamic organelle undergoing constant remodeling actually predict the fusogenic fragments identified in our studies. Labeling of Golgi structures in living cells with a fluorescent sphingolipid precursor first revealed that the trans-Golgi elements of separate stacks readily form and break tubular interconnections . Time-lapse photography of cells expressing green fluorescent protein–Golgi protein chimeras extended these studies and demonstrated dynamic extension/retraction of tubules that sometimes initiated novel and thicker connections between adjacent Golgi elements . These Golgi remodeling events have been proposed to contribute to Golgi traffic and help maintain the distribution of Golgi resident enzymes during rapid anterograde transport of secretory cargo through the organelle in vivo. Such observations suggest dynamic assembly of fusion sites such as uncoated bud tips and tubules readily observed by freeze-etch electron microscopy at the rims of cisternae that could be preferentially recovered in a subset of the Golgi fragments generated during homogenization. The transport active fragments identified here and in the studies of Love et al. could therefore represent intra-Golgi intermediates involved in maintaining the distribution of Golgi resident enzymes during rapid anterograde transport of secretory cargo through the organelle in vivo. Interestingly, Sciaky et al. also observed the retrograde transport of a small proportion of galactosyltransferase–green fluorescent protein chimeras to the ER. This latter event may very well be COP I dependent and could account for the observation of Love et al. that a small proportion of vesicular NAGT I can be generated from parent Golgi membranes in an ARF, COP I coat-omer-dependent fashion. However, such structures would only be a minor proportion of transport-active components in the cell-free transport assay and represent that proportion of Golgi resident enzyme recycling to the ER rather than to early Golgi compartments. A maturation model for secretory protein transport through the Golgi complex, with possible transient tubular connections between fusogenic elements to allow resident glycosyl transferase redistribution accommodates more readily the available data on the morphological characteristics and mechanisms of secretory cargo and resident enzyme dynamics in the Golgi complex from yeast to mammalian cells . The complete resolution of the enzymology of the Golgi cell-free transport assay and the precise identity of the fusogenic subcompartment identified here and in the studies of Love et al. should help define the molecular mechanisms and morphological framework for traffic within the secretory pathway . | Study | biomedical | en | 0.999996 |
10330399 | T84 colonic intestinal epithelial cells were maintained in culture medium (1:1 of Dulbecco-Vogt modified Eagle's medium and Ham's F12 ( GIBCO BRL ) supplemented with 10% newborn calf serum, 10 mM Hepes buffer, 40 μg/ml penicillin, and 90 μg/ml streptomycin. Polarized epithelial cell monolayers were prepared by seeding 5 × 10 5 cells on Transwell-Clear polyester membranes (3-μm pore size, 6.5-mm-diam Costar inserts; Costar Corp.). Integrity of tight junctions was monitored by transepithelial resistance to passive ion flow using a dual voltage ohmmeter (Millicell-ERS; Millipore Corp. ). Day 5 monolayers with transepithelial resistance of 1,500–2,000 Ω/cm 2 were used in this study. LPS was extracted and purified from invasive S . flexneri M90T according to Westphal . Lyophilized LPS was resuspended in tissue culture medium to the indicated concentration and sonicated lightly before use. Iron-saturated holo-transferrin, fatty acid-free BSA fraction V, FITC, and TRITC were obtained from Sigma Chemical Co. Transferrin-FITC, BSA-FITC, and BSA-TRITC were prepared essentially as described by Goding . Anti–CI-MPR (cation-independent mannose 6-phosphate receptor) antibody was a gift of Dr. Bernard Hoflack (IBL, Lille, France). Anti–Lamp 2 (lysosomal-associated membrane protein) (CD3) antibody was a gift of Dr. Minoru Fukuda (La Jolla Cancer Foundation, La Jolla, CA). Anti-canine rab7 antibody was raised against a synthetic peptide corresponding to the COOH terminus of the protein (residues 172–204) and covalently coupled to keyhole limpet hemocyanin. Antibodies to S . flexneri -5a LPS were a kind gift of Dr. Armelle Phalipon (Institut Pasteur, Paris, France). To quantitate LPS recycling at the apical surface in addition to transcytosis, 2 μg/ml of LPS was added to the apical pole and cells were incubated for 2 h at 37°C. The filters were then placed on ice and the apical and basal surfaces were washed extensively with cold culture medium to remove extracellular LPS. LPS was quantitated by ELISA (sensitive to 1 ng/ml) and confirmed by modification of the chromogenic limulus amebocyte lysate assay (LAL; sensitive to 10 pg/ml; Biowhittaker Inc.). The final wash revealed no LPS by ELISA (negligible by LAL assay). Prewarmed LPS-free medium was then added to both reservoirs and monolayers were incubated at 37°C. The integrity of intercellular tight junctions was maintained after LPS treatment as confirmed by transepithelial resistance and absence of paracellular passage of 3 H-mannitol. At the times indicated, the cells were rapidly cooled on ice, apical and basal supernatants were collected. Monolayers were subject to three cycles of freeze/thaw followed by light sonication. Corresponding LPS levels in these fractions were quantitated as described above. For initial description of LPS transcytosis, purified LPS was conjugated to 10 nm colloidal gold particles as previously described . Gold complexes were stabilized with 2% polyethylene-glycol (average mol wt = 6,000–8,000). LPS-gold was added to the apical side of polarized T84 cells for 2 to 6 h. Monolayers were then fixed with 1.6% glutaraldehyde in 0.1 M phosphate buffer for 1 h, post-fixed with 2% osmium tetroxide for 1 h, and embedded in Epon-Araldite. Thin sections were obtained, conventionally stained and observed with a Philips CM12 electron microscope. Subsequent analysis of LPS transcytosis was performed using differential immunogold labeling of LPS and of the transferrin receptor. LPS was added to the apical side of polarized T84 cells at a concentration of 25 μg/ml for 2 h. Cells attached on filters were fixed with 3% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 1 h at room temperature. Free aldehydes were quenched with PBS and 50 mM NH 4 Cl for 15 min. Cells were dehydrated with an increasing ethanol series at 4°C and infiltrated in LR White. Blocks were polymerized at 50°C for 24 h . Thin sections were incubated in drops of the following solutions: 10 min in PBS, 1% BSA, 1% NGS (normal goat serum), 0.2% Tween 20; then 10 min in a solution containing a rabbit immune serum against S . flexneri serotype 5a antigen at a 1/100 dilution and a mouse monoclonal antibody (H 68.4) directed against the human transferrin receptor (ZYMED Laboratories Inc.) at a dilution of 1/50 in PBS, BSA 0.1%; then 5 min in a solution containing an anti–rabbit IgG, gold-conjugated (10 nm beads) serum (British Biocell International) and an anti– mouse IgG (H+L) gold-conjugated (5-nm beads) serum (British Biocell International), both diluted 1/20 in PBS, 0.01% gelatin ( Sigma Chemical Co. ). Two 5-min washes were then performed in PBS and one 5-min wash was performed in PBS, 1% glutaraldehyde. Thin sections were then washed thoroughly with distilled water and counterstained with 1% uranyl-acetate (5 min). Observations were made at 60 kV with a Philips CM12 electron microscope. For uptake experiments, polarized cells were briefly rinsed with prewarmed culture medium. LPS (2 μg/ml) or BSA-TRITC (10 mg/ml) was added to the apical side of the epithelium for 20 min at 37°C. For analysis of longer internalization times, cells were further incubated in LPS- or BSA-free medium for an additional 40 min or 1 h and 40 min, corresponding to 1 and 2 h total internalization time, respectively. For basolateral uptake, transferrin-FITC (30 μg/ml) or BSA-FITC (10 mg/ml) was added simultaneously to the opposing basolateral pole of the polarized epithelium for the final 10 min of internalization. Apical and basolateral sides of cell monolayers were carefully washed with PBS containing 0.2% BSA at 4°C. After a final wash with PBS, cells were fixed for 30 min at room temperature in 3.7% paraformaldehyde in PBS, pH 7.4. Filters were then rapidly washed in PBS, incubated in 50 mM NH 4 Cl in PBS for 15 min and either directly mounted on coverslips in PBS containing 50% glycerol or further processed for immunofluorescence. For this purpose, cells were permeabilized with two rapid washes of PBS and 0.1% saponin and incubated for 30 min with primary antibodies in PBS and 10% horse serum and 0.1% saponin. Filters were extensively washed with PBS and 0.1% saponin, incubated for 30 min with secondary antibodies, washed, mounted on coverslips and viewed under a Leica TCS 4DA confocal microscope. Series of two plane sections of 1 μm thickness were monitored. For double-staining experiments, identical optical sections are presented. Superimposed images were treated with a pseudocolor scale. Colocalized structures are seen in a yellow color. A new multifluorescence image cytometry approach was used to estimate the proportion of vesicles containing internalized LPS and marked with antibodies raised against different intracellular markers. To identify all labeled vesicles, the high spatial frequency noise was removed from digital confocal images using a Gaussian convolution filter (SD of 2 pixels) followed by a top-hat based subtraction (radius of 9 pixels) and a grey level segmentation (20% of maximum intensity). To provide a statistical analysis of attributes of identified objects (size, fluorescence densities in channels FL1 and FL2), image cytometry file were created, stored in flow cytometry standard (FCS) and analyzed using CellQuest software ( Becton Dickinson ). For all objects, we determined the percentage of LPS-containing vesicles that were positive for the other marker on 2D-scatterplots representing the fluorescence FL1 vs FL2. Previous studies have shown that when 0.8 μg/ml of LPS, a concentration equivalent to the amount of LPS spontaneously released by 10 8 S . flexneri , is present at the apical surface of polarized T84 cells, LPS retaining endotoxic activity is recovered at the basolateral side of the epithelium . This process occurred in monolayers with properly established intercellular tight junctions, indicating that LPS was directed basolaterally via a transcellular route. To analyze the kinetics and quantify transcellular movements of LPS, LPS at a concentration of 2 μg/ml was added to the apical pole of polarized T84 cells for 2 h at 37°C. Monolayers were then washed extensively at 4°C to remove extracellular LPS and then reincubated at 37°C in LPS-free medium for the indicated times. As shown in Fig. 1 , 119 ± 17.8 ng of LPS was associated with the epithelial cell monolayer after the 2-h loading period. With subsequent culture in LPS-free medium, LPS was transcytosed to the basolateral pole, however, the bulk of LPS was released at the apical surface of the polarized monolayer. After 1 h, 17.7 ± 3.6 ng of LPS was released basolaterally corresponding to 15% of the total LPS initially loaded . This level is sufficient to stimulate clinically relevant biological activities. 45.4 ± 7.5 ng of LPS, equivalent to 38% of the initial cell-associated LPS, was detected in the apical reservoir. Apical LPS may correspond to aggregated LPS at the apical surface which was subsequently released without internalization. However, microscopic analyses revealed that under these conditions cell-associated LPS was primarily intracellular with a minute amount remaining membrane-associated. These results therefore suggest that after internalization, the LPS was recycled back to the apical pole, as well as transcytosed basolaterally. In addition, the total amount of detectable LPS preloaded into the epithelial cells declined by 38% after 30 min of subsequent culture in LPS-free medium. This loss of LPS, as detected by ELISA and the LAL assay, may reflect LPS transport and possible degradation in the lysosomal compartments. For conceptual purposes, the route of LPS movement through the polarized epithelium was analyzed by transmission electron microscopy. LPS-coated colloidal gold particles were placed on the apical side of polarized T84 cells. Analysis of LPS association with the epithelium revealed LPS within coated pits and coated vesicles at the apical membrane suggestive of uptake by receptor-mediated endocytosis . Intracellularly, LPS concentrated within apical multimembranous bodies immediately below the brush border . LPS was rarely observed in the perinuclear region and labeled particles were not present in the basolateral region below the nucleus. Because the intactness of tight junctions was maintained as monitored by electrical resistance and restriction of mannitol passage, the lack of any labeling in the basolateral region implicated the paracellular space below the tight junctions as a possible target for LPS transcytosis. Supporting this premise, labeled vesicular structures in close proximity of the paracellular space were observed with apparent exocytosis of LPS-coated particles into the paracellular space . To analyze the pathway traversed by apically internalized LPS and its mode of access to the basolateral medium, confocal microscopy and transmission electron microscopy were employed. As observed in confocal microscopy, the height of the polarized T84 cells was ∼30 μm. Three representative planes in each focal series were selected to analyze the distribution of LPS over the entire cell height. The three focal planes are illustrated in Fig. 4 A, scanning the cells through the apical, medial, and basolateral region of the polarized T84 cells. The basolateral surface of polarized T84 cells extends up to the paracellular tight junctions, ∼0.5 μm from the apical plasma membrane. Because ultrastructural analysis revealed that internalized LPS was confined to vesicles in the apical region, the accessibility of LPS to basolateral endosomes was analyzed. LPS was internalized from the apical side for 20 min. Transferrin receptors are located on the basolateral membrane of differentiated cells , therefore transferrin, a protein which is endocytosed and recycled at the basolateral surface, was subsequently added to the opposing basolateral pole for the last 10 min of internalization serving as a marker for basolateral early endosomal compartments. As shown in Fig. 4 A, transferrin was present in all focal planes. Consistent with ultrastructural analysis, LPS was evident only in the apical region. Superimposed images revealed striking colocalization of LPS to transferrin positive vesicles. Analysis of confocal micrographs by image cytometry indicated that >80% of LPS-containing vesicles also contain transferrin (Table I ). These results suggest that LPS trafficks to basolateral endosomes and may access the basolateral side of the epithelium via exocytosis into the paracellular space beneath the tight junctions. Transmission electron microscopy confirms confocal images, showing vesicles with internal multimembranous structures containing both apically internalized LPS (gold size 10 nm) and the transferrin receptor (gold size 5 nm), indicating that LPS has undergone a transcytotic process from the apical to the basolateral domain of T84 cells . These vesicles are essentially located at the upper part of T84 cells, close to the lateral membrane. To further define the basolateral transferrin compartment that was accessible to LPS, the fluid-phase marker BSA was analyzed. BSA was added to the apical surface of cells for 20 min and transferrin was subsequently added basolaterally for the final 10 min of internalization. As observed with LPS, apically internalized BSA was present only in the apical plane of T84 cells . In contrast, BSA failed to colocalize with basolaterally internalized transferrin. In addition, when LPS was added apically and the fluid-phase marker added to the opposing basal pole, a meager level of colocalization was observed . Thus within the first 20 min of internalization, LPS was sorted to an endosomal compartment accessible to basolaterally internalized transferrin and from which fluid-phase material was essentially excluded. To determine its subsequent fate, LPS was internalized apically for 20 min followed by a 40-min chase with LPS-free medium. As shown in Fig. 4 B, LPS remained in the apical plane with slight levels moving to the medial perinuclear region (not shown). At this internalization time, most of LPS no longer colocalized with basolaterally loaded transferrin. The distribution of LPS distinct from transferrin-containing compartments was even more evident after 2 h of internalization (20 min LPS internalization followed by 1 h 40 min chase) and only 10% of LPS-positive structures were shown to contain transferrin (Table I ). These results could indicate that LPS is exocytosed at the lateral surface remaining trapped within the paracellular space. Despite the likelihood of this possibility based on the kinetic analyses described above, and electron microscopy revealing exocytosis of LPS into the paracellular region , the punctate pattern of LPS labeling suggests that extended internalization of LPS results in the sorting of this molecule to vesicles independent of transferrin labeling. To further characterize compartments to which LPS is transported, markers of the endocytic network were incorporated into this study. When internalized apically for 20 min, LPS failed to colocalize with the late endosomal marker CI-MPR . However, a longer internalization of 1 h (20 min LPS internalization followed by 40-min chase) revealed dramatic colocalization of LPS with the CI-MPR, a well accepted marker for late endosomal compartments, in the apical region of these cells. Extension of LPS internalization to 2 h (20 min with 1-h 40-min chase) resulted in very low colocalization between these molecules. In addition, analysis of colocalization with rab 7 revealed a similar pattern of trafficking to late endosomes (not shown). Access of LPS to lysosomes was analyzed by colocalization studies with the lysosomal glycoprotein Lamp 2. Using the same time frame of internalization, LPS access to Lamp-containing vesicles was evident after 1 h, with striking localization of LPS to lysosomes after 2 h of LPS internalization . These results show that LPS is transiently associated with late endosomes and accumulates in lysosomes. It also indicates that the loss of detectable cell-associated LPS after internalization as shown in Fig. 1 , is likely a consequence of LPS processing in lysosomal compartments. The polarized intestinal epithelium possesses two functionally distinct plasma membrane domains that intercede opposing forces. The apical surface counters microbial threats and the presence of numerous commensals, being bathed in bacterial products such as LPS which has the capacity to stimulate proinflammatory immune responses. The basolateral surface interfaces the underlying immune system serving as a substrate for resident and emigrating cells that provide mucosal protection. Signaling between the apical and basolateral domains may occur in response to microbial-induced upregulation of cytokines or ligands of the epithelial cells allowing for emigration and enhanced interaction of immune cells at the basal pole. In addition, interaction between the two domains may occur via vectorial delivery of molecules across the polarized monolayer by transcytosis, a multistep vesicular transport pathway . Transcytosis reflects a dynamic role of the intestinal epithelium in attuning these opposing forces. After internalization at the apical or basolateral domain, much of the endocytosed fluid, macromolecules, and membrane components are recycled to the original plasma membrane surface . Endocytosed material may be sorted in early endosomes to late endosomes and lysosomes, or to transcytotic vesicles for delivery to the opposing surface. How molecules move from one early endocytic compartment to the opposite cell surface remains unclear. Numerous studies suggest that the apical and basolateral endocytic pathways interconnect at an apically localized transcytotic compartment . Basolaterally internalized ligands accumulate in this apical compartment, accessible to apically internalized membrane markers . Although this compartment can be accessed by basolaterally endocytosed transferrin that recycles back to the basolateral surface , it is thought to be distinct from the basolateral transferrin recycling pathway, as well as basolateral early endosomes and the lysosomal network . LPS released from S . flexneri present on the apical side of polarized epithelial cells was detected in the basolateral milieu under conditions in which the integrity of intercellular tight junctions was maintained . The uptake and intracellular endocytic processing of LPS was analyzed to elucidate the transcytotic pathway. LPS was internalized at the apical surface, clustered into coated pits and subsequently coated vesicles, suggestive of uptake via receptor-mediated endocytosis. Although a receptor for LPS in epithelial and endothelial cells has not been identified, binding studies have implicated the existence of such a receptor . In addition, the structural composition of LPS may provide the means for specific interactions with a variety of membrane components including glycoproteins, phospholipids, and glycosphingolipids . Evidence for LPS entry by receptor-mediated endocytosis provided in this study is purely morphological. Thus identification of an apical/brush border receptor remains a future prospect. The routing of LPS in polarized epithelial cells after apical interaction is summarized in Fig. 7 . Morphological studies identified the intracellular pathways that process LPS as consisting of peripheral compartments located just below the apical surface and in close proximity of the basolateral border directly below the intercellular tight junctions. By preloading endosomal compartments from the basolateral surface with transferrin, it was shown that apically internalized LPS was directly accessible, rapidly entering the same compartment. By intercepting the transferrin recycling pathway, this compartment provides LPS with an avenue to the basolateral side of the epithelium. This compartment was largely inaccessible to fluid-phase markers internalized at either the apical or basolateral pole. Complete colocalization of LPS with transferrin after 20 min of internalization indicated rapid entry of LPS into vesicles containing basolaterally applied transferrin but gave no indication of an intermediate compartment. The LPS transferrin-containing compartment may correspond to the apical transcytotic compartment described in the endocytic network of other cells . The fact that the LPS transcytotic compartment corresponds to large multimembranous bodies suggests that the transcytotic pathway may be related to a maturation process of transcytotic vesicles from apical to basolateral. In contrast to what has been observed in the maturation process of the MHC class II (MIIC) compartment and phagosomes , in which late phagosomes or multilamellar MIIC correspond to lysosomal-like compartments, late stages of transcytotic vesicle maturation would be characterized by the acquisition of molecules involved in the recycling pathway such as the transferrin receptor. The consequence of this specific property is the secretion of LPS in the lateral milieu. After accessing transferrin positive basolateral endosomal compartments, electron microscopic analyses revealed exocytosis of LPS into the paracellular space beneath intercellular tight junctions. In addition, analysis of the fate of this molecule indicated that after apical internalization a significant amount of LPS was recycled back to and released at the apical surface. It is unclear as to whether recycling of LPS occurred from the apical transcytotic compartment or was an event that ensues from a transferrin-negative apical endosome before this compartment. Longer internalization revealed that LPS could be delivered to the late endosomal and lysosomal compartments as shown by colocalization of LPS with CI-MPR, rab 7, and Lamp. In addition, a 38% loss in the total amount of LPS as detected by ELISA and the LAL assay supported the contention that some LPS accessed lysosomal compartments and was processed. However, direct demonstration that LPS that reaches the late lysosomal compartments was actually detoxified and further processed remains to be shown. It is unclear, for instance, if a lysosomal preparation from T84 cells does contain an enzyme equivalent to the acyloxyacyl hydrolase of neutrophils required for detoxification of lipid A . In addition, LPS extracted from lysosomal preparations of cells should be tested against crude LPS for proinflammatory potential. Depending on the cell type analyzed, a number of molecules transcytose polarized cell layers, with this process occurring from the apical to basolateral plasma membrane , or from the basolateral to apical domain . IgA binds to polymeric immunoglobulin receptor at the basolateral surface and is transcytosed to the opposing surface being released in mucosal secretions as a first line of defense against lumenal threats. By inhibition of attachment and invasion, inactivation of toxins, opsonization, and complement activation, this factor of the humoral immune response neutralizes microbes at the apical epithelial surface of mucous membranes. Because IgA directed against a serotype-specific epitope of S . flexneri LPS has been proposed as an important element in the protection against shigellosis , this study has important implications on the role of IgA in intracellular neutralization of Shigella LPS. Analysis of the epithelial cell processing of microbial products may also provide information concerning intracellular interactions with epithelial histocompatibility antigens. Such interactions emphasize the complexity of the epithelium in the immunophysiology of the intestinal mucosa. A question of considerable interest is the potential significance of nondetoxified LPS when allowed to transcytose the intestinal barrier. Transcytosed LPS may serve a physiological, as well as a pathological role. The physiological role of LPS may be restricted to the crypt area of the intestine. The crypt cells may channel a well-controlled quantity of LPS for two major purposes. (a) To maintain a low level of stress on the local innate immune system in order to hasten a nonspecific response to an infectious insult. (b) LPS may participate in intestinal differentiation. Previous studies have shown an inverse correlation between LPS transcytosis and epithelial cell differentiation with the most efficient passage of LPS occurring in cells of a state mimicking the less mature intestinal crypt cells . Expression of a putative apical receptor and/or access to a trafficking route may be arrested as cells differentiate on their way to the tip of the intestinal villi in the small intestine or to the higher part of the Lieberkühn crypts in the colonic and rectal mucosa. LPS may also have a pathological function if it is not properly contained. In inflammatory bowel diseases, initial inflammation is essentially in the intestinal crypts, indicating a potential role of some products of the intestinal flora in this region. In some infectious disease states such as shigellosis, initial inflammation also occurs in the crypts . It is possible that in certain serious conditions, the epithelial barrier to LPS is lifted, thus leading to an influx of LPS into the body with deleterious local and general effects (i.e., shock). The current study reflects the crucial aspect of the intestinal epithelium as a regulated barrier to LPS entry. | Study | biomedical | en | 0.999997 |
10330400 | A transgene construct consisting of the rat liver fatty acid binding protein (FABP) promoter (−596 to +21; kindly provided by Dr. Jeffrey Gordon, Washington University, St. Louis, MO), the full-length human PKC β II cDNA, and the SV40 large T antigen polyadenylation signal sequence was produced by conventional cloning methods. The resulting PKC β II transgene construct was confirmed by direct microsequencing before microinjection. The pFABP/PKC β II transgene construct was propagated in the mammalian expression vector pREP4 and the transgene insert was excised using NheI (5′) and XbaI (3′), purified, and microinjected into C57BL/6J × C3H/HeJ F 2 mouse oocytes as previously described . The microinjections and generation of transgenic founder mice were conducted at the University of Texas Medical Branch Transgenic Mouse Facility. Transgenic founder mice were identified by Southern blot analysis. In brief, genomic tail DNA (5 μg) was digested to completion with Taq I (Roche), resolved by agarose gel electrophoresis, transferred to nylon membrane ( Amersham ), and transgenic DNA detected with a radiolabeled probe corresponding to the SV40 polyadenylation sequence. Three transgenic founder animals were identified from a screen of 120 live births. Transgene copy number was determined for each transgenic line by quantitative Southern blot analysis as previously described . Genotype was confirmed by slot blot analysis using a radiolabeled probe corresponding to the polyadenylation sequences within the transgene . Founder mice were mated with C57BL/6J mice (The Jackson Laboratory ) to establish the transgene on a stable genetic background. Transgenic PKC β II mice and progeny were bred and housed in microisolator cages maintained at constant temperature and humidity on a 12-h on/12-h off light cycle in a pathogen-free barrier facility. Mice were provided a standard autoclavable chow and autoclaved water ad libitum. Total RNA was extracted from tissue samples using a Totally RNA kit (Ambion). Reverse transcription was carried out using 6 μg RNA, 1 μg oligo(dT) primer, 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 U SuperScript II reverse transcriptase ( GIBCO BRL ). Amplification of the transgenic RNA was carried out using 20 ng of the following primers, which amplify human PKC β II but not endogenous mouse PKC β II : forward, 5′ CGTCCTCATTGTCCTC 3′; reverse 5′ GACCTTGGTTCCCTGACTG 3′. An optimized amplification program of denaturation (94°C, 15 s), annealing (56°C, 15 s), and extension (74°C, 45 s) for 40 cycles using PCR Supermix ( GIBCO BRL ) was used. Human brain RNA was used as a positive control; mouse brain RNA and samples incubated without reverse transcriptase served as negative controls. Immunoblot analysis for PKC β II expression in mouse colonic epithelium was performed essentially as previously described . In brief, mice were killed by CO 2 asphyxiation, the colons were isolated and slit open longitudinally and rinsed well with PBS, and the colonic epithelium was scraped using a plastic coverslip. Total cell extracts were prepared in RIPA buffer [50 mM Tris, pH 7.2, 150 mM NaCl, 2 mM EDTA, 0.4 mM EGTA, 20 μM NaF, 0.5% deoxycholate, 1% NP-40, 0.1% SDS, 0.1 mM Na 3 VO 4 , 25 μg/ml aprotinin, 25 μg/ml leupeptin, 25 μg/ml pepstatin, 1 μg/ml soybean trypsin inhibitor, and 34.5 μg/ml 4-(2-aminoethyl) benzene sulfonyl fluoride]. Equal amounts (30 μg) of protein were subjected to immunoblot analysis using an isotype-specific antibody for PKC β II ( Santa Cruz Biotechnology ). Immunohistochemistry was performed using an enhanced biotinyl tyramide system ( New England Nuclear ) on sections from the proximal and distal colon fixed in ethanol, embedded in paraffin, and sectioned (5 μm) as previously described , with the following modifications. After deparaffinization and rehydration of tissues, sections were treated with 3% hydrogen peroxide in methanol to inhibit endogenous peroxidase, blocked with TNB reagent ( Dupont New England Nuclear ), and incubated with polyclonal antibody to PKC β II . Specificity was confirmed using antibody preincubated with excess antigen peptide as previously described . Sections were incubated with biotinylated secondary antibody followed by addition of streptavidin-conjugated peroxidase. Biotinyl tyramide amplification reagent was then added followed by a second streptavidin-peroxidase incubation. Visualization was with DAB chromagen. 12-wk-old mice were killed and their colons were dissected and measured for overall length. The distal colon (1 cm from rectal end) was fixed in 4% paraformaldehyde and processed for histology as described previously . Tissues were embedded in paraffin, sectioned (5-μm thickness) and stained with hematoxylin and eosin. 25 full-length, longitudinally cut crypts from each animal were analyzed for crypt height (micrometer) and number of cells per crypt height. 25 crypts cut on the cross-section at random height were counted to determine the average crypt circumference (in number of cells). These data were used to calculate cell size (crypt height in micrometer/crypt height in cell number) and estimate the total cells per crypt (mean cells per crypt column × mean crypt circumference). Cell proliferation was determined by immunohistochemical detection for proliferating cell nuclear antigen (PCNA) in distal colon sections. Primary antibody against PCNA (PC10 clone; DAKO) was diluted 1:50 in PBS and preincubated with 1:200 biotinylated anti–mouse IgG ( Santa Cruz Biotechnology ) overnight at 4°C. After deparaffinization, sections were processed for antigen retrieval as described by the manufacturer (DAKO), treated with 1% hydrogen peroxide for 10 min to inactivate endogenous peroxidases, and blocked with normal goat serum. The slides were then incubated with the PCNA/anti–mouse IgG antibody conjugate for 60 min at room temperature. Antigen–antibody complexes were detected with avidin and peroxidase–labeled biotin (ABC staining system; Santa Cruz Biotechnology ) and visualized with DAB. Slides were counterstained with hematoxylin to provide contrast. 20 full-length, longitudinally cut crypts were divided into thirds and scored visually for cells staining darkly for PCNA . The labeling index (percent of labeled cells) and proliferative zone (highest cell from the bottom of the crypt staining for PCNA divided by the total cells per crypt height) were calculated for each set of animals. The differentiation status of colonic epithelial cells was measured by detection of the specific binding of three different lectins. After deparaffinization, sections were incubated for 60 min at room temperature in normal goat serum. Three different biotinylated lectins (dolichos biflorus agglutinin [DBA], peanut agglutinin [PNA], and Ulex europaeus-I [UEAI]; Vector Labs.) were diluted to 10 μg/ml in PBS. Sections were incubated with one of the three lectin solutions for 60 min at room temperature. Sections were then washed in three changes of PBS and incubated with 5 μg/ml of rhodamine red-X–conjugated Streptavidin (Jackson Immunoresearch Labs.) in PBS for 30 min at room temperature. After three 5-min washes in PBS, sections were mounted in aqueous media containing 95% glycerol in PBS and analyzed by fluorescence microscopy. Sections were also analyzed histologically by Alcian blue/periodic acid Schiff (PAS) staining for detection of mature, mucin-producing goblet cells. The percentage of cells undergoing apoptosis (apoptotic index) was determined in paraformaldehyde-fixed distal colon tissue by the TdT-mediated dUTP-biotin nick end labeling of fragmented DNA (TUNEL) assay using the apoTACS kit from Trevigen. The tissue sections were counterstained with methyl green. 100 longitudinally cut, full-length crypts were scored for apoptotic cells based on a combination of positive staining and morphological criteria as previously described . 40 (20 transgenic PKC β II mice, 20 nontransgenic littermates) 6–7-wk-old female mice were injected intraperitoneally with azoxymethane (10 mg/kg body wt) or saline weekly for 2 wk as previously described . At 5 and 20 wk after the second injection, five animals per group were killed by CO 2 asphyxiation and the colons were removed. The colons were flushed with PBS to remove fecal pellets, slit open longitudinally, and fixed flat between two pieces of filter paper under a glass plate in 70% ethanol for 24 h. Fixed colons were stained with 0.2% methylene blue in PBS for 5 min before being mounted on a glass slide for observation at low magnification (×40) on a light microscope. Aberrant crypt foci (ACF) were scored blindly by a single observer (A.P. Fields) for total number and multiplicity (number of crypts/focus) using previously defined criteria . Colonic epithelia from transgenic and nontransgenic mice were scraped and equal amounts of protein from total tissue lysates were subjected to immunoblot analysis using a specific β-catenin polyclonal antibody ( Santa Cruz Biotechnology Inc. ) or a specific GSK-3β monoclonal antibody (Transduction Laboratories). For glycogen synthase kinase (GSK)-3β kinase assay, colonic epithelium scrapings were solubilized in lysis buffer [10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 25 μg/ ml pepstatin, 1 μg/ml soybean trypsin inhibitor, 34.5 μg/ml 4-(2-aminoethyl) benzene sulfonyl fluoride, 20 μM NaF, and 0.1 mM Na 3 VO 4 ]. Lysates containing 300 μg of protein were precleared with 75 μl of protein A agarose and then added to 75 μl of protein A agarose beads that had been preincubated with 5 μg of anti–GSK-3β monoclonal antibody (Transduction Labs.). Samples were incubated for 1 h at 4°C, and beads were pelleted and washed once with lysis buffer and once with kinase assay buffer (8 mM MOPS, pH 7.4, 0.2 mM EDTA, 10 mM Mg acetate, and 0.1 mM ATP). The washed and pelleted beads were then resuspended in 40 μl of kinase assay buffer containing 10 μCi [γ 32 P]ATP and 250 μmol of GSK-3β–specific substrate peptide (Upstate Biotechnology, Inc.). Reactions were incubated for 20 min at 25°C and stopped by pelleting the beads and adding the supernatant to 20 μl of 40% trichloroacetic acid. Reactions were spotted on P-81 filters and washed three times in 0.75% phosphoric acid and once with acetone. Incorporated radioactive phosphate was quantitated by Cerenkov counting. Nonspecific and background counts were calculated by performing parallel assays with a nonphosphorylatable GSK-3β substrate peptide. To investigate the role of PKC β II in colonic epithelial cell biology, we generated transgenic mice overexpressing PKC β II in the intestinal epithelium. For this purpose, we used the rat liver FABP promoter, which has been well characterized to target transgene expression to the intestinal epithelium . A schematic diagram of the transgene construct is presented in Fig. 1 A. The FABP promoter (−596 to +21) was fused to the cDNA for human PKC β II and the SV40 poly A signal sequence by conventional cloning. Southern blot analysis of tail DNA identified four potential transgenic founders (designated Nos. 54, 61, 78, and 92) from 120 live births . Animal 92 gave a reactive band of higher mol wt than the expected 453 bp Taq 1 fragment generated from the intact transgene . Further analysis using overlapping PCR primer sets demonstrated that this animal contained a truncated transgene, whereas animals 54, 61, and 78 contained multiple copies of the intact transgene construct. All three of the founder animals were fertile and subsequent analysis of progeny by quantitative Southern blot analysis demonstrated that they carried 6, 15, and 31 copies of the transgene, respectively. Furthermore, all three transgenic lines exhibit germline transmission of the transgene to subsequent progeny (data not shown). PKC β II transgenic RNA expression was detected by reverse transcriptase (RT)-PCR using primers specific for the human PKC β II transgene. A representative RT-PCR analysis of a litter of mice from the 54 transgenic line is shown in Fig. 1 C. As can be seen, transgenic PKC β II mRNA is detected in the colonic epithelium of all three transgenic mice, but not in nontransgenic littermates. Further analysis demonstrated that PKC β II transgene expression is fully penetrant, being detected in the colonic epithelium of all transgenic mice tested. Furthermore, no false positive RT-PCR products have been detected in nontransgenic animals, demonstrating the specificity of the RT-PCR primers for the human PKC β II transgene construct. Similar results were obtained in the 61 and 78 transgenic lines (data not shown). We next determined the level of PKC β II protein expression in the colonic epithelium of transgenic mice. Colonic epithelial cell lysates from transgenic and nontransgenic animals from the 54 transgenic line were prepared and subjected to immunoblot analysis using a PKC β II isozyme-specific antibody . Consistent with the presence of transgenic PKC β II mRNA, PKC β II protein levels in the colonic epithelium of transgenic mice are elevated relative to their nontransgenic littermates . Transgenic PKC β II exhibits a relative molecular mass of ∼85 kD, comigrating with mouse brain PKC β II used as a positive control. Similar results were obtained in the small intestine of these animals and from animals in the 61 and 78 transgenic lines (data not shown). Quantitation of PKC β II expression by densitometric analysis of the immunoblots indicated that 54 line transgenic mice express an average of fivefold more PKC β II protein than do nontransgenic littermates. Immunoprecipitation kinase assays showed an approximately fivefold increase in calcium- and phospholipid-dependent PKC β II activity in the colonic epithelium of transgenic mice, demonstrating that transgenic PKC β II is catalytically active and exhibits the same cofactor dependence of endogenous PKC/ β II (data not shown). Animals in the 54 transgenic line gave a consistently high level of transgene expression and therefore this line was selected for further analysis. We next assessed the pattern of transgenic PKC β II protein expression within the colonic epithelium by immunohistochemistry . For this purpose, tissue from the proximal colon of transgenic and nontransgenic mice was immunostained for PKC β II . Consistent with our RT-PCR and immunoblot results, the colonic epithelium from transgenic animals exhibits increased immunostaining for PKC β II when compared with nontransgenic littermates . In nontransgenic mice, PKC β II staining is observed in the mid-crypt regions and on the luminal surface of the epithelium. In transgenic animals, PKC β II staining is greatest in the mid-crypt region but is detectable throughout the entire crypt axis. Previous characterization of the transgene promoter demonstrated that the rat liver FABP promoter is active in both proliferating and postmitotic cells in the colonic epithelium of transgenic mice . The distribution of endogenous PKC β II overlaps that of the stem cell population, which is located in the mid-crypt region in the proximal colon . In the proximal colon, maturing colonic epithelial cells migrate from the proliferative mid-crypt region toward the base and the luminal surface of the crypt . The fact that endogenous PKC β II expression colocalizes with the stem cell population is consistent with the hypothesis that PKC β II plays a functional role in colonic epithelial cell proliferation. Transgenic PKC β II expression was detected in both the proximal and distal colon, indicating transgene expression throughout the colonic epithelium. To investigate the biological effects of overexpression of PKC β II in the colonic epithelium, we analyzed the following colonic morphometric parameters: colon length, colonic crypt height (in micrometer and cell number), crypt circumference (in cell number), and cell size (crypt height in micrometer/crypt height in cell number) (Table I ). This analysis revealed no statistical difference in the length of the colon, cell size, crypt height in micrometers, or crypt circumference between transgenic and nontransgenic littermates. However, colonic crypts from transgenic mice tended to be longer and have a larger circumference than those from nontransgenic mice. In addition, a highly significant increase in the number of cells per crypt height, and in the total number of cells per crypt, was observed in transgenic mice (Table I ). Similar results were obtained in a second transgenic mouse line (line 78; 22.2 cells per crypt height in transgenic versus 20.7 in nontransgenic mice, P = 0.009; and 356.4 total cells per crypt in transgenic versus 321.1 in nontransgenic mice, P = 0.007), indicating that this effect is due to the presence of the PKC β II transgene rather than an insertional mutagenic event. Both of these cytokinetic parameters are highly regulated and are determined by the balance among cell proliferation, differentiation, and apoptosis. These results demonstrate that increased expression of PKC β II disrupts one or more of the homeostatic mechanisms regulating cell number in the colonic epithelium. Elevated PKC β II could increase the number of colonic epithelial cells by increasing the level of proliferation, or by decreasing differentiation and/or apoptosis, in the colonic crypt. To distinguish between these possibilities, each of these cytokinetic parameters was measured. Immunohistochemical staining for PCNA revealed that the colonic epithelium from transgenic mice contain significantly more PCNA-positive cells than those from nontransgenic mice . Quantitation of PCNA-positive nuclear staining gave a labeling index of 28.3 ± 0.2% for transgenic mice compared with 21.4 ± 0.9% for nontransgenic mice (Table II ). This difference is highly significant and clearly contributes to the increase in crypt cell number observed in transgenic mice. The difference in labeling index was most pronounced in the bottom third of the crypts, the region containing the stem cell population in the distal colon. The size of the proliferative zone (calculated as the highest labeled cell in the crypt column) was also larger in transgenic colons; however, this difference was not statistically significant (Table II ). Taken together, these data demonstrate that elevated PKC β II expression stimulates hyperproliferation of the stem cell population residing within the base of the crypt, rather than stimulating postmitotic cells higher in the crypt to reenter the cell cycle. The differentiation state of the colonic epithelium was examined by staining with a panel of lectins and histochemical markers to identify the major differentiated colonic epithelial cell lineages. Fig. 4 , A and B, shows distal colonic epithelium from transgenic and nontransgenic mice stained with the two histochemical stains, Alcian blue and PAS, that detect goblet cells. The staining pattern seen in transgenic and nontransgenic animals is indistinguishable. Mucin production was detected by staining with several fluorescently labeled lectins . DBA binds fairly uniformly to mucin-producing cells in normal distal colonic epithelium . PNA gives a golgi (supranuclear) staining pattern on a subset of mucin-producing enterocytes and UEAI gives low level staining in normal mucosa of the distal colon . Analysis of the number and location of cells staining with the various lectins revealed no significant changes in the number of goblet cells or in the intensity or pattern of lectin labeling in transgenic PKC β II versus nontransgenic mice. These data indicate that increased expression of PKC β II has no demonstrable effect on the differentiation status of the major colonic enterocytic cell lineages. The level of apoptosis in the colonic epithelium was measured using an in situ TUNEL assay . An example of TUNEL staining of an apoptotic cell, which typically occurs near the top of the crypt, is shown in Fig. 5 A. As expected, we detected a very low level of apoptosis in the colon of transgenic PKC β II and nontransgenic mice. The apoptotic index in the distal colon of nontransgenic mice was not significantly different from that in transgenic PKC β II mice . Apoptosis is thought to contribute to the loss of cells required to maintain a balance with cell proliferation within the colonic epithelium . However, apoptotic cells are quickly eliminated in the colonic crypt, so that apoptosis is detected at a very low level . Our results are similar to the level of apoptosis in mouse colon reported by others , and demonstrate that increased expression of PKC β II has no significant effect on the level of apoptosis in the colonic epithelium. Increased cellular proliferation is a significant risk factor for development of colon cancer . Therefore, we assessed whether transgenic PKC β II mice exhibit an increased susceptibility to colon carcinogenesis. 1,2-dimethylhydrazine and its metabolite, azoxymethane (AOM), are organ-specific carcinogens that have been extensively characterized for their ability to induce colon cancer in rodents . AOM reproducibly induces colon tumors that exhibit many of the same genetic and signal transduction defects identified in human colon carcinomas . AOM also induces ACF, which represent well-established preneoplastic colonic lesions in both rodents and humans . Both the number and multiplicity (i.e., number of crypts per focus) of ACF are highly predictive of subsequent tumor development . Therefore, AOM-induced colon carcinogenesis is a highly relevant model for human colon cancer. To determine whether transgenic PKC β II mice differ from nontransgenic mice in their sensitivity to AOM-induced colon carcinogenesis, 6–7-wk-old transgenic PKC β II mice and nontransgenic littermates (five mice/group) received either AOM (10 mg/kg body wt) or saline by intraperitoneal injection once a week for 2 wk. At 5 and 20 wk after the second AOM injection, mice were killed and their colons were analyzed for the presence of ACF. In agreement with the literature , we observed no ACF in saline-injected animals, confirming that ACF arise as a result of AOM exposure. Colons from both transgenic and nontransgenic animals treated with AOM contained ACF exhibiting the distinguishing characteristics described by Bird and colleagues . Specifically, ACF appeared as enlarged crypts, often three or four times the size of adjacent crypts, that were raised above the surface of the surrounding mucosa. ACF characteristically stained darker than surrounding crypts, had thicker than normal intercryptal spaces, and exhibited thickening of the crypt wall, suggestive of epithelial stratification. The crypt lumens in ACF were elongated and often serrated, in contrast to the round, smooth lumens of normal crypts. ACF contained either a single aberrant crypt or involved two or more adjacent crypts. Fig. 6 Ashows the morphology of a typical ACF consisting of three crypts from an AOM-treated animal. The total number of ACF/colon and the multiplicity of ACF was determined at 5 and 20 wk after the last AOM injection . AOM-treated transgenic mice had a statistically significant increase in the total number of ACF/colon and in the number of ACF of higher multiplicity at both 5 and 20 wk . At 20 wk, the total number of ACF did not increase significantly from that measured at 5 wk; however, the number of ACF of higher multiplicity did increase in transgenic PKC β II mice . Interestingly, at 5 wk, although the total number of ACF and the number of ACF of higher multiplicity were greater in transgenic mice, the average multiplicity of ACF in these two groups did not differ . However, by 20 wk, transgenic mice exhibited an increase not only in the number of ACF but also in the average crypt multiplicity . Since the number of ACF, particularly those of higher multiplicity, are highly predictive of subsequent colon tumor incidence, these data demonstrate that transgenic PKC β II mice are more susceptible to AOM-induced colon carcinogenesis than nontransgenic littermates. Furthermore, these data suggest that elevated PKC β II is involved not only in the early promotive phase of ACF development but also in their progression to lesions of higher multiplicity and malignant potential. Colonic epithelial cell proliferation is under the control of the Wnt/APC/β-catenin proliferative signaling pathway . PKC has recently been demonstrated to play a key role in Wnt/wingless signaling in tissue culture cells . Selective PKC inhibitors can block Wnt-mediated inhibition of GSK-3β activity, whereas activation of PKC with PMA leads to inactivation of GSK-3β kinase activity in the absence of Wnt . GSK-3β is a constitutively active serine/threonine kinase that is a critical downstream target in the Wnt signaling pathway. GSK-3β–mediated phosphorylation of APC facilitates binding of β-catenin to APC, which targets β-catenin for degradation. The ability of PKC to inhibit GSK-3β activity is probably due to its direct phosphorylation of GSK-3β, since PKC has been shown to directly phosphorylate GSK-3β and inhibit its activity in vitro . To determine whether PKC β II activates the Wnt/APC/β-catenin pathway in vivo, we assessed GSK-3β levels and activity in the colonic epithelium of transgenic PKC β II mice . Immunoblot analysis reveals that GSK-3β protein levels are similar in transgenic and nontransgenic mice . However, immunoprecipitation kinase assays demonstrate that GSK-3β activity in transgenic mice is 50% of that observed in nontransgenic littermates . The observed decrease in GSK-3β activity is due to a decrease in the specific kinase activity of the enzyme since GSK-3β expression was unchanged in transgenic PKC β II mice . The extent of GSK-3β inhibition is similar to that observed in response to optimal concentrations of either soluble Wnt or PMA in fibroblasts in vitro . As another measure of Wnt pathway activation, β-catenin protein levels were assessed by immunoblot analysis . β-catenin levels are elevated in transgenic PKC β II mice when compared with nontransgenic littermates . Densitometric analysis of the immunoblot data indicate that on average β-catenin levels are ∼40% higher in transgenic PKC β II mice. These data indicate that the Wnt/APC/β-catenin signaling pathway can be stimulated by β II and provide a plausible molecular mechanism by which PKC β II causes hyperproliferation and increased susceptibility to colon carcinogenesis in these animals. Colon carcinogenesis is a multistep process involving the progressive loss of growth control mechanisms and accumulation of genetic mutations that result in an increasing level of neoplasia . The process of multistage carcinogenesis has been described as “a progressive disorder in signal transduction” . According to this model, nongenetic changes in normal signal transduction pathways which increase the susceptibility to further genetic “hits” and therefore play a critical role in the pathogenesis of colon cancer occur early in the carcinogenic process. However, the nature of these early cancer-promotive changes is not well understood. Members of the PKC family of enzymes have been implicated in the regulation of colonic cell proliferation, differentiation, and apoptosis. PKC β II plays a direct role in cellular proliferation in both human leukemia cells and colon cancer cell lines , and increases in PKC β II expression are early events in colon carcinogenesis in vivo . Our present data demonstrate that this increase in PKC β II expression plays a promotive role in colon carcinogenesis. To directly assess the role of PKC β II in colonic epithelial cell proliferation and colon carcinogenesis, we developed a transgenic mouse model in which PKC β II is overexpressed in the intestinal epithelium. Transgenic PKC β II mice exhibit hyperproliferation of the colonic epithelium characterized by an increase in the labeling index and an increase in the number of cells per colonic crypt. Interestingly, no significant changes were observed in colonocyte differentiation status or apoptotic index, indicating a selective effect of PKC β II on the proliferative program of the colonic epithelium. Although we cannot eliminate the possibility that subtle changes have occurred in the regulation of differentiation or susceptibility to apoptosis, our data clearly demonstrate that the change in proliferation is a major contributing factor to the increased colonic crypt cell number observed in transgenic PKC β II mice. Increased proliferation is an important risk factor for induction of colon cancer and is a key biomarker of preneoplastic events . Our data indicate that PKC β II acts early in the carcinogenic pathway to increase the proliferation of the colonic epithelium, perhaps making it more susceptible to further genetic mutations and formation of preneoplastic lesions, including ACF. The effect of increased PKC β II expression on the susceptibility to induction of colon cancer was tested using a well-characterized rodent carcinogenesis model . AOM-induced colon tumors are a good model for sporadic human colon cancer because they exhibit many of the same properties as human colon tumors, including increased proliferation, development of tumors predominantly in the distal colon, and the presence of many of the same genetic mutations found in human tumors. In addition, ACF, the earliest preneoplastic lesions observed in this model, are also thought to be preneoplastic lesions in humans . ACF exhibit many of the early phenotypic markers of colon cancer including increased proliferation and frequent mutations in the APC and ras genes . We demonstrate that increased PKC β II expression makes transgenic mice more susceptible to AOM-induced colon carcinogenesis as measured by an increase in the total number of ACF and in the number of ACF of higher multiplicity than nontransgenic mice. ACF are highly predictive of subsequent tumor formation and multiplicity in the rodent carcinogenesis model and of adenoma formation and colon cancer risk in humans . Our data indicate that elevated PKC β II expression not only promotes ACF formation, but also stimulates progression of these lesions. These results suggest that PKC β II plays a critical role at multiple stages in the colon carcinogenic pathway. Accumulating evidence suggests that PKC β II plays a direct role in intestinal epithelial cell proliferation and colon carcinogenesis in both rodents and humans. PKC β II levels and activity are elevated in preneoplastic and neoplastic colons, demonstrating that these changes precede colon carcinoma development . Here, we demonstrate that overexpression of PKC β II in the colonic epithelium leads to hyperproliferation and increased susceptibility to colon carcinogenesis. Furthermore, we demonstrate that elevated PKC β II leads to inhibition of GSK-3β activity and an increase in β-catenin levels. These observations are consistent with in vitro data demonstrating a requisite role for PKC in the Wnt proliferative signaling pathway , and suggest that PKC β II may play such a role in vivo. Further studies will be required to determine whether PKC β II –mediated activation of this pathway is required for its ability to stimulate proliferation and cancer susceptibility in the transgenic mouse setting. Taken together, the data lead us to propose a molecular mechanism by which PKC β II stimulates colonic epithelial cell hyperproliferation and increased colon carcinogenesis in transgenic mice . In this model, PKC β II either directly or indirectly leads to GSK-3β inactivation. PKC has been shown to phosphorylate GSK-3β and inactivate the enzyme in vitro , suggesting that PKC β II can inhibit GSK-3β by direct phosphorylation and inactivation. Inhibition of GSK-3β leads to an accumulation of β-catenin by decreasing the interaction of β-catenin with APC, which targets β-catenin for degradation. Accumulation of β-catenin causes Tcf-dependent transcriptional activation of growth-related genes to stimulate colonocyte proliferation . The APC/β-catenin pathway is a major site for mutation during colon carcinogenesis . Mutations in either APC or β-catenin that disrupt β-catenin degradation are present in the vast majority of colon cancers, providing strong evidence that elevated β-catenin levels are important in colon carcinogenesis . Furthermore, overexpression of a proteolytically-stable NH 2 -terminal truncated β-catenin in the intestinal epithelium of transgenic mice leads to hyperproliferation . Our data suggest that accumulation of β-catenin through PKC β II –mediated inhibition of GSK-3β may play an important promotive role in colon carcinogenesis before the acquisition of mutations in members of this critical signaling pathway. A major question is how PKC β II activity is modulated during the early stages of colon carcinogenesis. One attractive hypothesis arises from the finding that colonocyte PKC activity can be stimulated by cancer-promotive components of a high fat diet. Diets high in certain fatty acids have been shown to increase the proliferative activity of the colonic epithelium, stimulate colonocyte PKC activity, and increase susceptibility to carcinogen-induced ACF . This finding is of significance since increased colonic proliferation is a well-established risk factor and biomarker for colon cancer in individuals with familial adenomatous polyposis and ulcerative colitis, as well as in carcinogen-treated rodents . Cancer-promotive dietary fats function to increase the level of secondary bile acid and fatty acids in the intestinal lumen. Secondary bile acids can in turn activate colonic PKC by a number of mechanisms. First, secondary bile acids and fatty acids can directly activate PKC β II activity and stimulate cellular proliferation in the colonic epithelium . Second, bile acids can promote DAG production by intestinal bacteria, which in turn stimulate colonocyte PKC activity . Third, bile acids can stimulate phospholipid breakdown and DAG generation in colonic epithelial cells , leading to PKC activation. Therefore, we hypothesize that these dietary risk factors increase PKC β II activity in intestinal epithelial cells by multiple mechanisms, resulting in increased epithelial cell proliferation through activation of the APC/β-catenin signaling pathway in a Wnt-independent fashion . This model provides a plausible link between a critical intracellular signaling pathway that is known to be important in colon cancer, and known dietary risk factors for colon carcinogenesis. Our transgenic PKC β II mice will provide a valuable model to test the hypothesis that PKC β II is a relevant target for these cancer-promotive dietary risk factors, and to explore the mechanism by which these factors may impinge on the APC/β-catenin signaling pathway. | Study | biomedical | en | 0.999997 |
10330401 | cDNA coding for full-length human PKC isoforms α, βII, δ, and ε; the RD from PKCα, βII, δ, ε, η, and θ; or smaller fragments of PKCε were generated by PCR with introduction of appropriate restriction enzyme sites in the primers. The DNA fragments were introduced into the pEGFP-N1 vector ( Clontech Laboratories, Inc. ), thereby fusing the PKC cDNA with EGFP cDNA. The schematic structures of the protein products coded for by the different expression vectors are shown in Fig. 1 A. Templates for the PCR reactions were for PKCα, ε, and θ cDNA from SH-SY5Y cells; for PKCβII ATCC plasmid 80047 ; for PKCδ ATCC plasmid 80049 ; and for PKCη cDNA generated from human placenta mRNA ( Clontech Laboratories, Inc. ). The PKCε plasmids εPSC1aV3E and εPSC1bV3E ( εPSC1V3E with DNA coding for either the second or the first C1 domain deleted) were generated with primers designed to amplify the entire εPSC1V3E plasmid, excluding the DNA coding for the domain that should be deleted. An MluI site was introduced in each primer, the PCR product was cleaved with MluI, and ligated. Table I lists the primers used to generate the PKC fragments. All PCR reactions were performed with Pfu polymerase (Stratagene) to minimize introduction of mutations and all PCR-generated fragments used in this study were sequenced. The generation of the protein products of anticipated sizes were confirmed by transfecting the expression vectors into COS cells with the calcium phosphate method and subjecting the cell lysate to Western blot analysis . In addition, the NheI/SalI fragments from αFL and εFL (full-length PKC) were inserted into the CMS–EGFP vector ( Clontech Laboratories, Inc. ) to obtain expression of PKC and EGFP as two separate proteins. COS cells were transfected with different expression vectors, washed with PBS, and lysed in buffer (10 mM Tris, pH 7.2, 160 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM EGTA, 1 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 10 min at 15,000 g and 25 μg of protein was electrophoretically separated on an SDS polyacrylamide gel and thereafter transferred to Hybond-C extra nitrocellulose filter (Nycomed- Amersham, Inc. ). EGFP- or PKC-immunoreactivity was analyzed with antibodies directed against green fluorescent protein (GFP; Clontech Laboratories, Inc. ) or PKCα, βII, δ, or ε (Santa Cruz), and detected with an HRP-labeled secondary antibody using the SuperSignal system ( Pierce Chemical Co. ) as substrate. The chemiluminescence was detected with a CCD camera (Fuji Photo Film Co.). Human neuroblastoma SH-SY5Y, SH-SY5Y/TrkA, and SK-N-BE(2) cells were maintained in MEM supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin ( GIBCO BRL ). For transfection experiments, SH-SY5Y and SH-SY5Y/TrkA cells were trypsinized and seeded at a density of 350,000 cells/35-mm cell culture dish on glass coverslips in serum free medium. After 20 min the medium was changed to medium containing serum and antibiotics, and incubated for 24 h before start of the transfections. SK-N-BE(2) cells were seeded on glass coverslips in regular growth medium (300,000 cells per dish) and transfections were initiated 24 h after seeding. In experiments where cells were treated with 12- O -tetradecanoylphorbol-13-acetate (TPA; Sigma Chemical Co. ) for 4 d, or with growth factors for 40 h, the density at cell seeding was 250,000 cells/35-mm dish. SH-SY5Y cells were transfected using 3.5 μl Lipofectin ( GIBCO BRL ) and 1.8 μg of DNA/ml serum free medium and SK-N-BE(2) cells were transfected with 4 μl Lipofectamine ( GIBCO BRL ) and 2 μg DNA, essentially according the supplier's protocol. For differentiation studies, SH-SY5Y/TrkA cells were treated for 40 h with 100 ng/ml NGF ( Promega Corp. ), and SK-N-BE(2) cells with 10 μM retinoic acid (RA; Sigma Chemical Co. ) or 25 ng/ml ciliary neurotrophic factor (CNTF; Promega Corp. ). 16 h after the end of transfections (unless otherwise stated) cells were fixed in 4% paraformaldehyde in PBS for 4 min, mounted on microscopy slides using a PVA-DABCO solution (9.6% polyvinyl alcohol, 24% glycerol, and 2.5% 1,4-diazabicyclo[2.2.2]octane in 67 mM Tris-HCl, pH 8.0), and used for morphological studies. Digital images were captured with a Sony DKC 5000 camera system. The transfected cells were considered to have long processes if the length of the process exceeded that of two cell bodies. At least 200 transfected cells per experiment were counted. Cells were transfected, fixed, and mounted as for morphology studies. Cells expressing various PKCε–EGFP constructs and Texas red–phalloidin-stained F-actin were examined using a Bio-Rad MRC 1024 confocal system fitted with a Nikon Diaphot 300 microscope using a Nikon plan-apo 60 × 1.2 NA water immersion lens. Cells grown on glass coverslips were fixed with 4% paraformaldehyde as above. For detection of α-tubulin, synaptophysin, and neuropeptide Y (NPY), cells were permeabilized and blocked with 1% BSA/0.02% saponin in PBS. The primary antibody (monoclonal mouse anti–α-tubulin [ Sigma Chemical Co. ] diluted to 1:2,000; monoclonal mouse antisynaptophysin [clone SY38, DAKOPATTS] diluted to 1:10; or polyclonal rabbit anti–NPY [Biogenesis] diluted to 1:40, respectively) was incubated for 1 h in blocking/permeabilization solution. The secondary antibody (donkey anti–mouse IgG-TRITC [Jackson ImmunoResearch Laboratories, Inc.] diluted to 1:100 for α-tubulin and 1:20 for synaptophysin detection; or donkey anti–rabbit IgG-TRITC [Jackson ImmunoResearch Laboratories, Inc.] diluted to 1:300 for NPY staining) was incubated for 1 h in blocking solution. Extensive washing with PBS and blocking/permeabilization solution was done between all steps. For detection of neurofilament-160 (NF-160), cells were blocked for 30 min with 3% BSA in PBS and incubation with monoclonal mouse anti–NF-160 ( Sigma Chemical Co. ) diluted to 1:50 was performed for 3 h. Secondary antibody donkey anti–mouse IgG-TRITC (Jackson ImmunoResearch Laboratories, Inc.) was diluted to 1:300 and incubated for 1 h after extensive washing with PBS. For staining of F-actin, cells were fixed with 4% paraformaldehyde. Cells were treated for 5 min with 0.1% Triton X-100 in PBS and incubated for 10 min with 2 μg/ml TRITC–conjugated phalloidin ( Sigma Chemical Co. ) in PBS. For confocal studies, fixed cells were blocked and permeabilized with 5% donkey serum and 0.3% Triton X-100 in TBS, and stained for 20 min with Texas red–conjugated phalloidin (Molecular Probes, Inc.; 25 μl/ml blocking/permeabilization solution). Coverslips were mounted on object slides with 20 μl PVA-DABCO. To investigate whether increased levels of a specific PKC isoform are sufficient to induce neurites, expression vectors coding for four different PKC isoforms were transfected into neuroblastoma cells. The classical and novel isoforms consistently expressed in neuroblastoma cells, PKCα, PKCβII, PKCδ, and PKCε , were selected for this approach. The cDNAs coding for these isoforms were fused to cDNA coding for EGFP, generating a PKC–EGFP fusion protein when expressed. To confirm the generation of fusion proteins, COS cells were transiently transfected with these plasmids, and cell lysates were subjected to Western blot analysis using isoform-specific antibodies , which demonstrated the formation of proteins of the anticipated sizes. SH-SY5Y and SK-N-BE(2) neuroblastoma cells were transfected with the vectors and the morphology of transfected cells was visualized with fluorescence microscopy . When EGFP alone was expressed in SH-SY5Y and SK-N-BE(2) cells, the fluorescence was distributed throughout the cell. αFLE and βIIFLE (full-length PKC bound to EGFP) were mainly localized in the cytoplasm and were absent from the nucleus. δFLE localized throughout the entire cell, whereas εFLE localized mainly to the cell periphery and, in some cells, to perinuclear structures . All fusion proteins gave rise to fluorescence of similar intensity in the transfected cells, indicating that there were no major differences in the expression levels of fusion proteins in individual cells. The morphological effects of the overexpression of PKC isoforms were quantified by counting the number of transfected cells with cell processes longer than the length of two cell bodies. In SK-N-BE(2) cells, overexpression of εFLE induced long processes in 41% of the transfected cells, a substantially higher number than cells expressing EGFP only, where 6% of transfected cells had long processes. This effect was specific for PKCε, as overexpression of neither αFLE, βIIFLE, nor δFLE resulted in an increased number of cells with long processes . A similar, but less pronounced pattern was observed in SH-SY5Y cells where overexpression of εFLE lead to 23% transfectants with long processes compared with 12% for cells expressing EGFP only. As in the case of the SK-N-BE(2) cells, overexpression of other PKC isoforms did not induce processes . To exclude a potential role of EGFP in the PKCε effect, cDNA for PKCα and ε were transferred from αFL and εFL, respectively, to the CMS– EGFP vector as a control. In these constructs PKC and EGFP are expressed as separate proteins. SK-N-BE(2) cells were transfected with these vectors, and 5% of PKCα and 31% of PKCε overexpressing cells had processes. This demonstrates that the process induction of PKCε–EGFP is not dependent on EGFP. To investigate whether the changes in cell morphology provoked by overexpression of PKCε–EGFP can be blocked by inhibition of PKC, the transfectants were treated with GF109203X . This inhibitor did not cause a decrease in the percentage of transfected cells with long processes. The concentration used (2 μM) is in the range that inhibits the catalytic activity of classical and novel PKC isoforms in vitro and blocks TPA-induced expression of fos and jun genes in neuroblastoma cells . Thus, the induction of processes by PKCε appears to be independent of the catalytic activity of the enzyme. The fact that overexpression of full-length PKCε induced processes in the presence of GF109203X suggested an independence of the kinase activity. To analyze whether the PKC RD is sufficient for the effect, vectors coding for the RDs of PKCα, β, δ, and ε fused to EGFP, were created. The RDs of the remaining novel isoforms PKCη and PKCθ, which are not expressed in neuroblastoma cells, were also included as a comparison . All constructs were sequenced and found free of mutations. The constructs were expressed in COS cells where Western blot analysis confirmed formation of proteins of the anticipated sizes. SH-SY5Y and SK-N-BE(2) cells were transfected with the vectors and all fusion proteins gave rise to fluorescence of similar intensity in transfected cells , with the exception of θRDE . θRDE caused a weaker fluorescence suggesting lower levels of this protein. As in the case for full-length PKCα and PKCβII, their corresponding RD–EGFP fusion proteins localized mainly outside the nucleus with a tendency to perinuclear enrichment . Neither of these RDs induced a major increase in the number of cells with processes . In contrast, transfection with the δRDE , εRDE , and ηRDE constructs led to a drastic change in cell morphology, most prominent in εRDE transfectants. Overexpression of these proteins gave rise to 19% (δRDE), 32% (εRDE), and 25% (ηRDE) SH-SY5Y cells with long processes. The corresponding numbers for SK-N-BE(2) cells were 55% (δRDE), 56% (εRDE), and 46% (ηRDE). The fusion proteins seemed to be localized mainly to perinuclear structures and the cell periphery. θRDE seemed to localize to all parts of the cells and long processes were induced in 12% of the transfected SH-SY5Y cells and 20% of the SK-N-BE(2) cells. To clarify which parts of the RD that are essential for the induction of processes, a series of constructs coding for different parts of εRDE was created . The constructs were sequenced and transfected into COS cells, where proteins of expected sizes were detected in cell lysates with Western blot analysis using a GFP antibody . The PKCε subdomains were expressed in SH-SY5Y and SK-N-BE(2) cells , and proteins gave rise to bright fluorescence of similar intensity suggesting no major difference in intracellular concentration. All fusion proteins containing the two C1 domains were not detected in the nucleus, and displayed a tendency to enrich in perinuclear structures . Some fusion proteins, particularly εRDE and εPSC1V3E, also seemed to localize to the plasma membrane. C2-containing proteins without the C1 domains localized throughout the cell, and the smaller proteins (εPSE and εPSC1aE) were primarily present in the nucleus. When cell morphology was examined, it was evident that the fragment from PKCε containing the pseudosubstrate, the C1 domains, and the V3 region (εPSC1V3E) was necessary and sufficient to induce processes . 48% of the SH-SY5Y cells expressing this protein exhibited long processes. In SK-N-BE(2), the corresponding number was 59%. When the pseudosubstrate (εC1V3E) or the V3 (εPSC1E) was removed from the PSC1V3 fragment, no substantial induction of processes could be observed in either cell line. It is notable that in SH-SY5Y cells more εPSC1V3- than εRDE-expressing cells had processes (48% versus 36%), suggesting that removal of the C2 domain enhances the process-inducing capacity . It was also evident that the other constructs did not have a major effect on process induction. Fluorescence microscopy suggested that the PKCε fragments localized to different intracellular sites. To investigate a possible correlation between the localization and process-inducing ability of the fragments, transfected SH-SY5Y cells were analyzed with confocal microscopy . Full-length PKCε fused to EGFP localized uniformly outside the nucleus. The smallest fragment that induced processes, εPSC1V3E, displayed a distinct plasma membrane localization. Removal of the pseudosubstrate led to the complete loss of plasma membrane localization, as εC1V3E could only be seen in the perinuclear area of the cell. This suggests that the pseudosubstrate might be necessary for targeting of PKCε to the plasma membrane . Removal of the hinge region from the PSC1V3 fragment generating εPSC1E, which is incapable of inducing processes, did not cause a loss of plasma membrane localization . In conclusion, these data suggest that localization to the plasma membrane, for which the pseudosubstrate and the C1 domains are required, is necessary, but not sufficient for the process induction. The V3 region needs to be present for optimal function of the fragment. The previous results demonstrate that PKCε through the PSC1V3 fragment has the capacity to induce processes in neuroblastoma cells. To address the question of whether this capacity is a part of the molecular events driving neurite outgrowth in neuroblastoma cells differentiating in response to growth factors and RA, an attempt was made to find an εPSC1V3E-derived construct that could inhibit the process formation, putatively by acting in a dominant-negative manner. The two constructs that were most similar to εPSC1V3E , i.e., εPSC1E and εC1V3E , and did not display a process-inducing capacity, were initially evaluated for this purpose. Neither construct had a major effect on neurite outgrowth in RA-differentiated SK-N-BE(2) cells (data not shown). Thereafter, cDNA coding for either the first (C1a) or the second (C1b) C1 domain was deleted in the εPSC1V3E construct, forming εPSC1bV3E and εPSC1aV3E , respectively . SK-N-BE(2) cells were transfected with these vectors, and vector coding for EGFP only . Neither protein induced processes in untreated cells, demonstrating that both C1 domains are required for this effect. In fact, there was a slight suppression of the number of cells with processes in εPSC1aV3E-expressing cells . After treatment with RA, 57% of EGFP-expressing cells and 52% of εPSC1bV3E-transfected cells had neurites. In contrast, only 18% of εPSC1aV3E-expressing cells had processes, demonstrating a neurite suppressing effect of this protein. Treatment with CNTF gave results that followed the same pattern as in RA, albeit with generally fewer neurite extending cells . The constructs were also evaluated for NGF-driven neurite outgrowth of SH-SY5Y cells stably expressing the high affinity NGF receptor, TrkA . Also in this differentiation protocol, expression of εPSC1aV3E, but not εPSC1bV3E, caused a substantial decrease in the number of neurite-bearing cells, both in control and NGF-exposed cells. These results demonstrate that the protein lacking the second C1 domain (εPSC1aV3) inhibits neurite outgrowth in several neuronal differentiation protocols, whereas the protein with the first C1 domain deleted (εPSC1bV3E) has no such effect. To test whether the C1-deleted constructs have similar effects on processes induced by overexpression of PKCε or εPSC1V3E, εFLE and εPSC1V3E were cotransfected with εPSC1aV3E or εPSC1bV3E at a 1:3 ratio into SK-N-BE(2) cells . Cotransfection with εPSC1bV3E gave rise to fewer cells with processes than when either εFLE or εPSC1V3E alone was transfected, but substantially more process-bearing cells than when εPSC1bV3E alone was transfected into the cells. It is likely that the lower number of cells with processes in this cotransfection protocol could be due to a significant proportion of cells expressing only εPSC1bV3E , cells that will fluoresce, but will not have processes. On the other hand, cotransfection with εPSC1aV3E gave a lower number of cells with processes than did cotransfection with εPSC1bV3E . Thus, the εPSC1V3 fragment with the second C1 domain deleted (εPSC1aV3E) acts in a dominant-negative manner both suppressing processes induced by overexpression of PKCε and inhibiting neurite outgrowth in several neuronal differentiation protocols. This suggests that the effect of the PSC1V3 region from PKCε may be a common mechanism for these processes. All PKCε-derived, process-inducing constructs caused similar morphological changes of transfected cells. The outgrowth of processes was accompanied by a shrinkage of the cytoplasm and a rounding up of the cell body, which was most apparent in SK-N-BE(2) cells. Untreated SH-SY5Y cells generally have smaller cell bodies than SK-N-BE(2) cells, but a tendency towards rounding up of the cell body was observed in the SH-SY5Y cells, also. The overall morphology of the processes differed slightly between the two cell lines. In SH-SY5Y cells, generally one process per cell was observed, but this process frequently carried several branches of various lengths , but in some cells two or more processes extending from the same cell were seen . The SK-N-BE(2) cells generally had more than one process per cell, and these processes were frequently branched. To address whether the εPSC1V3E-induced processes have characteristics associated with neurites, expression of cytoskeletal components and synaptic markers were analyzed. The εPSC1V3E-induced processes in SH-SY5Y cells were compared with neurites obtained after 4 d of treatment with 16 nM TPA, a protocol that causes SH-SY5Y cells to differentiate neuronally . The experiments show that both εPSC1V3E-induced processes and the neurites of differentiated SH-SY5Y cells were composed of α-tubulin and NF-160 . The cells were also stained for F-actin , which besides staining of the main branches of the processes, also revealed an intense staining either at the tip of the processes or at sites where the processes have sharp bends (not shown). These actin-rich structures resemble the growth cones in TPA-differentiated cells , suggesting that εPSC1V3E-induced processes express growth cones. Staining for the presence of synaptic proteins NPY and synaptophysin in TPA differentiated SH-SY5Y cells was positive, while the processes of cells transfected with εPSC1V3E were negative . This shows that εPSC1V3E-induced processes are neurite-like, but lack important properties of functional neurites. Furthermore, no overall increase in the expression of NPY or synaptophysin could be detected in the εPSC1V3E - transfected cells, suggesting that this PKCε fragment does not induce complete differentiation of neuroblastoma cells. The characteristics of processes induced by εFLE were similar to εPSC1V3E-induced processes (not shown). An interesting issue is why overexpression of the RDs of both PKCε and PKCδ (εRDE and δRDE) induced processes, whereas for full-length isoforms the same effect only was obtained with PKCε (εFLE) and not with PKCδ (δFLE). A unique feature of PKCε, compared with other isoforms, is the presence of an actin-binding site between the C1 domains . Binding to F-actin via this site in vitro has been shown to maintain PKCε in an open conformation , which may result in exposure of structures in the RD essential for the process-inducing capacity of this isoform. If this interaction is important for the process induction of εFLE, it would be expected to detect colocalization of F-actin and εFLE. F-actin in εFLE -transfected SH-SY5Y cells was stained with Texas red–conjugated phalloidin and the colocalization of F-actin and εFLE was analyzed with confocal microscopy. Several processes were analyzed and it was evident that the proteins were colocalized in some parts of the processes . This result thus indicates that an interaction between εFLE and F-actin may take place in the processes. This study was designed to examine the role of PKC isoforms in neurite outgrowth regulation and identify structures in the PKC molecule of importance for its function in this process. For this purpose, we used neuroblastoma cell lines which have been extensively used to study neuronal differentiation. Of the classical and novel PKC isoforms that are consistently expressed in neuroblastoma cells , only overexpression of PKCε induced processes in these cells. PKCε has been suggested to be of importance for neurite outgrowth in PC12 cells where overexpression of PKCε, but not PKCδ, potentiated NGF-induced neurite outgrowth . The effect of PKCε in PC12 cells was suppressed by PKC inhibitors, which contrasts the results in the present study which demonstrates that the effect of PKCε was independent of its kinase activity. Furthermore, in PC12 cells, overexpression of PKCε did not by itself induce processes. It is thus likely that PKCε may regulate neurite outgrowth by a number of mechanisms. In neuroblastoma cells, several PKC isoforms are enriched in growth cones, but studies with phorbol ester treatment, which downregulates the classical isoforms, have suggested a role for PKCε or another novel isoform in supporting the growth cone . These facts, together with the results from the present study, highlight PKCε as one PKC isoform of importance in neurite outgrowth regulation. In this study, the PKC isoforms were fused to EGFP to visualize transfected cells and to facilitate an examination of the intracellular distribution of the expressed proteins. EGFP, in its native fluorescent form, is a highly condensed molecule . Approaches to fuse PKC isoforms with GFP variants have been successfully used to follow the translocation of PKCβII , PKCγ , PKCδ , and PKCε . When examined, this fusion has been shown not to influence the catalytic activity of the enzyme. GFP variants have also been fused to smaller proteins or isolated domains, such as histone 2B , pleckstrin homology domains , and PKC C1 domains without any obvious loss of function. Furthermore, as shown in this study, regardless if the position of EGFP was at the COOH terminus of intact PKCε or if it was placed COOH-terminally of the RD in constructs where the catalytic domain was deleted, processes were induced in neuroblastoma cells. This suggests that the effect on the process induction is independent of the position of EGFP. Processes were also induced when PKCε, without being fused to EGFP, was overexpressed. Several subdomains of PKCε that were fused to EGFP did not induce processes at all, further indicating that the effects observed in this study are not mediated by EGFP. As previously mentioned, the effect of PKCε was independent of enzymatic activity and of the presence of the catalytic domain, since expression of the RD was sufficient to induce processes. In fact, the RD could induce processes more potently than the full-length PKCε, suggesting that the catalytic domain may inhibit this function of the RD. The RD from PKCδ and η also induced processes in the transfected cells, despite the inability of full-length PKCδ to do so. Cells transfected with θRDE displayed less fluorescence than the other RD transfectants, probably indicating a lower level of expression of fusion protein in these transfectants. It is possible that the RD from PKCθ would have had the same effect if the protein levels in each individual cell had been higher. These results may suggest that the novel isoforms PKCδ and PKCη, and perhaps PKCθ, could have the capacity to induce processes under proper conditions. An interesting feature possibly explaining the selective effect of full-length PKCε, is the actin binding site which is present only in this isoform . When PKCε binds actin it is maintained in an open active confirmation exposing the catalytic domains and the RDs , which thereby can exert its activity. There was a colocalization of εFLE and F-actin in processes, a finding which may indicate that this interaction might be important for the selective effect of PKCε, although further experimentation is necessary to establish this interaction as crucial for process induction. The finding that the PKCε effect is insensitive to PKC inhibitors and could be mimicked by the RD is somewhat surprising. Since RDs of PKC isoforms have been suggested to act in a dominant-negative manner, the effects obtained in this study may be due to a dominant-negative effect of PKCε and its RD on another endogenous PKC isoform. If this were the case, it would be expected to see an induction of processes upon inhibition of this isoform with PKC inhibitors. However, treatment of the neuroblastoma cells with GF109203X did not cause an elevated number of processes. It could be argued that this lack of process induction is due to the fact that GF109203X also inhibits other kinases that are critical for the induction of processes. If so, it would be expected that GF109203X should suppress the processes also in PKCε-overexpressing cells, since the kinase of importance for processes also would be inhibited under these conditions. Furthermore, if the effects of the PKCε constructs are dominant-negative, the suppression by εPSC1aV3E of PKCε-induced processes, RA-, and NGF-induced neurites implies that this construct would act in a dominant-negative manner towards a dominant-negative effect in the first case, whereas in the latter protocols it would simply act in a dominant-negative way. Therefore, we think that the most plausible explanation for the effects observed in this paper is that PKCε RD induces processes through a mechanism that does not involve dominant-negative effects. There are other reports where parts of, or the entire PKC RD exert the same effects as the complete enzyme. PKCα was shown to activate phospholipase D in a PKC activator-dependent, but PKC activity-independent fashion, and phospholipase D was activated by PKCα regulatory, but not catalytic domain in vitro . Another example is the inhibition of Golgi-specific sulfation of glycosaminoglycan chains in cells overexpressing PKCε, which can be mimicked by overexpressing the PKCε C1 domains only . When examining the role of the different domains of PKCε RD in process induction, it was evident that a fragment centering on the two C1 domains was sufficient and necessary for this effect. Interestingly, the C2 domain, which is of importance for RACK binding , was not of importance for the process-inducing capacity. In fact, expression in SH-SY5Y cells suggested that removal of the C2 domain from the RD, generating PSC1V3, slightly increased the ability to induce processes. There are several examples demonstrating that protein interaction with the RD is mediated via the C1 domains. Beside the previously mentioned actin binding site in PKCε located between the C1 domains , a homologue of 14-3-3 has been shown to bind the Dictyostelium myosin II heavy chain-specific PKC through the PKC C1 domain . In addition, binding of the pleckstrin homology domain from the tyrosine kinase Btk was shown to be dependent on the pseudosubstrate and the C1 domain from PKCε . Using an overlay assay, it was shown that the second C1 domain from PKCγ bound several proteins from Xenopus laevis oocyte cytosol extracts . Taken together, these results indicate an important role for the C1 domains in PKC protein interactions. Thus, it is conceivable that the effects observed in this study are due to the C1 domains interacting with other proteins, thereby eliciting the observed morphological changes. However, there was also a dependence on the pseudosubstrate and parts of the V3 domain for the induction of processes. These structures have been shown to be of importance for localization of PKCε C1 domains to the plasma membrane in NIH3T3 fibroblasts . In line with that report, the process-inducing fragment, PSC1V3, localized almost exclusively to the plasma membrane, but this localization was lost when the pseudosubstrate was removed. This was accompanied with a loss of process-inducing capacity, which suggests that a plasma membrane localization is necessary for this effect. However, a plasma membrane localization per se of the C1 domains is not sufficient, since the PSC1 fragment to a large extent appeared to be present at the plasma membrane without inducing processes. Removal of the second, but not of the first, C1 domain generated a fragment that suppressed neurite outgrowth during RA-, CNTF-, and NGF-driven neuronal differentiation. Since this same fragment also acted in a dominant-negative manner towards processes induced by PKCε overexpression, these results suggest that the observed effects of PKCε is not only observed upon overexpression of the protein, but may indeed be of importance for neurite outgrowth that accompanies neuronal differentiation. However, given the abundance of proteins with C1 domains , it cannot be excluded that during neuronal differentiation effects reported in this study are mediated via other C1 containing proteins. The results obtained with the C1-deleted constructs also illustrate the different properties of the two C1 domains that have been described . From the present results, it is not possible to draw definite conclusions regarding the mechanisms whereby PKCε constructs elicit processes. To exclude the possibility that the increase in process-bearing cells is not due to a selection of cells with process-inducing capacity, the number of SH-SY5Y cells expressing EGFP or εRDE following transfection were counted. There was a lower percentage of εRDE-expressing cells (4.1 ± 0.5% of EGFP- versus 2.8 ± 0.6% εRDE-expressing cells), but this difference is too low to account for the increase in process-bearing cells (≤5% in EGFP- to 32% in εRDE-expressing cells). Furthermore, the few processes that could be observed in EGFP-expressing cells were much shorter than processes in cells transfected with PKCε constructs. This was also true for EGFP-expressing cells that were kept in culture for up to 4 d. This suggests that transfection with PKCε constructs does not result in an enhancement of a basal rate of process generation, but rather induces some events that eventually lead to the generation of neurite-like processes. This process generation may be mediated via cytoskeletal mechanisms, effects on the interaction of the cell with the substratum, or some other mechanism. It does not seem to involve altered expression of differentiation-coupled genes, since no increase in expression of NPY or synaptophysin, in the cell bodies or the processes, could be observed in εPSC1V3E-overexpressing cells. These proteins are elevated upon neuronal differentiation of neuroblastoma cells. Thus, it is likely that PKCε overexpression induces processes in undifferentiated cells and does not elicit a complete neuronal differentiation program. Both α-tubulin and NF-160 were present at apparently similar levels in the processes in εPSC1V3E-overexpressing cells and in neurites in neuronally differentiated neuroblastoma cells, indicating that the processes induced by the PKCε fragment to some extent display neuronal features. Such a dissociation between the physical induction of neurites and the accompanying increase in neuronal differentiation markers generally present in neurites has also been observed after overexpression of a constitutively active phosphatidylinositol 3 kinase in PC12 cells . In conclusion, this study demonstrates that PKCε, but not PKCα, βII, or δ, induces neurite-like processes in neuroblastoma cells and this effect can be ascribed to a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 domain. Identification of a dominant-negative construct derived from this region indicates that this effect of PKCε is of relevance for neurite outgrowth during neuronal differentiation. | Study | biomedical | en | 0.999997 |
10330402 | Anti–PI3-K p85 subunit antiserum was purchased from Upstate Biotechnology. Polyclonal antibodies against PKB(Akt), ERK, JNK, p38MAPK, MEK1, and MKK6 were obtained from Santa Cruz Biotechnology . Monoclonal antibodies against c-Myc and Flag were purchased from Santa Cruz Biotechnology and Sigma Chemical Co. , respectively. Construction of the caldesmon promoter plasmid, GP3CAT, was described previously . The expression vector containing the constitutively active form of the c-Myc–tagged PI3-K p110α subunit (pCMV5p110αact) was kindly provided by Drs. H. Kurosu and T. Katada (Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo). This cDNA was constructed by Hu et al. and was inserted downstream of the cytomegalovirus promoter, pCMV5. Expression vectors of constitutively active and dominant-negative forms of MEK1 and MKK6, and Flag-tagged ERK2 and Flag-tagged p38MAPK were kindly provided by Dr. K. Sugiyama ( Boehringer Ingelheim), Drs. M. Hibi and T. Hirano (Osaka University, Medical School), and Dr. E. Nishida (Graduate School of Science, Kyoto University). Mutant cDNAs of MEK1 and MKK6 were constructed as described elsewhere , and were inserted downstream of the cytomegalovirus promoter of pCS2+ or the SRα promoter of pcDLSRα296. In this study, we used expression vectors constructed in pCS2+ for active and dominant-negative forms of MEK1 (pCS2+MEK1act and pCS2+MEK1DN, respectively) and MKK6 (pCS2+MKK6act and pCS2+MKK6DN, respectively). A PKB(Akt) cDNA was amplified by reverse transcriptase PCR using human placental mRNA as a template, and the accuracy of its sequence was checked. A PKB(Akt) cDNA thus obtained was inserted downstream of the cytomegalovirus promoter of pCS2+c-Myc–tagged (MT) (pCS2+MT-PKB(Akt)wt for expression of c-Myc–tagged wild-type PKB(Akt). The expression plasmid of c-Myc–tagged constitutively active form of PKB(Akt), pCS2+MT-PKB(Akt)act, was constructed as described previously . Isolated gizzard SMCs were prepared from 15-d-old chick embryo gizzards as described elsewhere , and cultured on laminin-coated six-well plates with the indicated growth factors under kinase inhibited or stimulated conditions. Vascular SMCs were isolated from 5-wk-old rat aortae by enzyme-disperse methods as follows. Aortae were dissected under sterile conditions, minced well with scissors, and incubated at 37°C in 0.1% collagenase for 30 min, followed by incubation in the mixtures of 0.07% collagenase and 0.03% elastase for 90 min. Dispersed single cells were separated from undigested tissues by filtration, and were collected by centrifugation. The cells thus obtained were washed twice with growth factor–free basal medium (DME supplemented with 0.2% BSA), and were cultured in the medium containing IGF-I or PDGF-BB on laminin-coated culture plates. Treatment with specific inhibitors for ERK kinase (MEK1), PD98059 and/or for p38MAPK, SB203580, was performed as follows: gizzard or vascular SMCs were preincubated for 1 h in growth factor–free basal medium (DME supplemented with 0.2% BSA) containing the indicated amounts of inhibitors, and then stimulated with medium containing the indicated growth factors with or without inhibitors. Ligand-induced contractility of cultured SMCs was monitored as follows. The SMCs were cultured under indicated conditions for 3 d, and then washed with PBS, followed by stimulation with basal culture medium containing 1 mM carbachol for 1 min. Contractility of cultured SMCs was observed with an Olympus microscope, and the same fields before and after carbachol treatment were photographed. 2 μg of total RNA from precultured or cultured SMCs under the indicated conditions were separated on 1.0% agarose-formaldehyde denaturing gels, and then transferred to nylon membranes. A caldesmon cDNA fragment (nucleotides 286 to 810) and a calponin cDNA fragment (nucleotides 1 to 867) were used as probes to monitor the expression of respective mRNAs. This caldesmon cDNA fragment, which contains parts of exons 2 and 3a is a common probe for the h- and l- caldesmons . In our previous studies using specific probes for h- or l- caldesmon, we demonstrated that the full lengths of h- and l- caldesmon mRNAs are 4.8 and 4.1 kb, respectively . Probes were labeled with 32 P on the antisense strands and used for hybridization under the following conditions: 42°C for 16 h in 50% formamide, 6× SSC, 10× Denhardt's solution (1× Denhardt's solution is 0.02% polyvinylpyrrolidone and 0.02% BSA), 0.5% SDS, and 0.5 mg/ml denatured herring sperm DNA. The blots were washed in 0.1× SSC containing 0.1% SDS at 52°C, and visualized by autoradiography. To quantify the amount of RNA loaded, ribosomal RNAs were stained with 0.02% methylene blue. Total protein of the cell lysates from SMC cultures was separated by SDS-PAGE and transferred to nitrocellulose membranes. Detection of target proteins on the membranes was performed using an ECL Western blotting detection kit ( Amersham Pharmacia Biotech ) with the indicated polyclonal antibodies. Phospholipid mixtures (2 mg/ml) containing phosphatidylinositol (PI) and phosphatidylserine (PS) were dried under a stream of nitrogen, and sonicated in 10 mM Hepes (pH 7.4) in a bath sonicator at 0°C for 15 min. 10 μl of the resulting vesicles (PI/PS) were used as a substrate for PI3-K. The preparation of cell extracts and immunoprecipitation for PI3-K were performed at 4°C. The cultured cells were washed three times with ice-cold PBS, and then lysed in 550 μl of lysis buffer (20 mM Tris-HCl [pH 7.5], 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1% NP-40, 50 mM NaF, 1 mM Na 3 VO 4 , 50 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). After gentle shaking for 30 min, the cell extracts were obtained by centrifugation in a microfuge at 13,000 rpm for 5 min. The amount of PI3-K p85 subunit in the cell extracts was determined by Western blotting using antiserum against the PI3-K p85 subunit. The extracts containing equal amounts of PI3-K p85 subunit were precleaned with control rabbit IgG coupled protein A–Sepharose for 30 min. The PI3-K was immunoprecipitated with antiserum against the PI3-K p85 subunit followed by protein A–Sepharose. The immunoprecipitates were washed twice with lysis buffer, twice with 100 mM Tris-HCl (pH 7.5), 0.5 M LiCl, 1 mM DTT, and 0.2 mM Na 3 VO 4 , and three times with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 0.2 mM Na 3 VO 4 . All washes were performed at 4°C. The reaction mixtures (50 μl), containing the immunoprecipitates in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl 2 , 0.5 mM EGTA, 10 μM ATP, 5 μCi γ-[ 32 P]ATP, and 20 μg of PI/PS were incubated at 30°C for 10 min. The reactions were terminated and the lipids were extracted by the addition of CHCl 3 /MeOH (1:2). The mixture was then vortexed and cleared by centrifugation. The extracted products were separated by thin-layer chromatography in a developing solution composed of CHCl 3 / MeOH/4 M NH 4 OH (9:7:2). The production of phosphatidylinositol-3-phosphate was detected by autoradiography. Cell lysis buffers were as follows: 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 1 mM Na 3 VO 4 , 10 mM β-glycerophosphate, 50 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin for the ERK and PKB(Akt) assays; 20 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mM DTT, 120 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 1 mM Na 3 VO 4 , 10 mM β-glycerophosphate, 50 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin for the JNK assays; and 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 2 mM Na 3 VO 4 , 10 mM β-glycerophosphate, 50 μg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin for the p38MAPK assays. The cell extracts were immunoprecipitated with specific antibodies against individual protein kinases, and the immunoprecipitates were washed thoroughly with their lysis buffers and then kinase assay buffers, and incubated with their respective substrates and 5 μCi γ-[ 32 P]ATP for 30 min at 30°C. The reaction products were analyzed by 15% SDS-PAGE. Reaction mixtures for the kinase assays were as follows: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 μM protein kinase A inhibitor, 1 mM DTT, and 25 μg histone H2B for the PKB(Akt) assay; 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 μM protein kinase A inhibitor, 1 mM DTT, and 25 μg myelin basic protein (MBP) for the ERK assay; 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 μM protein kinase A inhibitor, 1 mM DTT, 1 mM Na 3 VO 4 , and 1 μg GST-Jun (1-79) for the JNK assay; and 20 mM Hepes (pH 7.4), 20 mM MgCl 2 , 20 mM β-glycerophosphate, 1 μM protein kinase A inhibitor, 2 mM DTT, and 1 μg GST-ATF2 (1-96) for the p38MAPK assay. The caldesmon promoter activity was analyzed using the chloramphenicol acetyltransferase (CAT) construct, GP3CAT, according to the method described previously . The SMCs prepared as described above were seeded onto laminin-coated six-well plates, and cultured in the indicated medium for 1 or 3 d. Transfection was carried out using Trans IT™-LT1, polyamine transfection reagents (Pan Vera Corporation). Complex mixtures composed of 10 μg of trans IT™- LT1 reagent and 2 μg of GP3CAT, 1 μg of control plasmid carrying the luciferase gene under the Rous sarcoma virus (RSV) promoter (RSV-luciferase), and 1 μg of either control expression plasmid (pCMV5, pCS2+, or pCS2+MT), expression plasmid carrying a c-Myc–tagged constitutively active form of PI3-K p110α subunit, a c-Myc–tagged wild-type or a constitutively active form of PKB(Akt) (pCMV5p110αact, pCS2+ MT-PKB(Akt)wt or pCS2+MT-PKB(Akt)act), or either or both of expression vectors carrying constitutively active and dominant-negative forms of MEK1 (pCS2+MEK1act and pCS2+MEK1DN) and MKK6 (pCS2+MKK6act and pCS2+MKK6DN), were added to the cells in Opti minimum Eagle's medium ( GIBCO BRL ). After a further 4-h incubation, the medium was replaced with DME supplemented with 0.2% BSA plus 2 ng/ml IGF-I or 20 ng/ml PDGF-BB, and the transfected cells were harvested 48 h later. Standardization of transfection efficiency was performed by measuring luciferase activity as described previously . The cell extracts containing equal amounts of luciferase activity were used for the CAT assay. The transfection experiments were repeated at least three times on duplicate cultures with two or three different plasmid preparations. The CAT activities were quantified by Scanning Imager (Molecular Dynamics). The effects of forced expression of MEK1 and MKK6 in cultured SMCs were analyzed as follows. The indicated amounts of control expression plasmid and either or both of expression plasmids carrying active or dominant-negative MEK1 and MKK6 were transfected into cultured SMCs together with 1 μg of a reporter plasmid carrying the β-galactosidase gene downstream from the SV-40 early promoter. Total RNA was isolated from the transfected cells and the expression levels of caldesmon and calponin mRNAs were analyzed by Northern blotting as described above. Transfection efficiencies were determined by staining for β-galactosidase activity from the reporter plasmid using 5-bromo-4-chloro-3-indolyl-β- d -galactoside (X-gal) as a substrate. Transfection was carried out as described above in Promoter Analysis and Transfection. In the cases of PI3-K and PKB(Akt) assays, SMCs were transfected with 3 μg of respective expression plasmids of constitutively active form of c-Myc–tagged PI3-K p110α subunit, pCMV5p110αact, or wild-type or constitutive active form of c-Myc–tagged PKB(Akt), pCS2+MT-PKB(Akt)wt or pCS2+MT-PKB(Akt)act. Two micrograms of expression plasmid of each Flag-tagged ERK2 or Flag-tagged p38MAPK was cotransfected with 2 μg of either expression plasmid of active or dominant-negative MEK1 or MKK6, or control plasmid. In both cases, SMCs were cultured under nonstimulated conditions after transfection. 2 d later, SMCs were stimulated under indicated conditions. The cell extracts containing the equal amounts of proteins were precleaned with control mouse IgG coupled protein G–Sepharose for 30 min and immunoprecipitated with monoclonal antibody against c-Myc or Flag followed by protein G–Sepharose. The kinase activities were determined as described above in PI3-K assay and other protein kinase assays. Conditioned medium obtained from SMCs transfected with both expression plasmids carrying active MEK1 and MKK6 was filtered through a 0.22-μm membrane. The conditioned medium was heated to 100°C for 15 min or treated with trypsin (30 μg/ml) for 3 h at 30°C, followed by the addition of trypsin inhibitor at a 10-fold excess. The heat- or trypsin-treated conditioned medium was dialyzed against DME supplemented with 0.2% BSA at 4°C for 16 h, and adjusted to the concentration of IGF-I to 2 ng/ml. The SMCs were cultured in these medium for 3 d. Heparin–Sepharose affinity chromatography was carried out as follows. One ml of 50% slurry of heparin–Sepharose ( Amersham Pharmacia Biotech ) equilibrated with PBS was added to 20 ml of the conditioned medium and gently agitated for 5 h at 4°C. The mixture was poured over a 1-ml Prep Column (Bio-Rad Labs.), and the follow through fraction (non–heparin-binding) was collected. The column was rinsed with 10 vol of PBS and eluted stepwise with 1 ml of PBC containing NaCl (0.5, 1.0, and 1.5 M). Each fraction was collected and desalted by dialysis against DME supplemented with 0.2% BSA through a membrane of 3-kD cutoff (Spectrum). Aliquots of each fraction were diluted (1:4) with DME supplemented with 0.2% BSA and then added to SMC cultures. Treatment with specific inhibitor of EGF receptor kinase, AG1478, was performed as follows: SMCs cultured for 2 d under IGF-I–stimulated conditions were preincubated for 1 h in DME supplemented with 0.2% BSA containing 1 μM AG1478, and then stimulated with the conditioned medium containing the same concentration of this drug for 3 d. Total RNAs from cultured SMCs were extracted and the expression patterns of caldesmon and calponin mRNAs were analyzed by Northern blotting. Control medium was obtained from culture supernatant of SMCs transfected with expression plasmid alone. We have recently established a novel culture system of gizzard SMCs in which they maintain a differentiated phenotype for a long culture period. Of growth factors and cytokines examined, IGF-I is the most potent for maintaining the differentiated SMC phenotype as defined by the expression of SMC-specific molecular markers, cell morphology, and function . On the other hand, PDGF-BB, bFGF, and EGF potently induce SMC de-differentiation . In 3- and 5-d–cultured SMCs stimulated with PDGF-BB, bFGF, or EGF, h- caldesmon mRNA converts to l- caldesmon mRNA, and total h- and l- caldesmon mRNAs decrease to 20% of the levels seen in precultured SMCs. Calponin mRNA is also downregulated to a negligible level . However, the levels of h- caldesmon and calponin mRNAs in cultured SMCs under IGF-I–stimulated conditions are identical with those seen in precultured cells . Similar results are obtained using α- and β-tropomyosins and α1 integrin , which are also SMC-specific molecular markers (data not shown). With regard to cell morphology and function, cultured SMCs under IGF-I–stimulated conditions showed a spindle-like shape, formed a meshwork structure, and displayed carbachol-induced contraction. In contrast, SMCs stimulated with PDGF-BB, bFGF, or EGF showed a fibroblast-like shape and lost the contractility , indicating that PDGF-BB, bFGF, and EGF are potent factors for inducing SMC de-differentiation. Our previous studies revealed that the PI3-K/PKB(Akt) pathway triggered by IGF-I plays a critical role in maintaining the differentiated SMC phenotype . To investigate the downstream signaling pathways involving in SMC de-differentiation triggered by PDGF-BB, bFGF, or EGF, we analyzed several kinases including ERK, JNK, p38MAPK, PI3-K, and PKB(Akt). PDGF-BB, bFGF, and EGF all activated ERK and p38MAPK . Their maximum activations were found at 10 min after stimulation. bFGF and EGF also activated JNK, whereas PDGF-BB did not . IGF-I had no effect on ERK , JNK , and p38MAPK . These data suggest that growth factors inducing SMC de-differentiation coordinately activate the ERK and p38MAPK pathways. As demonstrated previously , IGF-I potently activated PI3-K and PKB(Akt) ; the maximum activation of PI3-K was achieved at 10 min after IGF-I stimulation and this activation reduced thereafter, while the activation of PKB(Akt) by IGF-I (2 ng/ml) lasted for more than 180 min. The activation of PI3-K and PKB(Akt) by IGF-I was suppressed by specific PI3-K inhibitors, wortmannin or LY249002 (data not shown), indicating that the PKB(Akt) activation exclusively depends on the PI3-K activity. No significant activation of PI3-K and PKB(Akt) was observed in SMCs stimulated by either bFGF or EGF . Among the three growth factors inducing SMC de-differentiation, PDGF-BB was the only one that could activate PI3-K and PKB(Akt) . The PKB(Akt) activation by PDGF-BB was more potent than that by IGF-I at 15 min after growth factor stimulation, whereas this activation was transient, but retained at a substantial level for 180 min. By contrast, the PKB(Akt) activation by IGF-I was sustained at a high level for more than 180 min. These results suggest the possibility that in addition to PI3-K, PDGF-BB activates PKB(Akt) mediated through another unknown cascade. It is curious that PDGF-BB, which is a potent factor inducing SMC de-differentiation , triggered the dual signaling pathways mediated through both PI3-K/PKB(Akt) and two MAPKs, ERK and p38MAPK . To simplify the PDGF-BB–triggered signaling pathways, we examined the effects of specific MAPK inhibitors, PD98059 for ERK kinase (MEK1) and SB203580 for p38MAPK, on the PDGF-BB–stimulated SMC phenotype. Either PD98059 or SB203580 specifically inhibited the PDGF-BB–induced activation of ERK or p38MAPK, respectively, to near basal levels , but had no effect on PI3-K and PKB(Akt) (data not shown). Treatment with either PD98059 or SB203580 only slightly suppressed the PDGF-BB–induced isoform conversion of caldesmon mRNA and downregulation of caldesmon and calponin mRNAs . However, simultaneous treatment with both drugs strongly suppressed the PDGF-BB–induced SMC de-differentiation as monitored by the expression of caldesmon and calponin mRNAs . In addition to these molecular events, both drugs could also rescue the morphological alteration from a spindle-like shape to a fibroblast-like shape change and a loss of contractility induced by PDGF-BB . As a control, treatment with individual drug resulted in less significant effect on cell morphology and function. Table I shows the effects of PD98059 and/or SB203580 on carbachol-stimulated contractility of SMCs under various culture conditions. Further, both drugs only slightly delayed the induction of SMC de-differentiation by bFGF or EGF, but did not prevent SMC de-differentiation (data not shown). Promoter analyses of the caldesmon gene further support these findings . We used the caldesmon promoter/CAT construct, GP3CAT, which produces the high promoter activity in differentiated SMCs . The promoter activity in SMCs stimulated by PDGF-BB reduced to 30% of that by IGF-I . Even under PDGF-BB–stimulated conditions, inhibition of both the ERK and p38MAPK pathways by their specific inhibitors or the forced expression of active PI3-K or active PKB(Akt) could protect such reduction . These results suggest that PDGF-BB displays the dual function in maintaining the differentiated SMC phenotype mediated through the PI3-K/PKB(Akt) pathway and inducing SMC de-differentiation by the ERK and p38MAK pathways. Thus, changes in the balance between the strengths of the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine the SMC phenotype. The MAPK signaling cascades are involved in a variety of cell functions . Dual phosphorylation on Thr and Tyr within the Thr-Xaa-Tyr motif catalyzed by MAPK kinases is essential for MAPK activation . MEK1 and MKK6 are specific upstream kinases for ERK and p38MAPK, respectively . To investigate the direct involvement of ERK and p38MAPK in SMC de-differentiation, we examined the effects of active or dominant-negative MEK1 and/or MKK6 on the caldesmon promoter activity in SMCs under IGF-I–stimulated conditions. We determined the respective MAPK kinase activity by in vitro kinase assay . In this experiment, expression plasmids carrying active or dominant-negative MAPK kinases were cotransfected with expression plasmid carrying Flag-tagged ERK or Flag-tagged p38MAPK into cultured SMCs, and Flag-tagged proteins were immunoprecipitated from the cell lysates with anti-Flag monoclonal antibody. The ERK or p38MAPK activities were potently enhanced in SMCs cotransfected with active MEK1 or MKK6 under nonstimulated or PDGF-BB–stimulated conditions . Even when SMCs were stimulated by PDGF-BB, their enhancements were strongly abolished by the forced expression of dominant-negative MEK1 or MKK6 . The expressions of active and dominant-negative MEK1 or MKK6 proteins were confirmed by immunoblotting (data not shown). The caldesmon promoter activity in differentiated SMCs under IGF-I–stimulated conditions was analyzed by the forced expression of active or dominant-negative MAPK kinases . The promoter activity was not affected by either or both dominant-negative MEK1 and/or MKK6. Transfection with either active MEK1 or MKK6 significantly reduced the caldesmon promoter activity, while cotransfection with both active kinases further suppressed the promoter activity. Since the SV-40 promoter was not affected by the forced expression of MEK1 and/or MKK6 (data not shown), suppression of the caldesmon promoter activity by active MEK1 and MKK6 was specific. These results indicate that the ERK and p38MAPK pathways are directly involved in the induction of SMC de-differentiation. We further examined the effects of active and dominant-negative MEK1 or MKK6 on the endogenous expression of caldesmon and calponin mRNAs and on cell morphology. Transfection with either active kinase alone or cotransfection with dominant-negative kinases had less significant effects on caldesmon and calponin mRNAs in 2- or 4-d–cultured SMCs . Even under IGF-I–stimulated conditions, cotransfection of active MEK1 and MKK6 potently induced isoform conversion of caldesmon mRNA and downregulation of caldesmon and calponin mRNAs. These changes progressed during culture; the endogenous expressions of caldesmon and calponin mRNAs in 4-d–cultured SMCs after cotransfection were comparable to those seen in 4-d–cultured SMCs stimulated by PDGF-BB . These results indicate that after cotransfection with active MEK1 and MKK6, almost all of cultured SMCs alter their phenotype to de-differentiation. To compare transfection efficiency and cell morphology, cultured SMCs stimulated by IGF-I were transfected with control plasmid or both expression plasmids carrying active MEK1 and MKK6 together with β-galactosidase expression plasmid. As revealed by β-galactosidase staining, transfection efficiencies were ∼25%. The SMCs transfected with control vector remained to show a spindle-like shape . In the case of SMCs cotransfected with active MEK1 and MKK6, all of the β-galactosidase-stained and -unstained cells converted from a spindle-like shape to a fibroblast-like shape . The transfection efficiency of both active MEK1 and MKK6 was correlated with SMC-specific marker gene expression and cell morphology . These data suggest that SMCs cotransfected with active MEK1 and MKK6 secrete some factor(s) which induces de-differentiation of surrounding normal SMCs. To further characterize such a factor(s), conditioned medium (CM1) obtained from SMCs cotransfected with expression plasmids carrying active MEK1 and MKK6 was prepared. Fig. 8 A shows the expression of caldesmon and calponin mRNAs in 3-d–cultured SMCs stimulated with the conditioned medium. Even when SMCs were stimulated by IGF-I, the conditioned medium potently induced isoform conversion of caldesmon mRNA and downregulation of caldesmon and calponin mRNAs , whereas control medium (CM2) obtained from SMCs transfected with control plasmid alone did not . Further, the conditioned medium enhanced the ERK, JNK, and p38MAPK activities . These results exclude the possibility of retransfection with residual expression vectors, because MEK1 and MKK6 proteins derived from expression plasmids were not detected in cultured SMCs by immunoblotting (data not shown). Thus, this study revealed that the forced activation of both ERK and p38MAPK in SMCs induces the secretion of some factor(s) which initiates de-differentiation of surrounding normal SMCs in a paracrine manner. Heat or trypsin treatment of the conditioned medium completely abolished the activity inducing SMC de-differentiation , suggesting that the factor(s) is a protein in nature. The conditioned medium was further fractionated using a heparin–Sepharose column. The flow through (non–heparin-binding) fraction retained the potent de-differentiation activity, while the eluted fraction by 0.5, 1.0, and 1.5 M NaCl did not . PDGFs and bFGF show heparin-binding abilities; the former was eluted with 0.5 M NaCl and the latter with 1.5 M NaCl (data not shown). By contrast, EGF is known as a non–heparin-binding growth factor, suggesting a candidate for the active protein factor. A specific inhibitor for EGF receptor kinase, AG1478, only slightly inhibited the conditioned medium-induced de-differentiation, but this effect was less significant . Therefore, the active protein factor(s), which induces SMC de-differentiation, in the conditioned medium is considered to be different from PDGFs, bFGF, and EGF. To examine whether vascular SMCs could be regulated by the same signaling pathways as revealed in gizzard SMCs, we first applied our culture system of gizzard SMCs to vascular SMCs. We isolated rat vascular SMCs by the enzyme-disperse method, and cultured them on laminin-coated plates under IGF-I–stimulated conditions. Because of difficulty to obtain a lot of cell numbers from rat aortae, we observed cell morphology and ligand-induced contractility to determine the vascular SMC phenotype . Vascular SMCs could also maintain a spindle shape for more than 2 weeks under IGF-I–stimulated conditions, and showed ligand-induced contractility . A blockade of the PI3-K pathway by LY294002 resulted in a morphological change from a spindle shape to a fibroblast-like shape and a loss of contractility . In-consistent with the case of gizzard SMCs, PDGF-BB rapidly induced de-differentiation of vascular SMCs as monitored by cell morphology and ligand-induced contractility . Even under PDGF-BB–stimulated conditions, simultaneous treatment with PD98059 and SB203580 could retain a spindle shape and contractility . As a control, treatment with each drug individually was less significant effect on the PDGF-BB– induced de-differentiation (data not shown). Furthermore, the conditioned medium obtained from gizzard SMCs transfected with both active MEK1 and MKK6 remarkably induced de-differentiation of vascular SMCs . Therefore, these data also suggest that the PI3-K–mediated signaling pathway plays a vital role in maintaining a differentiated phenotype of vascular SMCs and the ERK and p38 MAPK pathways are coordinately involved in de-differentiation of vascular SMCs. Under pathological conditions, phenotype of SMCs can change from a differentiated state to a de-differentiated state in vivo and in vitro. During de-differentiation, SMCs show dramatic and irreversible alterations in their cell shape, function, and expression of SMC-specific molecular markers. Long spindle-shaped cells change to fibroblast-like cells, accompanied by losses in a ligand-induced contractility and SMC-specific molecular marker expression. Since there has not been a primary culture system available for SMCs or SMC-derived cell lines in which they can maintain a fully differentiated phenotype, the intracellular signaling pathways regulating the SMC phenotype have not been well characterized. Recently, we established a novel culture system in which gizzard SMCs can maintain a differentiated phenotype for a long culture time . In this culture system, IGF-I is the most potent for maintaining the differentiated SMC phenotype, and the IGF-I–triggered signaling pathway, PI3-K/PKB- (Akt), plays a critical role in this maintenance. Here, we investigated the signaling pathways inducing SMC de-differentiation and compared them with the PI3-K/PKB- (Akt) pathway. It has been reported that PDGF, EGF, bFGF, or angiotensin II enhance cell proliferation or hypertrophy through the activation of the ERK signaling cascade in passaged vascular SMCs . MAPKs such as JNK and p38MAPK are also activated in response to various cellular stresses . Angiotensin II and phenylephrine, which induce acute hypertension, enhance the ERK and JNK activities in aortic, carotid and femoral arteries , and endothelin activates both of these kinases in proliferative airway SMCs . p38MAPK, ERK, and/or JNK are also activated by balloon injury of carotid arteries . These findings suggest a close association of MAPK cascades with smooth muscle disorders. However, the direct involvement of these signaling cascades in regulating the SMC phenotype has been unknown. As a first step, we used a novel culture system of gizzard SMCs and demonstrated that activations of the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways are directly involved in maintaining SMC differentiation and inducing SMC de-differentiation, respectively. This conclusion is based on the following findings. First, the signaling pathways in regulating the phenotypic determination of gizzard SMCs were distinctly different; the PI3-K/PKB(Akt) pathway played a critical role in maintaining the differentiated SMC phenotype and the ERK and p38MAPK pathways triggered by PDGF-BB, bFGF, and EGF were closely associated with SMC de-differentiation . Second, among the three growth factors inducing SMC de-differentiation, PDGF-BB only triggered both the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways. When both the MAPK pathways were blocked by their specific inhibitors, PD98059 and SB203580, or when SMCs were transfected with active PI3-K or PKB(Akt), PDGF-BB in turn initiated to maintain the differentiated SMC phenotype . Third, even when SMCs were cultured under IGF-I–stimulated conditions, the forced activation of both the MAPK pathways by the coexpression of active MEK1 and MKK6 potently induced SMC de-differentiation . Fourth, SMCs cotransfected with active MEK1 and MKK6 secreted a nondialyzable and heat-labile protein factor(s), which induced de-differentiation of surrounding normal SMCs . Finally, the same signaling pathways as described above were observed to be involved in regulating the vascular SMC phenotype . Since IGF-I enhances the proliferation and migration of cultured vascular SMCs , it has been considered to be an important growth factor in the progression of atherosclerosis. These findings are, however, obtained using passaged SMCs. In this paper, we demonstrated using a novel SMC culture system that IGF-I solely triggers the PI3-K/PKB(Akt) pathway, but not the MAPK pathways . Further, IGF-I did not affect the proliferation of SMCs (data not shown). These properties of IGF-I signaling made it possible to maintain the differentiated SMC phenotype for more than 2 wk in primary culture. Since rapamycin had no effect on the differentiated SMC phenotype under IGF-I–stimulated conditions , p70 ribosomal S6 kinase (p70 S6K ) is unlikely to be a downstream target of PI3-K/PKB(Akt). Further study is required to identify the downstream targets of PI3-K/ PKB(Akt) in SMCs. It has been reported that IGF-I and its downstream signaling, PI3-K/PKB(Akt), play a role in protection against programmed cell death . We observed neither a significant decrease in cell numbers nor DNA fragmentation in our SMC cultures even under nonstimulated conditions or in the presence of IGF-I neutralizing antibodies (data not shown). Therefore, cultured SMCs might secrete anti-apoptotic factor(s) in the absence of IGF-I, and the maintenance of the differentiated SMC phenotype by IGF-I would be distinct from an anti-apoptotic action. In passaged SMCs, IGF-I enhanced the JNK and p38MAPK activities (data not shown). These results suggest that the downstream signalings of IGF-I might be modulated during cell passage. Actually, differentiated SMCs rapidly change their phenotype under serum-stimulated conditions . Since passaged SMCs do not represent a stable differentiated state, studies reported previously might not be able to reveal the IGF-I's function and signaling in differentiated SMCs. In this study, we used SMCs that showed well-characterized and stable phenotypes. We analyzed the relationship between the modulation of SMC phenotype and cell proliferation. Although serum-induced SMC de-differentiation was concomitant with cell proliferation, other growth factors that trigger SMC de-differentiation, such as PDGF-BB, bFGF, and EGF, did not significantly induce SMC proliferation (data not shown). This result also suggests that SMC de-differentiation is not essentially associated with cell proliferation. It has been reported that de-differentiated SMCs produce and secrete PDGF which further promotes cell proliferation and migration in an autocrine/paracrine manner . PDGF is also known to promote the activation of ERK and p38MAPK in passaged SMCs . In our culture system, PDGF-BB triggered the dual signaling pathways mediated by PI3-K/PKB(Akt) and two MAPKs, ERK and p38MAPK. Under these culture conditions, PDGF-BB stimulation resulted in SMC de-differentiation. When the two MAPK pathways were blocked by their specific inhibitors, PD98059 and SB203580, PDGF-BB stimulation, in turn, initiated to maintain SMC differentiation . bFGF and EGF are known to activate ERK and also to induce proliferation of SMCs . In our culture system, both growth factors activated ERK and p38MAPK and potently induced SMC de-differentiation . However, PD98059 and SB203580 could not prevent such de-differentiation (data not shown). This is because the signaling pathway mediated by PI3-K/PKB(Akt) was not activated by bFGF or EGF . In the present culture system, bFGF and EGF also activated JNK, but IGF-I and PDGF-BB did not . It is, therefore, unlikely that JNK is involved in regulating the SMC phenotype. PDGF-BB reduced the caldesmon promoter activity and this reduction could be overcome by the forced expression of active PI3-K or PKB(Akt), or by treatment with both PD98059 and SB203580 . Further, the activation of ERK and p38MAPK by the forced expression of both active MEK1 and MKK6 could overcome the PI3-K/ PKB(Akt) pathway triggered by IGF-I, resulting in the induction of SMC de-differentiation . These data support our hypothesis that the SMC phenotype would be determined by the balance between the strengths of the signaling pathways mediated by PI3-K/PKB(Akt) and by ERK and p38MAPK. Curiously, even though the transfection efficiencies of both expression vectors carrying active MEK1 and MKK6 were only 25%, almost all of SMCs came to de-differentiate as monitored by cell morphology and endogenous expression of SMC-specific molecular markers . These findings indicate that the production and secretion of some de-differentiation-inducing factor(s) occur in SMCs in which both the ERK and p38MAPK pathways are constitutively activated. We have not yet identified such a factor(s), but the conditioned medium from these cells activated three MAPK pathways (ERK, p38MAPK, and JNK). The activating factor(s) was a heat-labile, non– heparin-binding protein factor . From these biochemical properties , we excluded the possibility that PDGF-BB, bFGF, and EGF would be the main factor inducing SMC de-differentiation in the conditioned medium. Further study is required to identify such a factor(s). Anyway, this study provides a evidence that only a small portion of de-differentiated SMCs secretes a protein factor(s) for the surrounding normal SMCs to be de-differentiated. These findings could be helpful to understand the progression of smooth muscle disorders such as atherosclerosis. We then applied a culture system of gizzard SMCs to that of vascular SMCs and investigated the signaling pathways in regulating the vascular SMC phenotype . Like gizzard SMCs, IGF-I potently maintained a differentiated phenotype of vascular SMCs and a specific PI3-K inhibitors, LY24002, prevented this IGF-I's action. Treatment with two MAPK pathway inhibitors, PD98059 and SB203580, could rescue the PDGF-BB–induced de-differentiation of vascular SMCs. Further, the conditioned medium obtained from gizzard SMCs also induce de-differentiation of vascular SMCs. These results strongly suggest that a culture system of gizzard SMCs is applicable for that of vascular SMCs and that the distinct signaling pathways mediated by PI3-K/PKB(Akt) and two MAPKs are also involved in the phenotypic determination of vascular SMCs. Our results presented here are summarized in Fig. 10 . The signaling pathway mediated by PI3-K/PKB(Akt) is required to maintain a differentiated phenotype of visceral and vascular SMCs, while the activation of ERK and p38MAPK leads to SMC de-differentiation of both types of SMCs. Thus, the signaling pathways in regulating the phenotypic determination are considered to be essentially the same in visceral and vascular SMCs, and the SMC phenotype would be regulated by the balance between the strengths of these signaling pathways. Although visceral and vascular SMCs originate from different precursors, our present findings provide a further insight in the common molecular mechanism of phenotypic determination of two types of SMCs. This is because visceral and vascular SMCs have common characteristics with respect to cell structure, function, and expression of molecular markers as follows. The main function of both types of SMCs is contraction, which is Ca 2+ -dependent and controlled by a myosin-linked, actin-linked dual regulation . Both types of SMCs are rich in myofibrils and are organized in a three-dimensional direction with two prominent electron dense structures such as the dense body in the cytoplasm and the dense membrane (dense plaque) in cell–cell contact. Further, contractile and cytoskeletal proteins are also specifically expressed in and serve as specific molecular markers for differentiated SMCs. The expression patterns of these molecular markers are identical in both visceral and vascular SMCs and their expression mechanisms, including transcription and splicing, are also regulated in common ways . Further studies are required to understand the detailed signaling pathways in regulating the vascular SMC phenotype and the functional linkage between such signaling pathways and SMC-specific gene regulation machineries, and to apply these findings to SMC disorders. | Other | biomedical | en | 0.999997 |
10330403 | To express Myc-tagged forms of Axin, the coding region of mouse Axin (form 1) sequence was inserted downstream of the SP6 promoter in the vector pCS2-MT . DNA inserts amplified by PCR with Pfu DNA polymerase (Stratagene) were used for the construction of plasmids containing small fragments, such as Ax455-552, Ax497-672, etc. Full-length (FL) Xenopus APC cDNA was inserted downstream of the CMV/SP6 promoter in the pCS2 vector, with a 5′ 5× VSV-G (vesicular stomatitis virus glycoprotein) tag (YTDIEMNRLGK). Human Myc epitope-tagged APC constructs used for direct binding assays were described previously . pET32 vector (Novagen, Inc.) was used to produce the His·S tagged Axin fusion proteins. The reading frame of all constructs was confirmed by sequencing and detection of expected sized bands in Western blot or Coomassie brilliant blue R250 stained SDS–polyacrylamide gels. Plasmids for transfection and in vitro transcription/translation were isolated using the midi-prep kit (QIAGEN Inc.). Other constructs used included: Myc-tagged Xenopus β-catenin , hemagglutinin (HA)-tagged Xenopus β-catenin , HA-tagged Xenopus Dsh (gift of Dr. U. Rothbächer, University of Marseille, France), HA-tagged dominant negative human GSK3β (gift of Dr. X. He, Harvard Medical School, Boston, MA). Antibodies were purchased from the indicated sources: anti–β-catenin mouse mAb, clone 14, and anti-GSK3β mAb, clone 7, Transduction Laboratories; anti–VSV-G mAb P5D4, Boehringer Mannheim ; anti-Myc 9E10.2 mAb, Calbiochem-Novabiochem ; anti–HA-tag rabbit polyclonal antibodies (pAb), Santa Cruz Biotechnology ; and anti–β-galactosidase rabbit pAb, Cappel Laboratories and Organon Teknika Corp. Anti-Myc rabbit pAb was raised against the c–Myc-epitope tag (EQKLISEEDL) and affinity-purified. Anti-HA mAb 12CA5 was a gift from Dr. P. McCrea (M.D. Anderson Cancer Center, Houston, TX). 293 cells, obtained from the American Type Culture Collection, were cultured in DME/F12 medium (Mediatech) supplemented with 10% FBS (HyClone Laboratories Inc.) in humidified 6% CO 2 . Cells were transfected using a calcium phosphate mammalian cell transfection kit (5 Prime → 3 Prime, Inc.). The next day cells were collected and lysed and analyzed for transient expression of transfected DNAs. For IP and Western blot analysis, 293 cells were washed with PBS, pH 7.2, and lysed in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM NaF, 50 mM KH 2 PO 4 , 10 mM sodium molybdate, 20 mM Tris-HCl, pH 7.4, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 0.6 mM DTT, 2 mM sodium orthovanadate, 0.2 mM PMSF, 1% Triton X-100). After 20 min at 4°C with constant rotation, the lysate was centrifuged at 14,000 g for 15 min and the supernatant was saved. Protein concentration was measured by the Lowry method . For coimmunoprecipitation (coIP) of Axin/APC/ β-catenin/GSK3β complex, 150–300 μg (total protein) of cell lysate was incubated with 1–2 μg of an appropriate antibody in lysis buffer for 2 h at 4°C with constant rotation. 30 μl of protein A/G plus–agarose ( Santa Cruz Biotechnology ) was added and the incubation continued for an additional 1.5 h. Immunoprecipitates were pelleted and washed three times with lysis buffer. Immunoprecipitates were analyzed by SDS-PAGE (5% acrylamide for APC detection, 10% for detection of other proteins) and Western blot, using HRP-conjugated donkey anti–rabbit and sheep anti– mouse secondary antibodies ( Amersham Life Science) and the chemiluminescence system (RENAISSANCE™; NEN Life Science Products). Approximately 10–20 μg of protein was used to detect expression of the various constructs. Myc-APC constructs were produced with TNT coupled wheat germ extract system ( Promega ). These [ 35 S]Met-labeled proteins were incubated with 2 μg of bacterially expressed S-tagged Axin fusion proteins in 500 μl of buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 5 mM DTT, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 0.1 mM PMSF, 0.5% NP-40 at 37°C for 10 min and at 4°C for 10 min. The protein complexes were precipitated with 30 μl S-protein agarose (Novagen), washed three times with the same incubation buffer, and analyzed by SDS-PAGE (7.5% gel) and autoradiography. Lysates from cells transiently expressing VSV-G–tagged APC (VSV– APC) and Axin constructs were used for IP with anti–VSV-G P5D4 mAb. Immunoprecipitates were incubated with 1,000 U of λ-protein phosphatase ( New England Biolabs Inc. ) in 50 μl reaction buffer (50 mM Tris-HCl, pH 7.8, 5 mM DTT, 2 mM MnCl 2 , and 100 μg/ml BSA) at 30°C for 30 min. λ-Protein phosphatase-treated samples were separated by SDS-PAGE (5% gels) and VSV–APC was detected by immunoblot using the P5D4 mAb. mRNA was synthesized using SP6 polymerase ( Promega ) and dissolved in diethyl pyrocarbonate–treated water. For ventralization assays, 15 nl mRNA was injected in the subequatorial region of the two dorsal blastomeres of a four cell stage embryo. For axis duplication assays, 15 nl mRNA was injected in one ventral blastomere. Ventralization was scored at tailbud/tadpole stages according to the dorsoanterior index . Axis duplication was scored at neurula/tailbud stage, in four categories : (1) complete secondary axis (including cement gland), (2) partial axis (no cement gland), (3) vestigial axis (small remnants, blebs), and (4) normal. Only complete and partial axes were considered as bonafide secondary axes. Expression levels of Myc-tagged Axin constructs were determined from NP-40 extracts of late blastula–early gastrula embryos (stage 9–10 1/2) by SDS-PAGE and immunoblot, using the anti–Myc-tag 9E10.2 mAb. NP-40 buffer had the following: 1% NP-40, 100 mM NaCl, 10 mM Hepes, pH 7.4, 1 mM EDTA, with a cocktail of protease inhibitors (1 mM PMSF, 1 μg/ml pepstatin A, 2 μg/ml leupeptin, 4 μg/ml aprotinin, 10 μg/ml antipain, 50 μg/ml benzamidine, 10 μg/ml soybean trypsin inhibitor, 100 μg/ml iodoacetamide). Early cleaving embryos were coinjected with 1 ng Myc-tagged Axin (Myc– Axin) and 3 ng β-galactosidase mRNA. At stage 9–10, 10 embryos were homogenized in 500 μl 250 mM sucrose, 110 mM potassium acetate, 10 mM Hepes, pH 7.4, 2 mM magnesium acetate, 2 mM DTT, 1 mM EDTA supplemented with protease inhibitors. The homogenate was centrifuged for 5 min at 1,500 g , and the low speed pellet was extracted in NP-40 buffer. The low speed supernatant was fractionated further by centrifugation for 30 min at 100,000 g in a tabletop ultracentrifuge (TL-100; Beckman Instruments Inc. ) into a high speed pellet and supernatant. The fractions were analyzed for Myc–Axin and β-galactosidase by SDS-PAGE and immunoblot. FL Axin or various mutant constructs mRNA were injected into 4–8-cell stage embryos. At stage 9–10, pools of six embryos were extracted in 500 μl NP-40 buffer and each extract was incubated with 50 μl of Con A–agarose beads (75% slurry; Sigma Chemical Co. ) for 1–2 h at 4°C. The beads were spun down, the supernatant was collected (unbound fraction), the beads washed three times with 1 ml NP-40 buffer, and extracted by boiling in SDS-PAGE sample buffer (bound fraction). Levels of Myc–Axin constructs in bound and unbound fractions were analyzed by SDS-PAGE and immunoblot using the anti-Myc 9E10 mAb. HA-tagged β-catenin mRNA (75 pg) was coinjected with β-galactosidase mRNA (control) or various Axin mutant mRNAs. Amounts of mRNA injected were the following: β-galactosidase, 1 ng; FL Axin (Ax12-956), 1 ng; Ax12-531, 0.5 ng; Ax194-530, 0.5 ng; Ax194-672, 0.25 ng; AxΔ251-351, 1 ng; Ax331-956, 0.5 ng; and Ax531-956, 0.5 ng. Total amounts of injected mRNA were adjusted to 1.075 ng by addition of β-galactosidase mRNA. In some experiments , higher levels of β-catenin were tested using 0.75–1.5 ng mRNA. Embryos were extracted in NP-40 buffer at stage 9–10 and either directly analyzed by SDS-PAGE and immunoblot, or cleared from cadherin-bound β-catenin as follows: six embryos were extracted in 200 μl NP-40 buffer. 50 μl of Con A–agarose beads (75% slurry) were added, and the samples were incubated with constant mixing for 1–2 h. The beads were spun down and discarded and the supernatant was analyzed for β-catenin levels using an anti–HA-tag rabbit antibody ( Santa Cruz Biotech .), as well as for Axin mutant levels using the 9E10.2 mAb. Stage 9–11 embryos were fixed in 4% paraformaldehyde, 100 mM Hepes, pH 7.4, 100 mM NaCl for 1 h at room temperature, then in Dent's fixative (20% DMSO, 80% methanol) overnight at -20°C. They were rinsed in 100 mM Tris-HCl, 100 mM NaCl, and embedded in 15%, then 25% fish gelatin, and 10-μm cryosections were prepared as described . Sections were labeled with 9E10.2 mAb and anti–mouse Oregon green488 or Alexa488 secondary antibodies (Molecular Probes Inc.), the yolk counterstained with Eriochrome back, and nuclei with 4′,6-diamidino-2-phenylindole (DAPI) as described . For double staining, sections from embryos coinjected with Myc– Axin and HA-tagged Dsh (HA–Dsh) mRNAs (1 ng each) were stained simultaneously with anti-Myc rabbit pAb and anti-HA mAb 12CA5, followed by Alexa488 goat anti–rabbit and Cy3 donkey anti–mouse (Dianova) secondary antibodies. For localization of Myc–Axin in cultured cells, HeLa cells cultured in DMEM were transfected with pCS2-Myc–Axin using LipofectAmine ( GIBCO BRL ). 36-48 h after transfection, cells were fixed in 4% paraformaldehyde/PBS, permeabilized with 0.05% Triton X-100, and labeled with anti-Myc 9E10.2 mAb and Cy3 goat anti–mouse (Dianova) secondary antibodies. Nuclei were counterstained with DAPI. Samples were observed with an Axioplan epifluorescence microscope ( Zeiss ) using standard fluorescein and Cy3 filters, and digital images were collected using a camera (768x576 3CCD color video; Sony). Preembedding labeling was performed as described . In brief, embryos expressing FL Myc–Axin or Myc-AxΔ531-810 were fixed at stage 10 with 4% paraformaldehyde, 0.02% glutaraldehyde, and 100 mM Hepes-NaOH. Labeling was performed by incubating 100-μm vibrotome sections with the 9E10.2 mAb and a Nanogold-coupled anti– mouse secondary antibody, followed by silver enhancement. The reaction produced electron dense aggregates with a diameter of ∼20–60 nm . The sections were embedded in Spurr resin and ultrathin sections were prepared. Postembedding labeling was performed on small blebs obtained from the wounds of injected embryos. These blebs contained a large number of Axin-expressing cells, and their ultrastructure was better preserved than in whole embryos . Paraformaldehyde/glutaraldehyde-fixed samples were processed for Lowycryl embedding, ultrathin sectioning, and immunogold labeling (9E10.2 mAb and 15 nm gold-coupled protein G) according to standard procedures. When epitope-tagged full-length Axin (amino acids 12– 956) was expressed in 293 cells, endogenous GSK3β and β-catenin, as well as VSV-epitope tagged APC (VSV– APC), could be coimmunoprecipitated (coIP) with Axin. A variety of mutant forms of Axin were used next for coIP and direct binding assays to further delimit the regions of Axin required for these interactions and to compare binding abilities with activity in functional assays (see below). The results are summarized in Fig. 3 , which includes a schematic diagram of Axin, indicating the locations of the major binding sites for these proteins as well as PP2A binding and Axin self-binding . The region of rAxin corresponding to aa 561–630 of mAxin has been shown to contain a β-catenin binding site and our results confirmed that all Axin mutants containing this region could coIP with endogenous β-catenin. However, several mutants that lacked this region but included the RGS domain (APC binding site) were also able to coIP with β-catenin (Ax12-531, Ax194-531, and AxΔ531-810) . This may be due to indirect interaction with β-catenin via APC, because deletion of the RGS domain from this mutant Axin (Ax12-531Δ251-351), which eliminated APC binding (see below), also eliminated coIP with β-catenin . The 85-aa region of rAxin corresponding to mAxin 477–561 was reported to bind to GSK3β . We found that Ax497-600, Ax403-552, and Ax455-552 all showed strong coIP with GSK3β , indicating that the binding site is located in the 55-aa segment between aa 497 and 552. Mutants that terminate at aa 531 either coIP very weakly (Ax12-531 and Ax194-531) or fail to coIP with GSK3β (Ax12-531Δ251-351), suggesting that truncation at this site partially disrupts the GSK3β binding site . The RGS domain of Axin has been identified as a site for direct interaction with the 20-aa repeat region of APC . We confirmed that only Axin fragments including the RGS domain could bind to the 20-aa repeat region of APC (APC2 or APC25) . However, we also found that a second region of Axin, between aa 96–253, could bind directly to the NH 2 -terminal region of APC (APC21), which contains the Armadillo repeats and 15 aa repeats . coIPs of the Axin mutants with VSV–APC generally were dependent on the RGS domain: all mutants containing this domain were able to coIP with VSV–APC, whereas all but one of those lacking it failed to coIP with VSV–APC . However, Ax331-956 also was able to coIP with APC, probably via β-catenin . The second APC binding region (aa 96–253) was not sufficient for coIP with VSV–APC . Whether Ax12-355 or Ax12-167 could coIP with VSV–APC could not be determined because expression of these mutant Axins resulted in a strong reduction in the level of VSV– APC . When FL Axin and VSV–APC were cotransfected into 293 cells, the electrophoretic mobility of VSV–APC was reduced compared with control cells cotransfected with VSV–APC plus pCS2 vector. Ax12-810 and Ax12-531 caused a similar mobility shift, whereas AxΔ231-351, Ax12-355, and Ax497-672, which lack either the GSK3β or APC binding site, did not . It has been shown that phosphorylation of APC by GSK3β can be stimulated by Axin in vitro . To test whether this Axin-induced mobility shift was due to phosphorylation, the immunoprecipitated proteins were treated with λ-protein phosphatase before immunoblot analysis with anti-VSV. This treatment eliminated the mobility shift, indicating that it was due to phosphorylation . This suggests that binding of APC and GSK3β to Axin promotes the phosphorylation of APC in vivo, presumably by GSK3β . We previously have shown that the ability of Axin to inhibit dorsal axis formation, when expressed in early Xenopus embryos, is due to its inhibitory effect on the Wnt signaling pathway . Therefore, we used this assay to delimit the sequences in Axin required for its negative effects on signaling through the Wnt pathway. 22 mutant forms of Axin were expressed by mRNA injection on the dorsal side of 4-cell stage embryos that were cultured to the tadpole stage and examined for the extent of dorsal axis formation (fraction of embryos ventralized and dorso-anterior index). The amount of injected mRNAs was systematically titrated to obtain comparable levels of expression for the various mutants. The results are summarized in Fig. 3 , the data are listed in Table I and examples are shown in Fig. 4 . As we previously reported , an internal deletion of the RGS domain (AxΔ251-351) eliminated the ability to ventralize and instead caused dorsalization. Deletion of the GSK3β and β-catenin binding sites (AxΔ352-631) also abolished ventralizing activity. A small fragment containing the GSK3β and β-catenin binding sites (Ax497-672) was insufficient to ventralize the embryo, although similar Axin fragments were able to promote phosphorylation of β-catenin in vitro . A fragment containing only the RGS domain (Ax194-353) was also ineffective. Successive truncation from the NH 2 terminus of Axin confirmed the importance of the RGS domain for ventralization. Whereas removal of the first 193 aa had no significant effect, further truncation to aa 331 eliminated ventralizing activity and resulted in dorsalizing activity, similar to the internal RGS deletion . Truncation at aa 531, removing both the RGS domain and GSK3β binding region, eliminated all activity as did truncation to aa 810 (Ax810-956). Mutant Axins with NH 2 -termini at aa 194 were subjected to COOH-terminal truncation to examine the importance of the DIX, PP2A binding, β-catenin binding, and GSK3β binding domains. Removal of the DIX domain (Ax194-810) had little if any effect, whereas removal of the DIX and PP2A binding domains (Ax194-672) caused an increase in ventralizing activity . This observation is consistent with the hypothesis that the binding of PP2A to the Axin complex may negatively regulate the phosphorylation of β-catenin by GSK3β . Further truncation to aa 531, removing the β-catenin binding site, abolished ventralizing activity, and instead resulted in some dorsalizing activity . When injected at high concentrations, the other Axin mutants lacking the NH 2 -terminal and the COOH-terminal regions (Ax194-672 and Ax194-810) also showed dorsalizing activity, as discussed below. When the NH 2 terminus of Axin was left intact, truncation of the COOH terminus to remove the DIX domain, or both the DIX and PP2A domains, caused only a slight reduction, if any, in the ability to ventralize. Surprisingly, there was no further reduction in activity when the region including the β-catenin binding site was truncated (Ax12-531) or removed by an internal deletion (AxΔ531-810). Unlike the mutants with NH 2 -termini truncated at aa 194, no dominant negative effect (dorsalization) was seen when high concentrations were injected (Table II ). Further truncation, removing the GSK3β site, eliminated all activity (Ax12-355). Internal deletion of only the RGS domain, in the context of COOH-terminal truncations (mutant Ax12-810Δ251-351, Ax12-672Δ251-351, and Ax12-531Δ251-351), also eliminated ventralizing activity and instead cause weak dorsalizing activity. In contrast to the ability of Axin and other inhibitors of Wnt signaling (e.g., GSK3β) to ventralize when injected dorsally, factors that stimulate this pathway (e.g., certain Wnts, Dsh, dnGSK3β, or β-catenin) have dorsalizing activity; when injected dorsally, they can hyperdorsalize (i.e., they induce formation of a larger head, large or multiple cement glands, shorter axis, and double anterior axis). However, their activity is best seen in ventral injections, where they can induce a secondary axis . We previously showed that a mutant Axin lacking the RGS domain (AxΔ251-351) behaved as such a dorsalizing factor. This activity could be competed by coexpression of FL Axin, supporting the conclusion that it was due to a dominant negative effect . To identify the domains of Axin required for this activity, several additional mutant forms of Axin were also injected into the ventral side of the embryo to assay their ability to induce axis duplication. Sequences upstream from the RGS domain were not required, as an NH 2 -terminal truncation at aa 331 (Ax331-956) induced axis duplication as efficiently as the RGS deletion . However, the GSK3β binding site was required as truncation at aa 531 abolished the effect (Ax531-956). The COOH-terminal sequences were also important: in the presence of the RGS deletion, COOH-terminal truncation at aa 810, 672, or 530 strongly reduced the dorsalizing activity so that axis duplication was observed only when high concentrations of RNA were injected. When the amount of injected RNA was titrated down to yield expression levels at which AxΔ251-351 showed optimal activity, only Ax331-956 was active (Table II ). Modulation of the Wnt pathway has a striking effect on β-catenin levels; in the absence of a Wnt signal, constitutively active GSK3β phosphorylates β-catenin and causes its rapid turnover, whereas Wnt signaling induces stabilization of β-catenin. Therefore, we tested the effect of Axin on β-catenin levels in Xenopus embryos by coexpressing HA-tagged β-catenin with FL or mutant forms of Axin, or with β-galactosidase as a control. Low amounts of HA-tagged β-catenin mRNA were used to mimic the behavior of endogenous β-catenin. At the late blastula stage, when endogenous β-catenin signaling peaks , the levels of HA-tagged β-catenin in embryo extracts were analyzed. The membrane (cadherin-bound) pool of β-catenin is known to be very stable and changes in β-catenin levels by the Wnt signaling pathway affect mostly the unbound, soluble pool . Removing the cadherin-bound pool of β-catenin by Con A precipitation made it possible to obtain samples enriched in soluble β-catenin, allowing the effect of Axin on β-catenin levels to be analyzed more accurately. As shown in Fig. 5 B, FL Axin caused a dramatic decrease in exogenous β-catenin. Several mutant Axin constructs were coexpressed similarly; mutants with ventralizing activity (Ax12-531 and Ax194-672) also proved to be effective in reducing β-catenin levels. A mutant lacking ventralizing activity (Ax531-956) had no effect on β-catenin levels. On the other hand, mutants with strong dominant negative activity (AxΔ251-351 and Ax331-956), induced a clear increase in β-catenin levels. Unexpectedly, mutant Ax194-531, which failed to ventralize over a wide range of concentrations, but instead showed some dorsalizing activity, caused a strong decrease in β-catenin levels. The activity of the dominant negative mutant AxΔ251-351 was also tested on the ventral side, where the β-catenin degradation machinery is maximally active. Under these conditions, stabilization of β-catenin by AxΔ251- 351 could be observed even in total extracts . NH 2 terminally deleted β-catenin , which lacks the GSK3β-dependent phosphorylation site , was found to be insensitive to Axin overexpression , suggesting that Axin-induced destabilization of β-catenin requires phosphorylation by GSK3β. As the subcellular localization of endogenous Axin is so far unknown, we examined the distribution of the ectopically expressed Myc-tagged Axin in Xenopus embryos. As shown in Fig. 6 , FL Myc–Axin exhibited a striking and unusual pattern. The signal was mostly concentrated in very bright spots, which were found singly or in clusters of variable size, mainly, but not exclusively, at the cell periphery . The rest of the cytoplasm was devoid completely of the signal. In addition, some plasma membrane staining was also observed. However, the membrane staining was quite variable: absent in many cells, weak in others , and very strong in a few rare cells (not shown). The punctate pattern and the absence of diffuse cytoplasmic staining were observed at all mRNA concentrations used from 0.15 ng, the limit of detection by IF, to 2 ng. A similar pattern was observed in Axin-transfected cultured HeLa and A6 cells (not shown). Myc–Axin localization in Xenopus embryos was further studied at the EM level by two different techniques: on-section staining of Lowycryl sections and preembedding labeling using Nanogold and silver enhancement . Notwithstanding differences in ultrastructure preservation and labeling sensitivity (see legends), both methods gave similar results. Consistent with IF data, Myc–Axin was found to be concentrated highly in discrete areas of the cell. These areas were characterized by clusters of vesicles (asterisks) surrounded by gold-decorated electron dense material (arrows). Labeled clusters varied largely in size and density, apparently as a function of expression levels. Part of a loose cluster is shown in Fig. 7 C. Small groups of gold particles associated with a few vesicles and electron dense material could be resolved, probably corresponding to the individual spots detected by IF . On the other hand, Fig. 7 , D and E, shows very large dense Myc-positive areas, where vesicles were tightly packed and consequently the dense cytoplasm appeared less prominent. Fig. 7 B shows a cluster of intermediate size and vesicle density. Plasma membrane localization of FL Axin could not be detected unambiguously by EM, probably because it was generally too weak . However, strong plasma membrane staining could be observed for the mutant Axin AxΔ531-810 that is consistent with IF results . Consistent with the IF data, Axin was found to be largely particulate/sedimentable in differential centrifugation experiments . On the other hand, it was completely solubilized in the presence of a mild nonionic detergent, NP-40 . Thus, the sedimentation properties of Axin are not due to interaction with detergent-insoluble cytoskeletal elements. In the presence of NP-40, Axin could be partially precipitated using Con A beads , indicating that a pool of Axin is associated with a membrane glycoprotein. We believe that this Axin– membrane association involves the plasma membrane pool of Axin, but not that in the intracellular spots, because binding to Con A of all Axin deletion mutants tested strictly correlated with plasma membrane localization (as detected by IF, see below). The punctate distribution of Axin strongly was reminiscent of the localization pattern of ectopically expressed Dsh (the distribution of endogenous Dsh in Xenopus is not known). Thus, we compared the localization of coexpressed Myc-tagged Axin and HA–Dsh by double IF. We observed a very good colocalization of these two proteins : HA–Dsh was detected at all sites positive for Myc–Axin (arrowheads), although some other spots were positive for Dsh but negative for Axin (arrows). The Myc– Axin pattern in these embryos was indistinguishable from the pattern observed in the absence of exogenous Dsh, suggesting that Dsh does not influence Axin localization. In contrast, Myc–Axin overexpression clearly affected HA–Dsh distribution: when HA–Dsh was expressed alone, it localized exclusively in single cytoplasmic spots, or small clusters of spots, distributed throughout the cell . No membrane staining was observed. However, when coexpressed HA–Dsh and Myc–Axin colocalized in a pattern typical for overexpressed Axin (enrichment of spots at the cell periphery, presence of large clusters, and plasma membrane staining). These results suggest that Dsh may bind, directly or indirectly, to the Axin complex. To examine the sequences in Axin that target it to its specific locations, and the functional significance of this localization, we examined the intracellular distribution of the same mutant forms used above. Internal deletion of the RGS domain had little or no effect on localization; the mutant protein localizing primarily in the spots and less at the plasma membrane . Deletion of the GSK3β and β-catenin binding sites (AxΔ352-631) also had no effect on localization to the spots, but eliminated the membrane staining . Deletion of the COOH-terminal 146-aa resulted in localization mainly at the membrane, with little or no labeling of the spots, e.g., Ax12-810 (not shown) Ax12-672 , Ax12-531 , and Ax12-355 . Forms of Axin lacking the NH 2 -terminal half displayed a mostly diffuse cytoplasmic localization, e.g., Ax497-672 . When the APC and GSK3β-binding domains were left intact, there seemed to be some enrichment at the cell periphery , although it was difficult to assess the extent of membrane enrichment, because of the high cytoplasmic signal. Thus, the NH 2 terminus of Axin appears to be required for the characteristic pattern of localization, both in the cytoplasmic spots and at the membrane. The presence of the normal COOH terminus tends to cause localization to the spots, although it is not absolutely required for this. The COOH terminus, which includes a dimerization domain, might bind to endogenous Axin or to other cellular components. The APC and GSK3β binding sites appear to have a weaker effect on localization at the membrane. The membrane localization of mutants containing the NH 2 terminus correlates very well with Con A binding . For instance, Ax12-531 binds very efficiently to Con A, whereas Ax531-956 does not bind at all. However, localization to the spots appears to depend on a different mechanism, only a small fraction of FL Axin and an even smaller fraction of AxΔ351-630 (found mostly in spots) bind to Con A . Axin has been shown to negatively regulate signaling through components of the Wnt pathway. Coinjection experiments in Xenopus embryos previously suggested that it acts downstream of GSK3β and upstream of β-catenin. Subsequent studies have shown that Axin is part of a complex including these two proteins as well as APC and that it promotes the phosphorylation of β-catenin by GSK3β and its subsequent degradation . The aims of the experiments reported here were to understand the relationship between Axin's ability to bind to these and other proteins and its capacity to function in the regulation of this pathway. To this end, we have examined a series of Axin mutants for their ability to (1) bind to APC, GSK3β, and β-catenin; (2) ventralize or dorsalize Xenopus embryos, an established assay for effects on β-catenin signaling; and (3) alter the stability of β-catenin expressed from coinjected mRNA. In addition, we have examined the intracellular localization of FL Axin and a series of Axin mutants. Through direct binding in vitro and coIP from mammalian cell extracts, we have confirmed that Axin forms a complex with APC, GSK3β, and β-catenin, and we have further delimited some of the binding sites for these proteins. Based on coIP, the region of mAxin required for interaction with GSK3β lies between aa 497 and 531. The COOH-terminal boundary of the minimal binding region appears to lie between aa 526 and 531. Whereas Itoh et al. did not detect coIP of GSK3β with Axin 12-526, we detected weak interaction of GSK3β with some Axin mutants terminating at aa 531. We confirmed that the RGS domain (aa 220–340) includes a major binding site for APC and interacts with the 20-aa repeat region of APC. Furthermore, we identified a second region of Axin, between aa 96–253, that can bind directly to the NH 2 -terminal region of APC containing the Armadillo and 15-aa repeats. This region of Axin was neither necessary nor sufficient for coIP with VSV-G–tagged APC, suggesting that it plays a secondary role to the RGS domain in vivo. We observed good agreement between the presence of the direct binding site for GSK3β and the ability of Axin mutants to coIP with GSK3β . Axin mutants lacking this region, some of which were able to coIP with APC and/or β-catenin, all failed to coIP with GSK3β, suggesting that GSK3β must bind directly to Axin to join the complex. However, several forms of Axin that lacked the direct binding site for either APC or β-catenin were able to coIP with both of these proteins. APC was found to coIP not only with all forms of Axin containing the RGS domain, but also with Ax331-956, which lacks any direct binding site for APC. Similarly, β-catenin could coIP not only with all Axin mutants containing aa 600–622, but also with Ax12-531 and Ax194-531. Both of these discrepancies likely are due to the ability of APC and β-catenin to bind to each other . Since each of these three components can interact directly with the other two, we suggest that they form in vivo a triangular complex . The GSK3β binding domain was dispensable for direct binding to APC and β-catenin, but required for indirect binding to β-catenin (presumably via APC). Indeed, a small fragment of Axin containing only the RGS domain (Ax194-353), while able to coIP with APC, did not coIP with β-catenin. Conversely, Ax531-956 was found to coIP with β-catenin but not with APC. We also found that Axin induced a mobility shift in APC that appeared to be due to phosphorylation. This activity required the GSK3β-binding domain as well as the APC-binding region, suggesting that GSK3β is responsible for this modification. It previously has been shown that GSK3β can phosphorylate APC in vitro , that Axin promotes this event , and that this phosphorylation enhances the ability of APC to bind to β-catenin . Our observations argue that Axin performs a similar function in vivo. In general, we found a good correlation between the ability of Axin mutants to ventralize frog embryos and to lower the levels of coinjected HA-tagged β-catenin, presumably by promoting its degradation. The RGS domain and GSK3β binding site were both required, although not sufficient, for Axin activity. In addition, either the β-catenin binding site or the NH 2 -terminal region upstream of the RGS domain (but not necessarily both) was required. The activity of mutant forms of Axin lacking the β-catenin binding site (e.g., Ax12-531 and AxΔ531-810) is consistent with the observation that such forms can coIP with β-catenin in 293 cells, apparently via an indirect interaction. Why the NH 2 -terminal sequence can substitute for the β-catenin binding site is not clear, but this could be related to its ability to bind to APC or its influence on the intracellular localization of Axin. Fragments of Axin containing the GSK3β and β-catenin binding sites, but lacking the RGS domain, can promote the phosphorylation of β-catenin by GSK3β in vitro as well as the degradation of endogenous β-catenin in cultured SW480 cells . However, in our experiments, all forms of Axin lacking the RGS domain either had no effect on axis formation and β-catenin levels or they dorsalized rather than ventralized the frog embryo and raised β-catenin levels. This indicates that direct binding to APC plays a critical role in the ability of Axin to promote the degradation of β-catenin. These discrepancies may be due to the peculiar properties of SW480 cells that lack FL APC and have high levels of soluble β-catenin. In contrast, we used very low levels of exogenous β-catenin that can still be effectively regulated by the Wnt pathway . In other experiments , we found that FL Axin was able to downregulate even very large amounts of coinjected β-catenin. However, under these conditions several Axin mutants gave results that were inconsistent with their effects at more physiological β-catenin levels and with their activity on axis induction. In particular, both dominant negative mutants AxΔ251-351 and Ax331-956 failed to stabilize β-catenin expressed at high levels, but rather caused some destabilization (not shown). Thus, Axin is capable of stimulating β-catenin degradation independently of the RGS domain, but only provided high levels of free β-catenin and/or absence of APC. Consistent with this observation, the Axin-like protein Conductin/Axil/Axin-2 appears to behave similarly. Indeed, a ΔRGS Conductin construct could downregulate β-catenin levels in SW480 cells, yet acted as a dominant negative (i.e., increased β-catenin levels) in Neuro2A cells, which have low levels of endogenous β-catenin and FL APC . It is likely that high levels of β-catenin are flooding the system, bypassing normal regulatory mechanisms. Nevertheless, it is also possible that two different mechanisms exist, one APC-dependent, and one APC-independent, active at low and at high β-catenin concentrations, respectively (see below). Surprisingly, in SW480 cells, truncation of the NH 2 terminus, including the RGS domain, increased the activity of Axin that led to the proposal that the RGS domain may repress Axin activity in the absence of APC . However, this mutant construct could not discriminate between a role of the RGS domain itself and an effect of upstream sequences. In contrast, ΔRGS Conductin, a mutant with an internally deleted RGS domain and an intact NH 2 terminus, showed weaker activity than FL Conductin in SW480 cells, arguing that the RGS domain is not responsible for the apparent repression reported by Hart et al. . In fact, SW480 cells are not null for APC, but still contain an NH 2 -terminal fragment, which can bind β-catenin , and can also interact with the NH 2 terminus of Axin upstream of the RGS domain (our data). Thus, it is conceivable that the truncated APC may interfere with Axin activity. In embryos, deletion of the RGS domain caused a strong dominant negative effect. The self-binding region (including the DIX domain) appeared to play a role in this activity, because deletion of this region from AxΔ251-351 substantially reduced activity. As the COOH-terminal 100 aa of Axin can mediate multimerization, the strong dominant negative forms of Axin may act by binding to endogenous Axin. However, forms of Axin lacking the DIX domain also showed weak or moderate dorsalizing activity (Ax12-810Δ251-351 and Ax12-531Δ251-351), suggesting that dominant negative activity may be generated in more than one way. One possibility is that these forms of Axin bind to GSK3β but not to APC, thus, interfering with the formation of the complete complex. Also, two mutants lacking both the NH 2 and COOH termini (Ax194-672 and Ax194-810) efficiently ventralized at low concentrations, but dorsalized at high concentrations. This dual activity might be related to their subcellular localization (see below). The NH 2 - and COOH-terminal portions of Axin were not required for its ventralizing activity in our assays, as also observed by Itoh et al. , although they may modulate its function. Deletion of the DIX domain had little or no effect on activity. Deletion of the PP2A binding region caused some increase in activity of the forms of Axin that initiated at aa 194, although this difference was not apparent when the NH 2 terminus of Axin was left intact. We have hypothesized that the binding of PP2A to the Axin complex might counteract the phosphorylation of β-catenin by GSK3β that could account for the increased ventralization activity in the absence of this domain. FL Myc–Axin expressed in Xenopus embryos was found primarily in characteristic spots, singly or in clusters of variable size. Ultrastructural analyses indicated that the spotty distribution is not merely due to aggregation of an overexpressed protein, but corresponds to particular (though as yet ill-defined) subcellular structures, consisting of clustered vesicles associated with dense cytoplasm. Several arguments suggest that endogenous Axin has a similar distribution including: (1) identical spots were observed over a wide range of Axin expression levels. (2) HA–Dsh accumulated in similar spots in the absence of overexpressed Axin. (3) Myc–Axin colocalized with HA–Dsh . However, the formation of large clusters of spots very likely is due to Axin overexpression, as it was not observed for HA–Dsh in the absence of Axin expression. However, it may reveal the ability of Axin to act as a scaffold through multiple interactions with other cytoplasmic proteins. A small, variable fraction of Myc– Axin was also associated with the plasma membrane, and Con A-binding showed that Axin interacts with a cell surface glycoprotein. The NH 2 terminus is sufficient for membrane targeting, although some other internal sequences may also confer weaker binding. Interestingly, the sequences of Axin crucial for its subcellular localization (i.e., the NH 2 and COOH termini) do not bind any of the core components related to its activity (APC, β-catenin, and GSK3β), and are apparently dispensable, at least under conditions of overexpression . Clearly, additional molecular interactions must take place at both ends of the molecules. The occurrence of two well-defined locations for Myc– Axin may reflect the existence of two functionally distinct pools, possibly an active and an inactive one. Ectopically expressed Dsh shows a similar dual localization at spots/ membrane, which can be manipulated by overexpression of Wnt/Frizzled, and that may correspond to different functional states . However, which would be the active and inactive sites remains unclear. The comparison of various Axin mutants did not reveal any simple correlation between their activity in functional assays and their intracellular distribution, although all active mutants can to some extent localize at the plasma membrane. However, it is quite possible that localization per se is not required for activity, but that regulation is achieved by sequestering various components of the signaling pathway in different compartments of the cell. It is then easy to conceive that overexpression may bypass such regulation and allow Axin/Axin mutants to interact with other components of the complex independently of upstream signals. Our observations provide some hints for a role of Axin localization. For instance, the concentration-dependent dual activity of Ax194-672 and Ax194-810 may be related to their diffuse distribution; at high concentrations, they could act as dominant-negatives by affecting the balance between various endogenous components otherwise strictly compartmentalized. This dual activity was also found for the Axin-like protein Axil/Conductin/Axin-2 that has a similar diffuse distribution (Zhang, T., F. Fagotto, and F. Costantini, manuscript in preparation), but was never observed with Axin constructs showing a well-defined localization (spots and/or plasma membrane). Our functional data emphasize the essential role of the RGS domain for Axin activity . They demonstrate that binding to GSK3β and to β-catenin, which was reported to stimulate β-catenin phosphorylation in vitro , is not sufficient in vivo either for β-catenin degradation or for inhibition of its signaling. Although the RGS domain may interact with other yet uncharacterized molecules, its importance most likely resides in its ability to bind APC, thus inducing the formation of a trimeric complex Axin•β-catenin•APC . Note that the RGS domain of Axin is significantly diverged from the sequences of bona fide RGS proteins , and does not appear to bind to G-proteins (our unpublished data) or to have RGS activity . Apparently, it has diverged toward other interactions and functions. The apparent discrepancy with other data that suggests, under certain conditions, Axin can function without the RGS domain , may be best reconciled by postulating two mechanisms, active at different levels of free β-catenin: When β-catenin levels are low, β-catenin degradation would depend primarily of an Axin• β-catenin• APC complex, whereas when β-catenin levels are high, β-catenin• Axin complexes may form and function in the absence of APC. Both mechanisms may be physiologically important: in the absence of Wnt signal, very low levels of free β-catenin might be maintained by the combined action of Axin and APC. However, after a burst of Wnt signal, excess β-catenin would be first downregulated by an APC-independent coarse mechanism, before fine tuning by APC could eventually restore normal levels. The occurrence of an Axin-based complex has further potential implications on the regulation of the pathway by Wnt. It had been assumed that Wnt caused β-catenin stabilization by inhibiting GSK3β activity . However, GSK3β inhibition could hardly account for the specificity of the pathway, considering the many other substrates and pleiotropic functions of GSK3β. We now know that binding of GSK3β to Axin is required for phosphorylation of Axin, APC, and β-catenin, and ultimately for activity of the complex . Therefore, inhibition of Axin-GSK3β binding, or in fact any other interactions within the complex, could be a far more specific way to regulate this pathway. This might be precisely the function of Dsh. In this context, the distinct cellular pools of Axin may reflect the existence of different, active and inactive, complexes. The challenge will be to characterize the nature of these complexes and their regulation by upstream components of the pathway. | Study | biomedical | en | 0.999998 |
10330404 | S. cerevisiae strain KFY437 ( MAT a cdc48 :: URA3 his4 -619 leu2 -3,112 ura3 -52 YEp52/cdc48 S565G containing mutant allele cdc48 S565G on vector YEp52), and the corresponding control strain KFY417 ( MAT a cdc48 :: URA3 his4 -619 leu2 -3,112 ura3 -52 YEp52/CDC48) with a wild-type CDC48 on YEp52 have been described . Strain YPH98gsh1 was constructed from yeast wild-type YPH98 by deletion of the complete GSH1 reading frame . Cell division cycle mutants used as controls were Hartwell strains LH370 ( cdc2 ts ) and LH12021 ( cdc31 ts ), and rE24-15 . Plasmids pSD10.a-Bax and pSD10.a-Bcl-X L contain murine bax, respectively, bcl-X L under the control of a hybrid GAL1-10/CYC1 promoter in a pRS316-based vector with a URA3 marker . Plasmid pL009 was constructed by replacing the URA3 marker of plasmid pSD10.a-Bcl-X L by LEU2 . Strain WCG4 ( MAT α his3 -11 ura3 -52 leu2 -3,112) was transformed with plasmid pSD10.a-Bax (strain WCG4bax), or with both pSD10.a-Bax and pL009 (strain WCG4bax/bcl-X L ), or with vector pRS316 as a control. YEPD consisted of 1% yeast extract (Difco), 2% Bacto peptone (Difco), and 4% glucose. Synthetic complete medium (SC) consisted of 0.67% yeast nitrogen base (Difco) and 2% glucose (SCD) or 2% galactose (SCGal) supplemented with amino acids and nucleotide bases. Strains KFY437 and KFY417 were pregrown on YEP plates with 4% galactose and inoculated in liquid YEPD. WCG4bax, WCG4bax/bcl-X L , and the vector control strain were pregrown in synthetic medium with 2% glucose to a cell density of about 0.5 × 10 6 cm −3 . To induce the expression of Bax or Bcl-X L , cells were washed 3× and resuspended in synthetic medium with 2% galactose. For growth under anaerobic conditions, strains were incubated in 100 ml culture medium in wash bottles under flow of N 2 (Messer Griesheim GmbH) at 100 ml/min. Viability was determined as the portion of cell forming visible colonies on YEPD plates after 3 d at 28°C. Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously . For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described , and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD ( Boehringer Mannheim ). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence . The slides were rinsed with PBS and incubated with 0.3% H 2 O 2 in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim ) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution ( Boehringer Mannheim ) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared. Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine; Sigma Chemical Co. ). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS ® Calibur ( Becton Dickinson ) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively. Free spin trap reagents N -tert-butyl-α−phenylnitrone (PBN; Sigma -Aldrich) and 3,3,5,5,-tetramethyl-pyrroline N -oxide (TMPO; Sigma -Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d. To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated. Cycloheximide was added to exponentially growing yeast cultures at 5, 15, 50, 200 μg/ml, or 0 μg/ml as a control. After 30 min, 10 μCi 4,5-[ 3 H]- l -leucine (Movarek Biochemicals Inc.) was added to a 500-μl aliquot. After further incubation of 200 min at 30°C with shaking, 100-μl aliquots were spotted onto glass fiber filters and incubated in 10% TCA for 15 min in a boiling water bath. Filters were washed twice with 5% TCA, twice with ethanol and once with methanol, dried and counted with 5 ml Ultima Gold scintillation cocktail (Packard BioScience B.V.). Exposure to H 2 O 2 triggers apoptosis in numerous mammalian cells. To examine its effect in yeast, various amounts of H 2 O 2 were added to wild-type cultures (strain YPH98) growing exponentially on YEPD. After a 200-min incubation, cells were examined for markers of apoptosis. DNA cleavage was assayed using the TUNEL test , chromatin was visualized by fluorescence microscopy after DAPI staining as well as by electron microscopy. We found that low concentrations of H 2 O 2 result in a TUNEL-positive phenotype, which vanishes at higher concentrations. Incubation with 3 mM H 2 O 2 produces a strongly TUNEL-positive phenotype (intense black nuclear stain) in 70% of the cells , indicating massive DNA fragmentation. With 0.3 or 1 mM H 2 O 2 , only 20– 40% of the cells are stained . TUNEL-positive cells are often remarkably enlarged compared to TUNEL-negative cells from the same batch . Cells incubated in the absence of H 2 O 2 showed unstained or only slightly grayish nuclei . Increasing the H 2 O 2 concentration above 5 mM did not intensify the TUNEL reaction. Instead, with 15 mM H 2 O 2 the TUNEL staining is even much less intense and occurs in fewer cells than with 3 mM H 2 O 2 . Incubation with 180 mM H 2 O 2 results in no detectable TUNEL staining . To demonstrate that the DNA fragmentation is not a result of cell necrosis, membrane integrity was tested by incubating an aliquot of the protoplasts used for TUNEL staining with 23 μg/ml propidium iodide. Only 3–5% of the protoplasts from cultures treated with 0–5 mM H 2 O 2 are stained (not shown). In cultures incubated with 180 mM H 2 O 2 , ∼80% of the protoplasts are stained with propidium iodide, indicating the loss of plasma membrane integrity. Cells from stationary cultures tolerate higher concentrations of H 2 O 2 . After incubation with 5 mM H 2 O 2 or less, no TUNEL staining is observed (not shown). On the other hand, incubation with 180 mM H 2 O 2 results in a strong TUNEL staining . Analysis of isolated chromosomal DNA from H 2 O 2 cells by agarose electrophoresis showed only a smear at low molecular weights, but not the DNA ladder pattern (not shown) that is found in many apoptotic systems as the result of DNA cleavage between nucleosomes. This confirms our result with the cdc48 mutant showing an apoptotic phenotype and is probably caused by the S. cerevisiae chromatin structure with little or no linker DNA between the nucleosomes . In addition, apoptosis without the occurrence of a DNA ladder has been described for several metazoan cell types . DAPI staining of cells incubated with 3 mM H 2 O 2 shows chromatin fragments arranged in a half-ring or distributed nuclear fragments in 10– 50% of the cells. In some cells, nuclei seem to degenerate, showing protruding tubes . In untreated cultures, all nuclei appear as single round spots in the cells. In contrast to nuclei or most nuclear fragments, mitochondria are predominantly located near the periphery of the cells and appear as dots of far less intensity and size . Electron microscopic investigation of cells incubated with 3 mM H 2 O 2 revealed extensive chromatin condensation along the nuclear envelope typical for apoptosis , cells containing multiple nuclear fragments , as well as tubular structures protruding from the nucleus that probably correspond to the tubular structures observed in DAPI-stained cells . Some condensation is already visible after 30 min H 2 O 2 treatment increasing gradually with time . Nuclei of untreated cells are homogeneous in shape and density . In addition, some cells show tiny vesicles on the outer side of the plasma membrane . This could be an equivalent of membrane blebbing that is a characteristic marker of apoptosis and has not been observed in yeast cells before. At higher concentrations of H 2 O 2 , most intracellular structures are destroyed , indicating necrotic cell death. This corresponds to the diminished TUNEL reaction in these cultures . Apoptosis needs participation of the cell and can be prevented in many cell types by the inhibition of protein synthesis . To address the question whether the H 2 O 2 -induced DNA breakage, chromatin condensation, and cell death are the result of basic radical chemistry or depend on participation of the cellular metabolism, we investigated the effect of cycloheximide on H 2 O 2 -treated yeast cells. To establish which concentration of cycloheximide is sufficient for complete inhibition of protein synthesis, incorporation of labeled leucine into TCA precipitable material was determined. 5 μg/ml cycloheximide reduced leucine incorporation to 23% of the untreated control, 15 μg/ml cycloheximide, or higher concentrations reduced the incorporation to <8% of the untreated control. Exponentially growing wild-type cultures were preincubated with 15 μg/ml cycloheximide for 30 min. After adding 3 mM H 2 O 2 , cultures were incubated for another 200 min. DNA fragmentation was visualized by TUNEL staining . The resulting TUNEL assay stain is only slightly more intense than in untreated controls and far weaker than in cells treated with 3 mM H 2 O 2 in the absence of cycloheximide . Incubation only with cycloheximide for 230 min results in approximately the same enhancement of TUNEL staining as the combination of cycloheximide and 3 mM H 2 O 2 . This minor increase in DNA fragmentation compared to untreated controls could be due to a reduction of DNA repair as a result of the inhibited protein synthesis. Under the electron microscope, neither chromatin condensation, margination, nor nuclear fragmentation is detected in cells incubated with H 2 O 2 in the presence of cycloheximide. However, numerous cells show vacuoles accumulating autophagic bodies, increased intracellular vacuolization, or misformed nuclei indicating necrotic damage . To investigate the effect of cycloheximide on yeast cell survival during oxygen stress, we incubated exponentially growing wild-type cultures with 15 μg/ml cycloheximide for 30 min, added various concentrations of H 2 O 2 , and incubated the cultures for another 200 min. Survival was determined as the portion of cells forming colonies after 3 d of incubation on YEPD plates. Cycloheximide considerably increased the cell survival rate in the range of 0.03 to 0.3 mM H 2 O 2 . To investigate whether cycloheximide affects intracellular glutathione concentration by the inhibition of protein synthesis, the level of intracellular glutathione was determined as described . Untreated wild-type cells contained 1.55 μM glutathione per gram of wet twice-washed cells. Incubation with 15 μg/ml cycloheximide for 230 min increased the glutathione level to 140% of the control. The increase is probably caused by accumulation of cysteine. However, even an increase of the glutathione level to 250% of the wild-type level by overexpression of GSH1 has no significant effect on H 2 O 2 tolerance (Grey, M., unpublished result). Also, after 200 min incubation with 3 mM H 2 O 2 , cells retained a glutathione level of 1.24–1.36 μmol/g cells (∼80% of the control level) indicating that the intracellular glutathione pool cell is far from exhaustion. Therefore, the observed increase of glutathione by cycloheximide can not be responsible for its protective effect. Glutathione plays a major role in the protection against ROS. Strain YPH98gsh1 lacks glutathione due to a deletion of the GSH1 gene coding for γ-glutamylcysteine synthetase. It grows on YEPD due to the significant amounts of glutathione contained in the yeast extract, as well as on synthetic media supplemented with glutathione, but dies after 3 d on glutathione-free media . Strain YPH98gsh1 and the isogenic wild-type strain YPH98 were transferred from YEPD plates to glutathione-free synthetic medium plates and incubated for 3 d. Cells were examined for the phenotypic markers of apoptosis: DNA breakage, chromatin condensation and margination, and the exposition of phosphatidylserine. The deletion strain shows strong staining (black nuclei) with the TUNEL test indicating massive DNA fragmentation in >80% of the cells , while the control strain shows no TUNEL staining . When the deletion strain is grown on YEPD or on synthetic medium supplemented with 20 μg/ml glutathione, nuclear staining is not observed with the TUNEL assay (not shown). An aliquot of the protoplasts used for TUNEL staining was also tested for membrane integrity by incubation with 23 μg/ml propidium iodide. 10–20% of the protoplasts of both wild-type and glutathione deficient strain are stained (data not shown), confirming that the majority of the cells is intact and that the DNA fragmentation is not a result of cell necrosis. The deletion strain exhibits an enhanced sensitivity towards H 2 O 2 . Incubation with 0.1 mM H 2 O 2 induces a strong TUNEL reaction, as well as condensation and fragmentation of chromatin, whereas little or no TUNEL staining is observed after incubation with 3 mM H 2 O 2 or higher concentrations (not shown). Analysis of isolated chromosomal DNA from the deletion strain by agarose electrophoresis showed no indication of DNA laddering (not shown). DAPI staining of the deletion strain shows a strong chromatin condensation with fragments arranged in a ring, a half-ring , or as several randomly distributed nuclear fragments . Electron micrographs show intense chromatin margination and fragmentation of the nucleus . In cells of the deletion strain grown on YEPD medium or on synthetic medium supplemented with 20 μg/ml glutathione, no chromatin condensation or fragmentation occurs (not shown). In mammalian cells, phosphatidylserine exposure at the outer leaflet of the cytoplasmic membrane is an early marker of apoptosis . The exposure can be detected with annexin V that specifically binds phosphatidylserine in the presence of Ca 2+ . Like mammalian cells, S. cerevisiae has an asymmetric distribution of phospholipids within the cytoplasmic membrane with 90% of the phosphatidylserine on the cytoplasmic side of the membrane . We have recently described the exposure of phosphatidylserine in a yeast CDC48 mutant showing characteristics of apoptotic cells . Spheroplasts of the GSH1 deletion strain and the isogenic control strain grown on glutathione-free synthetic medium for 3 d were examined for exposure of phosphatidylserine by incubation with FITC-labeled annexin V and for membrane integrity by incubation with propidium iodide. Approximately 20% of the protoplasts, both wild-type and mutant strain, take up propidium iodide indicating membrane damage. These protoplasts often exhibit annexin V staining of the whole cell. More than 15% of the protoplasts from the deletion strain show a strong fluorescence around the circumference of the cell of which none stain with propidium iodide, demonstrating that phosphatidylserine is indeed exposed at the outer layer of the cytoplasmic membrane . All intact protoplasts of the isogenic control show little or no FITC staining in the cell lumen . When the deletion strain was grown on YEPD or on synthetic medium supplemented with 20 μg/ml glutathione, no annexin V fluorescence is visible either (not shown). To investigate whether oxygen stress is involved in other apoptotic scenarios described in S. cerevisiae , we tested apoptotic mutant strain KFY437 for the occurrence of oxygen radicals. Strain KFY437 expresses cdc48 S565G , a mutated allele of CDC48 responsible for the homotypic fusion of ER-derived vesicles, and spontaneously develops an apoptotic phenotype during exponential growth at 28°C while other conditional alleles of CDC48 result in cell death at the restrictive temperature without exhibiting any of these morphological markers . Strain KFY437 was tested for the production of ROS during apoptosis by incubation with dihydrorhodamine 123. This substance accumulates in the cell and is oxidized to the fluorescent chromophore rhodamine 123 by ROS . Strain KFY437 growing exponentially on YEPD was incubated with 5 μg/ml dihydrorhodamine 123 for 2 h at 28°C. More than 50% of the cells show a rhodamine 123 fluorescence . Most of the cells of the corresponding wild-type strain show no fluorescence, appearing dark against the faint background fluorescence. Marginal fluorescence occurs in <1% of the cells . Incubation for 2 h with 10 μg/ml dichlorodihydrofluorescein diacetate, which is deacylated to dichlorodihydrofluorescein and oxidized by ROS to fluorescent dichlorofluorescein , or with 5 μg/ml dihydroethidium for 10 min, which is oxidized specifically by superoxide ions to the fluorescent ethidium , gave similar results (not shown). Flow cytometric analysis of dihydrorhodamine 123–stained cells confirms that strain KFY437 accumulates oxygen radicals. A majority of the mutants cells show an increased signal , while all wild-type cells display a low fluorescence . We found that elevated temperature (37°C) induces apoptotic cell death in most cells of strain KFY437. After 4 h at 37°C, >80% of the KFY437 cells show a strong TUNEL reaction as well as condensed and fragmented chromatin. The elevated temperature induces a higher intensity of TUNEL staining as compared to cells grown at 28°C . When strain KFY437 is incubated at 37°C for 4 h, more than 80% of the cells show a strong rhodamine 123 fluorescence of a significantly higher intensity than at 28°C. The majority of cells also show intense fluorescence after incubation with dichlorodihydrofluorescein or dihydroethidium . The isogenic wild-type strain shows no increased fluorescence at 37°C with either dye . To verify that the accumulation of ROS is not an effect of any cell cycle arrest, temperature sensitive mutants in cell division cycle genes were arrested at 37°C for 4 h, incubated with dihydrorhodamine 123 and tested for fluorescence. Neither a non-apoptotic allele of CDC48 ( cdc48 -3), nor mutants in cdc2 (arresting as large-budded cells like cdc48 mutants) or cdc31 (arresting with an abnormal spindle-like cdc48 mutants) show an increase of intracellular fluorescence (not shown). To discriminate whether the accumulation of ROS is just a byproduct of the induction of apoptosis in the cdc48 mutant or whether it is essential for the process, oxygen radicals were scavenged with free radical spin traps . Strain KFY437 and the corresponding wild-type strain KFY417 were grown on YEPD with shaking at 28°C and then incubated with various concentrations of PBN or TMPO for 4 h at 37°C. To evaluate effects on survival, cell viability was determined as the number of colonies formed after plating a defined number of cells. Aliquots of the same sample were used for TUNEL reaction and electron microscopic investigation. 5 mM PBN and 0.5 mM TMPO suppress TUNEL staining dramatically, whereas 0.5 mM PBN and 5 mM TMPO have less effect (not shown). Electron microscopy shows that the intense chromatin margination of strain KFY437 at 37°C is prevented almost completely by 5 mM PBN . These observations correspond to the protective effect of the spin traps on cell viability at 37°C, which is strongest with 0.5 mM TMPO or 5 mM PBN. Viability of the wild-type control is inhibited by the spin trap substances, indicating a certain cytotoxicity . At some concentrations of TMPO, proliferation of the control strain is even lower than that of the apoptotic strain. This might be due to neutralization of radicals and spin traps in the apoptotic cdc48 mutant. A similar protective effect was observed after incubation in an anaerobic environment. Mutant cultures and controls were incubated in a nitrogen atmosphere at 28°C for 30 min and further incubated in a 37°C waterbath for 4 h under nitrogen. Their viability was tested in a plating assay. While the control was not affected by the anaerobic conditions, the mutant strain showed significant survival after anaerobic conditions . Incubation in a nitrogen atmosphere also prevented accumulation of DNA strand breaks, as shown by TUNEL staining . Expression of bax in S. cerevisiae results in cell death with an apoptotic phenotype that is suppressed by the coexpression of bcl-X L . To determine whether Bax-induced apoptosis in yeast is also accompanied by oxygen stress, strain WCG4bax carrying a bax gene controlled by the GAL1 promoter was used. Bax expression was induced on galactose medium for 4 h or 13 h. 5 μg/ml dihydrorhodamine were then added and the fluorescence determined after 2 more hours of incubation. After a total of 6 h induction, strain WCG4bax shows little fluorescence (not shown). After 15 h, >80% of WCG4bax cells show an intense rhodamine 123 fluorescence , while the control strain (empty vector) shows no detectable fluorescence (not shown). Coexpression of bcl-X L suppresses the bax-induced accumulation of radicals. The strain WCG4bax/bcl-X L shows almost no rhodamine 123 fluorescence after 15 h incubation with galactose . Incubation with dichlorodihydrofluorescein diacetate or dihydroethidium gave similar results (not shown). Flow cytometric analysis of dihydrorhodamine 123–stained cells confirms that a majority of bax-expressing cells show an increased signal, while all control cells display a low fluorescence (not shown). To determine whether spin traps can prevent Bax-triggered cell death WCG4bax and the corresponding wild-type strain (empty vector) were incubated with the free radical spin traps PBN or TMPO at 28°C on galactose media for 15 h. Viability was determined as the portion of colony-forming units. While spin traps have a toxic effect on the control strain, WCG4bax survival is partially restored. As with the apoptotic cdc48 mutant, 5 mM PBN has the strongest protective effect on WCG4bax . Accordingly, 5 mM PBN prevents DNA strand breakage and chromatin condensation occurring in bax-expressing WCG4bax in the absence of spin traps . Anaerobic conditions also strongly reduce cell death and DNA breakage after induction of bax. Strain WCG4bax and the vector control strain were incubated in galactose medium in a nitrogen atmosphere at 28°C for 15 h. Viability of the bax-expressing strain is restored to control levels under these anaerobic conditions . The corresponding TUNEL test shows no indication of strand breaks . In metazoan apoptosis, ROS have been shown to participate in both early and late steps of the regulatory chain. In several apoptotic models, for example in ischemia-induced apoptosis , ROS act upstream of bax and caspases. In these models, radical trapping prevents the activation of caspases, and an inhibition of ensuing steps, e.g., with caspase inhibitors, prevents cell death indicating that the radicals act as signal molecules and do not simply cause lethal damage to DNA, lipids or proteins . However, radicals have also been shown to play a role in the late steps of apoptosis. K + deprivation induces apoptosis in cerebellar granule neurons via an accumulation of ROS. ROS production is prevented by actinomycin D, cycloheximide, and caspase inhibitors Ac-YVAD-CMK, suggesting that ROS act downstream of gene transcription, mRNA translation, and activation of caspases . Two scenarios connecting yeast with metazoan apoptosis have been described previously. The expression of some metazoan proapoptotic genes (bak, bax, ced-4) results in cell death accompanied by an apoptotic phenotype . The mutant allele cdc48 S565G induces the appearance of typical phenotypic markers of apoptosis . We found that in both cases ROS accumulate in the cell. Radical trapping or growth under strictly anaerobic conditions prevent cell death and the accompanying apoptotic effects, demonstrating that the radicals are not a byproduct but a promoter of the apoptotic-like features in these cells. In addition, apoptotic cell death could be induced by exposing yeast cells to oxidative stress, either with low concentrations of exogenous H 2 O 2 or by growing a gsh1 deletion mutant in the absence of glutathione. These results illustrate a central role of ROS in all cases of apoptosis in yeast known to date. We observed that with low concentrations of H 2 O 2 , cycloheximide enhances the survival rate of S. cerevisiae cells. This phenomenon has been observed before, but at the time remained unexplained . Our electron microscopic investigation and TUNEL test show that cycloheximide prevents both apoptotic chromatin condensation and DNA fragmentation, and increases the number of necrotic cells. The prevention of cell death by inhibition of protein synthesis is a specific indicator to distinguish apoptosis from necrosis in metazoan systems . Our findings indicate that yeast cell death triggered by low H 2 O 2 concentrations is not caused by cell damage but involves an active cooperation of the cell. Massive corruption of cellular structures and metabolism prevents the cooperation of the cell in its death process. As we have shown, high concentrations of H 2 O 2 result in cell death associated with a disintegration of intracellular structures but without the phenotypic markers of apoptosis. Stationary cells of S. cerevisiae are much less sensitive to oxidative stress than exponentially growing cells . We find that they show no detectable effect at concentrations of H 2 O 2 that cause an apoptotic phenotype in exponentially growing cells and give strong TUNEL staining at high concentrations of H 2 O 2 . The observation that numerous cytotoxic substances that cause necrosis at high concentrations induce apoptosis at lower concentrations give a clue to the origin of apoptosis. ROS are natural inducers of fatal cell damage, aging, and cell death. A likely evolutionary mechanism for the development of apoptosis might be based on that phenomenon. Even if a cell survives the damage caused by radicals, it will probably become a useless mouth to feed, consuming resources but producing no or impaired offspring. For the total clonal cell population, the altruistic death of these cells spares energy sources for the undamaged cells and therefore poses an evolutionary advantage. A potentially altruistic death has been described for stationary cultures of S. cerevisiae that can survive for very long times in pure water, but quickly lose viability in nutrient depleted media, perhaps in order to keep the few resources for the best adapted isogenic relatives. Bcl-2 delays the loss of viability . In evolution, more complex regulatory pathways probably developed upstream of the basic mechanism, resulting in a complex signaling network with a seemingly contradictory behavior in different apoptotic models . The phenomenon that glutathione is actively extruded during apoptosis of human monocytic cells may be a strategy to enhance ROS signaling– induced apoptosis. Some apoptotic pathways retain the usage of ROS in early regulatory steps, other pathways still use it in late steps. In addition, alternative pathways lacking the involvement of ROS developed that cannot be blocked by radical trapping. But even in the case of the ROS-independent apoptotic pathway via CD95 , the expression of the proapoptotic CD95 ligand is induced by ROS . While none of the established apoptotic proteins are encoded in the S. cerevisiae genome, some of them will perform similar functions when expressed in yeast cells. In mammals, one of the effects of Bax expression is a caspase-independent production of ROS . Our finding that the expression of Bax protein in yeast also results in an accumulation of ROS that is prevented by the coexpression of Bcl-X L indicates a mechanism for the induction of cell death and apoptotic phenotype by Bax in yeast. Anorganic ROS are therefore the first regulators of apoptosis found to be shared between yeast and higher animals . Future research should address whether the mechanism downstream of this signal uses conserved elements. | Study | biomedical | en | 0.999998 |
10330405 | Specimens were obtained in accordance with the tenets of the Declaration of Helsinki, and all donors provided informed consent for biopsy. Permission was obtained for specimens taken from an organ donor. In preliminary experiments, ocular keratinocytes were cultivated from several biopsies taken from conjunctiva, cornea, and limbus of patients undergoing penetrating keratoplasty. Results of growth rate experiments and serial cultivation were inconsistent, probably because the epithelium was suffering from the original pathology. Therefore, samples were obtained only from patients undergoing cataract, strabismus, and keratoconus surgery and presenting with undamaged anterior ocular epithelium. In one case, biopsies were taken (within 9 h from death) from the eye of a 54-yr-old organ donor woman with no history of ocular surface disorders. Biopsies (1–2 mm 2 ) were taken from different areas of the eye, as indicated in Fig. 1 . 3T3-J2 cells were a gift from Prof. Howard Green (Harvard Medical School, Boston, MA). The keratin 3-specific AE5 mAb was a gift from Dr. Tung-Tien Sun (New York University Medical Center, New York). The keratin 19-specific RCK108 mAb was purchased from Dako Corp. Ocular keratinocytes were cultivated on a lethally irradiated feeder layer of 3T3-J2 cells as described previously . In brief, samples were treated with trypsin (0.05% trypsin and 0.01% EDTA) at 37°C for ∼80 min. Cells were collected every 20 min. We obtained an average of 17.3 × 10 3 cells/mm 2 , a value lower than that obtained with skin biopsies, which yield an average of 30 × 10 3 cells/mm 2 (our unpublished data). Cells were plated (1.5 × 10 4 /cm 2 ) on lethally irradiated 3T3-J2 cells (2.4 × 10 4 /cm 2 ) and cultured in 5% CO 2 and humidified atmosphere in: DME and Ham's F12 media (2:1 mixture) containing FCS (10%), insulin (5 μg/ml), adenine (0.18 mM), hydrocortisone (0.4 μg/ml), cholera toxin (0.1 nM), triiodothyronine (2 nM), glutamine (4 mM), and penicillin-streptomycin (50 IU/ml). Epidermal growth factor (10 ng/ml) was added at 10 ng/ml beginning at the first feeding, 3 d after plating. Cultures were then fed every other day. Subconfluent primary cultures were passaged at a density of 6 × 10 3 cells/cm 2 and cultured as above. For serial propagation, cells were passaged as above, always at the stage of subconfluence, until they reached senescence. Single cells, isolated under the microscope, were inoculated onto multi-well plates already containing a feeder layer of 3T3 cells . After 7 d of cultivation, clones were identified under an inverted microscope, photographed and their area was measured. Each clone was then transferred to three dishes. Two dishes (3/4 of the clone) were used for serial propagation and further analysis. The third (indicator) dish (1/4 of the clone) was fixed 12 d later and stained with rhodamine B for the classification of clonal type. The clonal type was determined by the percentage of aborted colonies formed by the progeny of the founding cell. When 0–5% of colonies were terminal the clone was scored as holoclone. When all colonies formed were terminal (or when no colonies formed), the clone was classified as paraclone. When >5% but <100% of the colonies were terminal, the clone was classified as a meroclone . Selected clones (see Results) were serially propagated to determine the number of cell generations. The entire procedure of cloning and subcultivation was done under strict timing conditions identical for each clone. Cells (300–2,000) from each biopsy and from each cell passage of serially cultivated mass and clonal cultures were plated onto 3T3 feeder layers and cultivated as above. Colonies were fixed 12 d later, stained with rhodamine B and scored under a dissecting microscope. Values are expressed as the ratio of the number colonies on the number of inoculated cells. All colonies were scored whether progressively growing or aborted. The number of cell generations was calculated using the following formula: x = 3.322 log N/No, where N equals the total number of cells obtained at each passage and No equals the number of clonogenic cells. Clonogenic cells were calculated from the colony-forming efficiency data (see above), which were determined separately in parallel dishes at the time of cell passage. Confluent sheets of epithelium generated by either mass or clonal cultures, were detached from the vessels with Dispase II . Specimens were fixed in paraformaldehyde (4% in PBS) overnight at 4°C and embedded in paraffin. Sections were either stained with hematoxylin-eosin or double-immunostained with K3-specific AE5 mAb and K19-specific RCK108 mAb (DAKO). AE5-immunoreaction was detected with the HRP-dextran-anti–mouse complex (EnVision Plus/HRP system; DAKO), using 3,3′-diaminobenzidine tetrahydrochloride (FAST DAB; Sigma Chemical Co. ) as a chromogen. RCK108 immunoreaction was detected with the alkaline-phosphatase-dextran-anti–mouse complex (EnVision/AP system; DAKO), using Fast Red TR/Naphtol AS-MX (Fast Red; Sigma Chemical Co. ) as a chromogen. Double-immunostained sections were washed, counterstained with hematoxylin, and mounted in an aqueous mounting media. Goblet cells were stained with the Alcian blue-periodic acid-Schiff reaction as described . Dissociated cells obtained from either mass or clonal cultures were centrifuged on a coverslip , fixed in methanol/acetone, and immunostained as above. Parallel coverslips were fixed in 4% paraformaldehyde and stained with Alcian blue-periodic acid-Schiff reaction for goblet cell quantification. Ocular keratinocytes were isolated from 1-mm 2 biopsies taken only from donors with no history of ocular surface disorders and cultivated as described under Materials and Methods. In one case, biopsies were freshly harvested from a cadaver. Mimicking the growth behavior of epidermal keratinocytes , limbal, bulbar, and forniceal keratinocytes founded colonies, each colony being the progeny of a single cell . The shape and overall appearance of colonies and of cells within the colonies were similar to those observed with epidermal keratinocytes (not shown). The doubling time of both limbal and conjunctival cells was ∼20 h. Colonies eventually fused and generated a stratified epithelium . Corneal keratinocytes isolated from central and paracentral cornea usually did not form colonies. Occasionally, keratinocytes isolated from paracentral cornea formed scattered colonies which, however, could not be serially cultivated. Epithelia from different parts of the body express keratin pairs that are unique for each location . For instance, the epidermis expresses the K1/K10 keratin pair whereas the corneal epithelium expresses the K3/K12 keratin pair . The conjunctival epithelium expresses K19 but not K3/K12 . Fig. 2 shows that cultured corneal-limbal epithelium is K3 + and K19 − (C), while cultured conjunctival epithelium is K3 − and K19 + (D), suggesting that the site-specific differentiation program is maintained under these culture conditions. 12 1–2-mm 2 biopsies were taken from the eye of a female, 54-yr-old organ donor. Biopsies were taken from different areas of the eye, as indicated in Fig. 1 . Four limbal biopsies (from the four quadrants of the eye: g, h, i, and l), four bulbar conjunctival biopsies (from the four quadrants of the eye: c, d, e, and f), and biopsies from paracentral cornea (m), central cornea (n), superior fornix (a), and inferior fornix (b) were processed simultaneously within 24 h from death. To evaluate the clonogenic ability of the different areas of the ocular surface, 300 cells from each area were plated onto lethally irradiated 3T3-J2 cells and stained 12 d later with rhodamine B. As shown in Fig. 3 , the limbus was the only area of the corneal-limbal epithelium able to form large and smooth colonies, while cells from paracentral and central cornea were not clonogenic. In contrast, keratinocytes isolated from the superior and inferior fornix, as well as from the four quadrants of the bulbar conjunctiva, displayed a comparable colony forming ability . It is worth noting that values obtained from the limbus and from the different areas of the conjunctiva were comparable. Clonogenic ability and growth potential of epithelial cells are two very different concepts. The former indicates the capacity of a basal cell to found a colony, the latter deals with its capacity of producing cell generations, hence it deals with its self-renewal potential. Therefore, the proliferative capacity of cells isolated from different areas of the eye was evaluated by serial cultivation. As shown in Fig. 4 A, keratinocytes from the limbus (four quadrants: g, h, i, and l), from either the superior and inferior fornix (a and b) and from bulbar conjunctiva (four quadrants: c, d, e, and f) could be cultivated up to 14 passages (2–3 mo) and underwent 80–100 divisions before senescence. As clearly shown by Fig. 4 , B and C, conjunctival cells with very high capacity for cell division were uniformly distributed on the ocular surface, whereas corneal cells with high proliferative capacity were segregated in the limbus. Again, it is worth noting that the values obtained from the limbus and from different areas of the conjunctiva were similar. These results were confirmed by serially cultivating ocular keratinocytes obtained from 42 biopsies of unrelated donors of different ages. As shown in Table I , cells from 12 forniceal biopsies underwent an average of 79 doublings; cells from 21 bulbar biopsies underwent an average of 82 doublings; cells from 9 limbal biopsies underwent 85 doublings before senescence. Cells from central cornea could not be serially cultivated. These data demonstrate that clonogenic cells endowed with high capacity for cell division (typical of stem cells) are segregated in the limbal region of the corneal-limbal epithelium and are evenly distributed in the conjunctival epithelium covering the eye bulb and the fornix. To investigate whether holoclones, meroclones and paraclones, previously identified in human skin , were also present in the ocular epithelium, single cells were isolated from 15 different sub-confluent primary cultures obtained from 7 different donors. After 7 d of cultivation, each single clone was photographed and its area was measured. Each clone was then transferred to three dishes. Two dishes (3/4 of the clone) were used for serial propagation and further analysis. The third dish (1/4 of the clone) was fixed 12 d later and stained with rhodamine B for the classification of clonal type, which is based on the relative number of aborted colonies . As shown in Table II , we analyzed 339 clones (129 from the inferior and superior fornix, 152 from the bulbar conjunctiva and 58 from the superior limbus). The majority of clones were classified as meroclones. Holoclones were identified, in similar percentage, in limbus, fornix, and bulbar conjunctiva (Table II ). Confluent sheets of epithelium or dissociated cells, both obtained from randomly chosen clones, were analyzed using anti-K3 or anti-K19 antibodies. Immunohistochemistry confirmed that all clones from forniceal and bulbar conjunctiva were K19 + and K3 − . Although conjunctival cells are usually present in a limbal biopsy, the majority of clones from limbal epithelium was K3 + and K19 − (not shown). Only clones expressing the proper differentiation markers were selected for further experiments. Cultures from 60 conjunctival (33 bulbar and 27 forniceal) clones (4 holoclones, 43 meroclones, and 13 paraclones) obtained from the same donor were then serially cultivated to evaluate their proliferative capacity. In agreement with the number of doublings of the original mass cultures (97 doublings for the bulbar mass culture and 96 doublings for the forniceal mass culture), holoclones isolated from both bulbar and forniceal conjunctiva were able to produce 91.7 and 84.5 (mean values) cell generations before senescence, respectively. Instead, meroclones from both areas displayed a high heterogeneity in their growth potential, with clones able to undergo as many as 60 and as few as 25 cell doublings . As expected, paraclones underwent a very limited number of cell divisions . The three types of clones were similarly represented in bulbar and forniceal epithelium. We also serially cultivated five holoclones isolated from the superior limbus of a different donor. As with conjunctival holoclones, limbal holoclones underwent 95 ± 8 doublings before senescence, accounting for the entire proliferative potential of the original mass culture. Previous studies have demonstrated that the relative percentage of epidermal holoclones declines in vivo during aging , suggesting that newborns start their life with a high content of keratinocyte stem cells which are programmed to undergo a fixed number of cell divisions before senescence. Since holoclones are found also in the adulthood , it is conceivable that recruitment of stem cells, to generate transient amplifying and differentiated cells, occurs in “waves” during life and that this recruitment is somehow accelerated by emergency situations as wound healing or cultivation . Accordingly, stem cell-derived transient amplifying cells are expected to have a quite variable proliferative potential. As shown in Fig. 5 , conjunctival meroclones are highly heterogeneous in their capacity for cell division, hence we consider them as transient amplifying cells endowed with a higher proliferative potential than paraclones. Taken together, these data fulfill the criteria for the existence of ocular holoclones arising from stem cells and demonstrate that holoclones, meroclones, and paraclones, previously identified only in skin, constitute the proliferative cell compartment also of the human anterior ocular surface; corneal stem cells are located exclusively in the limbus; conjunctival stem cells are uniformly distributed in the clonogenic layer of both bulbar and forniceal epithelium; the proliferative potential of both corneal and conjunctival stem cells is of 80–100 doublings, a value considerably lower than that characterizing the epidermal (and hair follicle) stem cell compartment (120–160 doublings); and the transient amplifying cell compartment is formed by cells with highly variable proliferative potential. The conjunctival epithelium is populated by goblet cells, which are essential for the maintenance of ocular surface integrity. PAS staining of confluent sheets prepared from secondary cultures of bulbar conjunctival keratinocytes showed that several goblet cells were present in suprabasal layers , while, as expected, goblet cells were absent in the epithelium cultured from limbal biopsies . PAS-staining performed during serial cultivation of forniceal and bulbar keratinocytes (growing colonies) from 11 different donors indicated that goblet cells were present during the entire life span of the cultures. The relative content of goblet cells was investigated during the exponential phase of conjunctival cell growth (growing colonies) and after generation of cohesive epithelial sheets (2–3 d after confluence). As shown in Fig. 6 D, an increasing number of goblet cells was present during serial cultivation. However, while growing colonies displayed a goblet cell content of 200–500 cells per cm 2 , confluent conjunctival sheets had >5,000 goblet cells/cm 2 . We have calculated that this value corresponds to a goblet/keratinocyte ratio of ∼1/30, a ratio not far from that found in vivo in resting bulbar conjunctiva . These data could be explained by either proliferation of differentiated goblet cells in culture; existence of an unidentified goblet cell precursor, able to proliferate and differentiate in vitro ; differentiation of bipotent conjunctival stem cells able to give rise to two different cell types . Therefore, we investigated whether the progeny of conjunctival clones was populated by goblet cells. As shown in Fig. 6 , E–H goblet cells were present in cultures of both holoclones (E and F) and meroclones (G and H) isolated from both forniceal (E–G) and bulbar (F–H) conjunctiva. PAS-staining of mass cultures from the same 339 clones shown in Table II , revealed that 100% and 93% of holoclone- and meroclone-derived cultures contained goblet cells, respectively. Paraclones were usually goblet-negative, but a few paraclones, notably those able to be passaged once , were also able to produce scattered goblet cells. Cultures from limbal holoclones and meroclones were invariably goblet-negative. This set of data proves that conjunctival keratinocytes are bipotent since they can generate also goblet cells. Moreover, goblet cells were found in cultures from meroclones, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. To investigate whether the generation of a goblet cell by a conjunctival keratinocyte was related to a specific time of its life, we selected (from the same donor) 14 conjunctival clones endowed with significant proliferative potential and analyzed the formation of goblet cells during their serial cultivation (PAS reactions were carried out at each cell passage on exponentially growing colonies). Based on data shown in Fig. 5 , we arbitrarily defined as “young” transient amplifying cells those meroclones still able to undergo 35–60 doublings, and “old” meroclones those cells undergoing 20–35 cell divisions before senescence. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at a specific time of their cycles of cell duplication. As shown in Fig. 7 A (first peak), a first generation of goblet cells occurred at 45–50 cell doublings. A second, and more substantial, bulk of goblet cells was generated very late in the life of the clones, at 10–20 doublings before senescence . As shown in Table II and Fig. 5 , holoclones (and young meroclones) are usually present in low abundance. Also, it is not possible to determine a priori whether a cell will generate a holoclone, a young meroclone or an old meroclone. This explains the low number of cells with high proliferative potential, hence able to generate two peaks of goblet cells, that we were able to analyze in a single experiment . Therefore, to substantiate the observation of a cell doubling-dependent mechanism for goblet cell generation, we decided to perform clonal analysis of sub-confluent primary cultures initiated from new conjunctival biopsies taken from a different donor. 53 new clones were analyzed by serial cultivation. 12 clones had a significant proliferative capacity and 4 clones were classified as holoclones . Serial cultivation of these clones confirmed that conjunctival keratinocytes with high proliferative capacity give origin to a bulk of goblet cells at precise times of their life history. As with clones analyzed in Fig. 7 A, a first generation of goblet cells occurred at 45–50 cell doublings . A second generation of goblet cells occurred very late in the life of the clones, at 10–20 doublings before senescence (second peak). Holoclones in Fig. 7 A produced ∼90 cell generations, while holoclones in Fig. 7 B produced an average of 104 cell generations before senescence. In both cases, however, goblet cells were generated at 45–50 cell doublings and at ∼15 cell doublings before senescence. This suggests that generation of goblet occurs at times precisely set for the number of cell doublings and explains the longest interval observed between the two peaks in Fig. 7 B as compared with Fig. 7 A. The contemporary presence of transient amplifying cells with very different residual proliferative potential explains why the overall goblet cell content of the conjunctival epithelium tends to remain constant in growing colonies of mass cultures , even though the bulk of goblet cells generated by a single clone occurs only at precise times in its life history . It is worth noting that conjunctival keratinocytes consistently generate a higher overall number of goblet cells late in their life . In vivo, this is the time when clones are approaching the end of their life, are close to their postmitotic state and are therefore preparing themselves for migrating in the suprabasal layers. This might explain the strong and sudden increase in the number of goblet cells observed when growing colonies reach confluence and stratify , and fits with the suprabasal location of goblet cells. This set of data is summarized in Fig. 7 C and demonstrate that forniceal and bulbar conjunctival stem cells are bipotent, since they can give rise to conjunctival keratinocytes and goblet cells; both young and old transient amplifying conjunctival keratinocytes are able to produce goblet cells; transient amplifying cells generate goblet cells at precise times of their life related to an intrinsic cell doubling clock; and the total amount of goblet cells generated by old transient amplifying cells is consistently higher than that generated by young transient amplifying cells. Analysis of goblet cells in selected clones began after 20–30 doublings in culture , the preceding interval being devoted to the processing of the biopsy, the isolation of the clones and their growth to suitable large populations. Therefore, we cannot exclude generation of goblet cells also during the first 20–30 doublings. Also, scattered production of goblet cells can be observed between the two peaks. It has been reported that goblet cells can duplicate in vivo . Thus, we cannot exclude that goblet cells can undergo additional cycles of duplications . We have identified cells with extensive capacity for cell division (holoclones) in cultured limbal, forniceal, and bulbar human ocular epithelia. One might argue that, since holoclones have been taken from their natural “niche” and forced to undergo rapid proliferation, they have irreversibly lost their “stem-ness,” hence they should not be considered as representative of the in vivo stem cell compartment. However, permanent epithelial regeneration obtained with cultured keratinocytes in massive full-thickness burns and in severe lining epithelial defects is the best available proof that stem cells are indeed preserved in culture. Recently, this was further confirmed by long-term analysis of cultured retrovirus-transduced porcine and human keratinocytes after grafting onto syngeneic or athymic animals . Since holoclones are endowed with the highest proliferative potential in vitro and account for the entire proliferative capacity of the original mass culture destined to transplantation, we feel quite confident in considering them as keratinocyte stem cells. Interestingly, ocular holoclones have a lower proliferative potential (80–100 doublings) than epidermal holoclones . This might reflect the fact that human epidermis is renewed monthly, while the ocular epithelium is renewed every year, and suggests that holoclones can adjust their proliferative potential according to the needs of the tissue of origin. We have shown here that corneal stem cells are segregated in the limbus, whereas conjunctival stem cells are evenly distributed in the epithelium covering the eye bulb and the fornix. Our data fully confirm the location of corneal stem cells suggested by the slow-cycling properties (quiescence) of mouse limbal cells , but are in contrast with data (also based on [ 3 H]TdR-retaining experiments) suggesting that murine conjunctival stem cells were concentrated in the fornix . Usually, in vivo, stem cells are slow-cycling, hence they stay in the G0 phase of the cell cycle and enter in the S phase very rarely. However, at variance with the extensive capacity for cell division, quiescence is not an obligatory property of stem cells . For instance, stem cells inhabiting the mouse limbus are induced to rapid division under wound healing stimuli , while stem cells of the human intestinal crypts and of the Drosophila ovary have been estimated to divide every 24 h, even during normal homeostasis . However, it is worth noting that in rabbits, forniceal keratinocytes have a much higher proliferative capacity in vitro than bulbar keratinocytes , further suggesting a segregation of stem cells in the fornix of some animals. Whether these differences between species reflect divergent mechanisms of normal tissue homeostasis or a different behavior of the epithelium in wound healing remains to be determined. The discrete location of corneal stem cells in the limbus and the absence of cells with proliferative capacity in the central cornea, suggests that corneal epithelium is formed mostly by transient amplifying cells. This gradient of distribution of cells with different capacity for multiplication fits well with the hypothesis of a continuous centripetal migration of limbal stem cell–derived transient amplifying cells, which is governed by a circadian rhythm and is strongly increased in wound healing . It is worth noting that murine corneal cells are still able to divide (at least twice) in vivo . Therefore, it is conceivable to speculate that in order to keep the integrity of the ocular surface, human corneal cells must also undergo some rounds of division in vivo in the central region of the cornea. These transient amplifying cells are not clonogenic under our culture conditions. This strongly resembles a similar situation in the human hair follicle, where a second population of non-clonogenic transient amplifying cells has been postulated to exist in the hair bulb . The differentiated progeny of a stem cell can be represented by a single cell type or by distinct cell types . For instance, epidermal stem cells give rise to basal and subrabasal keratinocytes at different level of differentiation whereas, in the hemopoietic tissue, a pluripotent stem cell can generate committed lymphoid or myeloid progenitors which, in turn, give rise to several distinct blood cell types. The origin of conjunctival goblet cells has been controversial. Experiments by Tsai and colleagues suggested that conjunctival keratinocytes and goblet cells derive from different precursors. A first indication on the possibility of a bipotent common progenitor came from experiments by Wei et al. . These authors isolated epithelial cells from the fornix of rabbits and plated them at low density onto a 3T3 feeder-layer. When they implanted primary cultures into the flanks of BALB/c mice they observed the development of epithelial cysts bearing variable amounts of goblet cells. Our data clearly settle this controversy and confirms data by Wei et al. by showing that, indeed, clones of conjunctival keratinocytes give rise to the mucin-producing goblet cells and that both are therefore derived from a common bipotent progenitor . It is worth noting that the differentiation of a keratinocyte into a goblet cell is more drastic than the differentiation of a basal into a suprabasal keratinocyte. It amounts to a thorough revision of cytoplasmic structure and function from a filament-rich cell whose function is to confer strength and resistance into a cell secreting proteoglycan (mucin), which conditions the surface formed by the keratinocytes. Commitment to differentiate into goblet cells occurs relatively late, so that goblet cells are preferentially generated by old transient amplifying cells. This is consistent with the suprabasal location of goblet cells in vivo, the low but reproducible number of goblet cells found in growing conjunctival cell colonies, and the spectacular increase of the number of goblet cells at a specific time of cell culture (when colonies fuse and generate a stratified epithelium). Indeed, goblet cells elicit their function in the suprabasal layers, where they secrete the mucin responsible for the formation of the tear film . We show here that the rate of formation of goblet cells from conjunctival keratinocytes depends upon the number of cell doublings. In particular, we show that conjunctival keratinocytes give origin to a bulk of goblet cells at least twice in their life. A first generation of goblet cells occurs at 45–50 cell doublings, while a second generation of goblet cells occurs at ∼15 doublings before senescence. The second peak is usually greater than the first peak , suggesting that a young transient amplifying cell tends to generate less goblet cells than an old meroclone. Because of the heterogeneity in the clonal composition of the epithelium, this cell doubling-dependent form of differentiation would not be observable in the intact epithelium. The precision of the conjunctival cell doubling clock argues in favor of a deterministic way of generating a differentiated goblet progeny. However, as shown in Fig. 7 , clones generate a highly variable number of goblet cells (sometimes the second peak is very low), even when they reach the right number of doublings, suggesting a role for probabilistic events. Whether this reflects a flexibility of fate decisions within the same cell, or the presence of distinct predetermined progenitor cells remains to be determined. In a recent review, Morrison et al. addressed the question concerning control of stem cell differentiation and regulation of the repertoire of stem cell fate. Usually, instructive or selective actions of external factors are evoked to explain the decision of a multipotent cell to enter a particular differentiation pathway. A nice example of an instructive mechanism has been reported for the neural crest stem cell, whose differentiation is promoted by members of the TGF-β superfamily . A selective action of environmental factors implies that the initial choice of differentiated fate by a multipotent cell is controlled by a cell autonomous mechanism, which, however, has never been shown in mammalian stem cells . Generation of goblet cells by conjunctival keratinocytes can also occur in serum free medium, in the absence of 3T3 feeder-layer and in the absence of EGF, cholera toxin and insulin (not shown), suggesting a cell-autonomous mechanism. Alternatively, it might be argued that instructive (non–cell-autonomous) mechanisms might arise through secretion of growth factors that induce a specific set of sister cells to differentiate into goblet cells. Future experiments will clarify these issues and will hopefully shed light on the molecular mechanisms responsible for the conjunctival cell fate decision. The unambiguous identification and characterization of stem and transient amplifying cells in lining epithelia is of paramount importance for cell therapy and gene therapy . Indeed, improvements of cell culture techniques allow the preparation of cohesive sheets of stratified epithelia suitable for autologous transplantation in patients suffering from life-threatening or highly disabling epithelial defects . It is evident that the long-term persistence of the regenerated epithelia requires engraftment of stem cells. Moreover, any attempt at using keratinocytes for gene therapy of genetic disorders , requires identification and stable transduction of stem cells . Cultured limbal cells can generate cohesive sheets of authentic corneal epithelium, which have already been used to restore the corneal surface of patients with complete loss of the corneal-limbus epithelium . The data presented in this paper suggest that severe bilateral destruction of the conjunctival epithelium from alkaline burns could be cured by the engraftment of cultures of autologous conjunctival cells initiated from a tiny biopsy which can be taken not only from the fornix, but from any spared area of the conjunctival surface. Goblet cell–dependent mucin deficiency has been implicated in various disorders, including ocular cicatricial pemphigoid, Stevens-Johnson syndrome, xerophthalmia, and certain sicca syndromes as a result of chronic keratoconjunctivitis. The engraftment of cultured sheets of conjunctival epithelium bearing goblet cells may accomplish for these diseases what has already been accomplished for corneal epithelium by the engraftment of limbal cultures. | Other | biomedical | en | 0.999997 |
10330406 | Metatarsal rudiments were isolated from 15.5 d p.c. ICR/B6D2 mouse embryos or embryos from crosses of PTHrP+/− mice and were used as noted. Noon on the day of the vaginal plug is 0.5 d p.c. The three central metatarsal rudiments were cultured in each well of a 24-well plate in 1 ml of chemically defined medium containing α-MEM supplemented with 0.05 mg/ml ascorbic acid ( Sigma Chemical Co. ), 0.3 mg/ml l -glutamine ( Sigma Chemical Co. ), 0.05 mg/ml gentamicin 90, 1 mM β-glycerophosphate ( Sigma Chemical Co. ), and 0.2% endotoxin-free fraction V BSA ( Sigma Chemical Co. ) as previously described . Explants were grown at 37°C in a humidified 5% CO 2 incubator. TGF-β1 (1 or 10 ng/ml) in 4 mM HCl (R&D Systems) or PTHrP (1–34) in 10 mM acetic acid containing 1% BSA (Bachem) at varying concentration was added to cultures 12–16 h after dissection. Medium was changed on the third day of culture. Cultures were observed and photographed with an Olympus SZH10 dissecting microscope at 24 h, 3 d, and 5 d of treatment. The length of metatarsals was calculated by measuring the length of rudiment from photographs taken on the dissecting microscope and dividing by the magnification factor (usually ×5). The length of several bone rudiments (at least three for each condition) was calculated, and the data are shown as the mean ± SD. Metatarsal rudiments were fixed overnight at 4°C in fresh 4% paraformaldehyde and then decalcified 2 h to overnight at 4°C in 1 mM Tris, pH 7.5, 10% EDTA tetrasodium salt, 7.5% polyvinyl pyrolidone, and 1 μl/ml diethyl pyrocarbonate (DEPC). The explants were dehydrated through a series of ethanols and xylene and then embedded in paraffin and cut into 5-μm sections. Sections were stained with hematoxylin and eosin as noted using standard procedures. Metatarsal rudiments were treated with 10 μM bromo deoxyuridine (BrdU; Boehringer Mannheim ) for 2.5 h. Metatarsals were then washed twice in PBS at 37°C, fixed in paraformaldehyde at 4°C overnight, embedded in paraffin, and cut into 5-μm sections. Sections were deparaffinized, denatured in 2 N HCl for 20 min at 37°C, and neutralized in 1% boric acid/ 0.285% sodium borate, pH 7.6. Next, the sections were treated with 0.005 mg trypsin/ml 0.05 M Tris, pH 7.6, for 3 min at 37°C and washed three times in PBS. Immunostaining was then performed using components and directions from the Vectastain Elite staining kit (Vector Laboratories). A rat mAb directed to BrdU (Harlan) was used as the primary antibody at a 1:200 dilution. Cy3-conjugated avidin (Vector Laboratories) was substituted for the avidin-biotin-peroxidase complex. Excess Cy3-conjugated avidin was removed from the sections by washing three times for 10 min each in PBS at room temperature, and the sections were immediately mounted with Aquapoly mount (Poly Sciences). Fluorescence was observed and imaged using a Zeiss Axiophot microscope and a Princeton Instruments CCD camera with Sellomics imaging software. Immunohistochemical staining of type X collagen was performed using polyclonal antibodies to mouse type X collagen (a generous gift from Bjorn Olsen, Harvard Medical School, Boston, MA). Sections were dewaxed, rehydrated, and incubated with 1 mg/ml hyaluronidase ( Sigma Chemical Co. ) in PBS at 37°C for 30 min. Immunohistochemistry was then performed following the directions supplied with the Vectastain Elite immunoperoxidase staining kit (Vector Laboratories). The color reaction was performed using the DAB substrate kit, also from Vector Laboratories. Sections were very lightly counterstained with toluidine blue, and photographs were taken under bright field illumination with a Zeiss Axiophot microscope. The percentage of the bone rudiment stained for type X collagen was calculated as follows: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}\frac{length\;of\;area\;stained\;for\;type\;X\;collagen}{length\;of\;total\;bone\;rudiment}{\times}100=\%\;of\;total\;cartilage\;containing\;type\;X\;collagen.\end{equation*}\end{document} This measurement takes into account changes in the total length of the bone rudiment. Immunofluorescent staining for the TGF-β type I and type II receptors was performed using polyclonal antibodies obtained from Santa Cruz Biotechnology (type I, cat. no. sc 398; type II, cat. no. sc 220). Sections were dewaxed, rehydrated, and treated with 0.05% Saponin in water for 30 min at room temperature. Saponin was removed by washing three times for 5 min each in TBS with 0.1% Tween 20 at room temperature. Immunostaining was then performed using components and directions from the Vectastain Elite staining kit; however, Cy3-conjugated avidin (Vector Laboratories) was substituted for the avidin-biotin-peroxidase complex. Excess Cy3-conjugated avidin was removed from the sections by washing three times for 10 min each in TBS with 0.1% Tween 20 at room temperature, and the sections were immediately mounted with Aquapoly mount (Poly Sciences). Fluorescence was observed and imaged using a Zeiss Axiophot microscope and a Princeton Instruments CCD camera with Sellomics imaging software. In situ hybridization was performed as described . Metatarsal rudiments were fixed overnight in paraformaldehyde at 4°C, then decalcified in 1 mM Tris, pH 7.5, 10% EDTA tetrasodium salt, 7.5% polyvinyl pyrolidone, and 1 μl/ml DEPC at 4°C for 2 h to overnight. The metatarsals were then dehydrated in alcohol and embedded in paraffin. Sections (5 μm) were hybridized to 35 S-labeled antisense riboprobes. The Ihh plasmid (a kind gift from Andy McMahon, Harvard University, Cambridge, MA) was linearized with XbaI, and riboprobe was synthesized using T7 polymerase. The PTHrP plasmid (a generous gift from Henry Kronenberg, Harvard Medical School, Boston, MA) was linearized with EcoRI, and riboprobe was synthesized using T3 polymerase. Slides were exposed to photographic emulsion at 4°C for 2 wk, then developed, fixed, and cleared. Sections were counterstained with 0.02% toluidine blue. Bright field and dark field images were captured with a Princeton Instruments CCD camera. Bright field and dark field images were merged using the electronic photography imaging program from Sellomics. RNA was extracted from cartilage rudiment cultures by lysis in guanidine thiocyanate using the Ambion RNaqueous kit and the manufacturer's instructions with the modification that the cartilage rudiments were first homogenized in the guanidine solution in a microcentrifuge tube with a small pestle. RNA was treated with RNase-free DNase ( Promega Corp. ) for 1 h at 37°C, phenol/chloroform extracted, and ethanol precipitated. Precipitation of RNA was facilitated with 20 μg glycogen per sample ( Boehringer Mannheim ). RNA concentration was determined spectrophotometrically. For reverse transcription (RT)-PCR analysis, cDNA was synthesized from 1 μg of total RNA as described in the GeneAmp RNA PCR kit ( Perkin-Elmer ) using the oligo dT primers. For each sample, 5 μl cDNA was amplified with 0.2 μM primers and 0.2 mM nucleotides for 20 cycles. These conditions were determined to fall within the linear range of PCR product formation for both PTHrP and glyceraldehyde-6-phosphate dehydrogenase (GAPDH). Samples incubated without reverse transcriptase were used to determine if there was DNA contamination in the RNA samples. DNA contamination was not detected in the RNA from the three separate experiments performed. PCR products were blotted to nylon membrane as described and probed with 32 P-labeled cDNA probes for PTHrP (obtained from H. Kronenberg, Harvard Medical School, Boston, MA) and GAPDH. GAPDH was used as an internal control for the amount of cDNA used in each reaction. Relative levels of mRNA were quantified using a Molecular Dynamics PhosphorImager. GAPDH primers were purchased from Clontech . The PTHrP primers used were as follows : PTHrP5′, TGG TGT TCC TGC TCA GCT A, and PTHrP3′, CCT CGT CGT CTG ACC CAA A. Previously, we showed that expression of a dominant-negative mutation of the TGF-β type II receptor in skeletal tissue in transgenic mice resulted in increased hypertrophic differentiation , suggesting that TGF-β regulates terminal differentiation in vivo. To determine the effects of exogenously added TGF-β1 on endochondral bone development, mouse metatarsal rudiments isolated from embryos at 15.5 d of gestation and grown in a chemically defined medium were used in the following experiments . Organ culture allows for the study of complex biological processes in a three-dimensional structure in the context of native cell–cell and cell–extracellular matrix interactions. Over 5 d, cartilage organ cultures grew longitudinally and several stages of endochondral bone formation were detected . The zone of hypertrophic cartilage (clear area plus dark area in the center of the rudiment), calcified zone (dark area), and proximal and distal bone ends representing resting and proliferating zones of cartilage have been described previously . Treatment with 1 ng/ ml TGF-β1 (data not shown) and 10 ng/ml TGF-β1 for 5 d inhibited longitudinal growth of the cartilage rudiment . In addition, mineralized matrix was not detected in TGF-β1–treated cultures . To further characterize the effects of TGF-β1 on bone development, the histology of untreated and TGF-β1– treated cartilage rudiments was examined. In untreated cartilage rudiments grown in culture for 5 d, histologically defined resting, proliferating, and hypertrophic zones were clearly demarcated in hematoxylin and eosin–stained sections . These zones of cartilage were not easily recognized in cultures treated with 10 ng/ml TGF-β1, and treatment appeared to result in a decreased fraction of histologically hypertrophic cartilage . In addition, the perichondrium in TGF-β1–treated cultures contained up to eight layers of cells, while the perichondrium in control cultures was only two to three cell layers thick . The morphology and histology of TGF-β1–treated metatarsal bone rudiments suggested that TGF-β could have effects on both cell proliferation and differentiation. The effect of TGF-β1 on bone length could be in part due to effects on cell proliferation. To test this hypothesis, bone rudiments were either treated with the TGF-β1 vehicle or were treated with 10 ng/ml TGF-β1 for 24 h followed by treatment with BrdU for 2.5 h. BrdU incorporation was assayed using immunofluorescence . In control cultures, BrdU-labeled cells were detected throughout the histologically defined zones of resting and proliferating cartilage . Cell proliferation in the histologically defined resting zone is not unusual in fetal bones. Treatment with TGF-β1 dramatically inhibited chondrocyte proliferation as measured by the lack of BrdU-labeled cells in the cartilage , suggesting that inhibition of chondrocyte growth participates in the decrease in bone length observed after treatment with TGF-β1. In contrast, TGF-β1–treated rudiments had an increase in the number of BrdU-labeled cells in the perichondrium compared with the untreated cultures , suggesting that TGF-β1 stimulated growth of perichondrial cells. Immunolocalization of type X collagen was used to determine the effects of TGF-β1 on hypertrophic differentiation in the organ culture model. Type X collagen is a well-documented marker for hypertrophic cartilage . Expression of Ihh, a marker for cells committed to become hypertrophic , was also used to determine the effects of TGF-β1 on hypertrophic differentiation. In untreated metatarsal rudiments grown in culture for 5 d, the matrix of the histologically hypertrophic cartilage contained type X collagen . Ihh expression was localized to prehypertrophic chondrocytes in the lower zone of proliferation (transition cells) and continued into the zone of hypertrophy . Treatment with TGF-β1 resulted in a decrease in the amount of histologically hypertrophic cartilage as well as a decrease in the fraction of the cartilage area stained for type X collagen . This measurement takes into account changes in the total length of the bone rudiment and suggests that inhibition of hypertrophic differentiation occurs independent of the inhibition in longitudinal growth. Ihh mRNA was restricted to a small population of cells in the center of the TGF-β1–treated rudiment , suggesting that treatment with TGF-β1 resulted in a decrease in the number of cells committed to hypertrophic differentiation in the metatarsal organ cultures. The data taken together suggest that TGF-β1 acts to inhibit several points of endochondral bone formation, including cell growth, hypertrophic differentiation, and matrix mineralization. TGF-βs signal through heteromeric serine/threonine kinase receptors . Both type I and type II receptors are required to generate a response to TGF-β . The current model is that TGF-β ligand binds to the TGF-β type II receptor on the cell surface . The type II receptor is then able to recruit the type I receptor to form a heterotetrameric complex of two type I and two type II receptors. The type II receptor, which is a constitutively active kinase, phosphorylates the type I receptor, activating the type I serine/threonine kinase. Downstream targets of the type I receptor then transduce the signal to the nucleus. To determine which cell types in the cartilage rudiment cultures potentially respond to the TGF-β1 signal, expression of the TGF-β type I and type II receptors in embryonic mouse metatarsal rudiments isolated at 15.5 d of gestation and kept in culture for 24 h (data not shown) or 5 d was examined by immunofluorescence. The receptor expression pattern was similar at 24 h and 5 d of culture. Staining for the TGF-β type II receptor was detected at varying levels in all the cell types in the cartilage rudiment . The highest levels of staining were detected in the perichondrium, the resting cartilage at the most distal ends of the metatarsal, the cells in the portion of proliferating zone closest to the hypertrophic zone (transition zone), and hypertrophic cells in the center of the rudiment. Strong staining was also observed in small round cells within the osteoid seam between the zone of hypertrophy and the perichondrium/periosteum. These cells are presumably osteoblasts. Immunofluorescent staining with an antibody directed to the TGF-β type I receptor demonstrated an expression pattern for the type I receptor similar to that observed for the type II receptor . The pattern of expression for the receptor proteins suggests that all cells are potentially capable of responding to TGF-β1, but also suggests that cells in the perichondrium, distal tips of the rudiment, lower proliferating, and hypertrophic zones could be more sensitive to treatment with TGF-β1. To provide evidence for the model that TGF-β and PTHrP act in a common signal cascade to regulate the rate of chondrocyte differentiation, the hypothesis that TGF-β1 regulates expression of PTHrP mRNA in metatarsal cultures was tested. RNA extracted from cartilage rudiments either untreated or treated with 10 ng/ml TGF-β1 for 8 h or 5 d was analyzed using RT-PCR analysis . Conditions were determined such that PCR product formation would be in the linear range. PTHrP mRNA levels were normalized to the expression of GAPDH, a constitutively active housekeeping gene. In three separate experiments, treatment with TGF-β1 resulted in an approximately threefold increase in PTHrP mRNA levels after 8 h, and the increased levels of PTHrP mRNA persisted for up to 5 d of treatment . In situ hybridization was used to localize expression of PTHrP in cartilage rudiments untreated or treated with TGF-β1 for 6 h or 5 d . PTHrP mRNA was localized to the periarticular region in both untreated and TGF-β1– treated cultures at 6 h and 5 d . Cultures treated with TGF-β1 also demonstrated ectopic PTHrP mRNA expression in the perichondrium . These data support the model that TGF-β1 acts to positively regulate PTHrP expression. If TGF-β1 functions via increasing PTHrP expression, we would predict that PTHrP would have some of the same effects as TGF-β1 on bone development. Previously, it was shown that transgenic mice that misexpress PTHrP from the type II collagen promoter and mice that express a constitutively active PTH/PTHrP receptor show a delay in hypertrophic differentiation . The hypothesis that PTHrP inhibits development of metatarsal rudiments grown in organ culture was tested, and the effects of PTHrP were compared with those of TGF-β1 . Similar to treatment with TGF-β1 , treatment with as little as 10 −8 M PTHrP inhibited mineralization of the cartilage matrix in the explant cultures relative to the untreated control . In contrast to TGF-β1, PTHrP at a concentration as high as 10 −5 M did not result in significant inhibition of longitudinal growth in the explants when compared with the untreated cultures . The hypothesis that PTHrP did not affect chondrocyte proliferation was confirmed using the BrdU incorporation assay . Cultures were untreated or treated with 10 −6 M PTHrP for 24 h followed by treatment with BrdU for an additional 2.5 h. BrdU was detected using immunofluorescence. There were no detectable differences in BrdU labeling in control and PTHrP-treated cultures. The effects of PTHrP on metatarsal organ cultures were further characterized by staining with hematoxylin and eosin and immunostaining for type X collagen (data not shown). Histological examination of organ cultures revealed a decrease, relative to the untreated control , in the fraction of the cartilage area that contained histologically hypertrophic cartilage and stained with type X collagen in both PTHrP- and TGF-β1– treated cultures (control = 30 ± 3.7%, n = 11; TGF-β1 = 19.3 ± 3.9%, n = 10; PTHrP = 16.7 ± 3.2%, n = 3). The histology and BrdU labeling of perichondrium were not affected by PTHrP . These data indicate that PTHrP and TGF-β have some distinct and some overlapping functions. Both PTHrP and TGF-β1 inhibit hypertrophic differentiation and matrix mineralization in the assays described above; however, in contrast to TGF-β1, PTHrP did not affect longitudinal growth, chondrocyte proliferation, or the perichondrium. A model whereby TGF-β could act through PTHrP to regulate hypertrophic differentiation and/or matrix mineralization is suggested. Metatarsal organ cultures from embryos with targeted deletion of the PTHrP gene were used to test the hypothesis that PTHrP is required for TGF-β–mediated effects on endochondral bone development. Two separate experiments were performed to compare the effects of 0 , 1 , and 10 ng/ml TGF-β1 on rudiments from PTHrP-positive and PTHrP-null embryos. There were no detectable differences in cartilage rudiments from PTHrP-positive and PTHrP-null cultures at the time of dissection (15.5 d p.c.) or at the time TGF-β was added (16 h after dissection; data not shown). By 5 d of culture, rudiments from mice homozygous for the PTHrP deletion demonstrated increased matrix mineralization relative to PTHrP-positive controls . This observation is consistent with the reported phenotype of PTHrP-null mice . PTHrP-positive cultures treated with TGF-β1 for 5 d demonstrated a dose-dependent decrease in length and mineralized matrix . Cultures from PTHrP-null embryos demonstrated similar effects , suggesting that PTHrP is not required for TGF-β1–mediated effects on growth or matrix mineralization. The effects on longitudinal growth were predicted in the previous experiment where addition of PTHrP to explant cultures did not alter growth. To determine if PTHrP is required for the effects of TGF-β1 on metatarsal histology, hematoxylin and eosin– stained sections of organ cultures from PTHrP-positive and PTHrP-null embryos either untreated or treated with TGF-β1 were examined. Consistent with the assertion that PTHrP normally inhibits hypertrophic differentiation , all of the chondrocytes in untreated PTHrP-null rudiments were histologically hypertrophic , whereas only a fraction of the cartilage in PTHrP-positive cultures is hypertrophic . In PTHrP-positive cultures, TGF-β1 treatment resulted in a dose-dependent decrease in the fraction of chondrocytes in the histologically hypertrophic and proliferating zones . In contrast, most of the chondrocytes in rudiments from PTHrP-null mice remained histologically hypertrophic despite treatment with 1 or 10 ng/ml TGF-β1 . Treatment with 1 and 10 ng/ml TGF-β1 resulted in an increase in the thickness of the perichondrium relative to control cultures in both PTHrP-positive and PTHrP-null cultures. The data suggest PTHrP is not required for the effects of TGF-β on the perichondrium but indicate that PTHrP may be required for TGF-β–mediated effects on hypertrophic differentiation. To specifically determine the role of PTHrP in TGF-β–mediated effects on hypertrophic differentiation, sections from PTHrP-positive and PTHrP-null organ cultures untreated or treated with TGF-β1 were immunostained for type X collagen, a marker of hypertrophic differentiation . Treatment of PTHrP-positive cultures with 1 ng and 10 ng/ml TGF-β1 resulted in a decrease in the fraction of cartilage stained for type X collagen relative to untreated cultures . In contrast, TGF-β1 had no effect on the fraction of cartilage stained for type X collagen in PTHrP-null cultures . Since treatment with TGF-β1 did not inhibit hypertrophic differentiation in PTHrP-null cultures, the data indicate that PTHrP is required for the effects of TGF-β1 on hypertrophic differentiation and suggest TGF-β acts upstream of PTHrP in a common signaling cascade to regulate differentiation. The data also suggest that TGF-β1 has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation. Embryonic metatarsal bone rudiments grown in organ culture were used to test the hypothesis that TGF-β1 acts upstream of PTHrP to regulate chondrocyte differentiation. TGF-β1 acted at several check points during endochondral bone formation, inhibiting longitudinal growth, hypertrophic differentiation, and matrix mineralization. TGF-β drastically reduced BrdU labeling in chondrocytes of the histologically defined resting/proliferating zone by 24 h of treatment, suggesting that TGF-β affects longitudinal growth by regulating chondrocyte proliferation. However, additional effects of TGF-β on apoptosis or extracellular matrix production cannot be ruled out by the experiments presented here. PTHrP also inhibited chondrocyte hypertrophy and matrix mineralization but did not affect cell proliferation. TGF-β1 stimulated expression of PTHrP mRNA, suggesting that TGF-β1 and PTHrP could be part of the same signaling cascade to regulate hypertrophic differentiation and/or matrix mineralization. The hypothesis that PTHrP is required for TGF-β1–mediated effects on endochondral bone formation was tested using organ cultures from PTHrP-null mice. TGF-β1 did not inhibit hypertrophic differentiation in PTHrP-null cultures, suggesting that inhibition of hypertrophic differentiation occurred through a PTHrP-dependent mechanism. In contrast, longitudinal growth and matrix mineralization were inhibited by TGF-β1 in both PTHrP-expressing and PTHrP-null cultures, suggesting TGF-β has PTHrP-independent effects as well. A model where longitudinal growth, hypertrophic differentiation, and matrix mineralization can be regulated independently is supported by data presented here and by others . Inhibition of hypertrophic differentiation by TGF-β1 is not solely a consequence of reduced proliferation. Treatment with TGF-β resulted in a decrease in the fraction of the bone area that stained for type X collagen. This measurement took into account differences in the total length of the bone rudiment and suggest that TGF-β inhibits hypertrophic differentiation in addition to inhibiting longitudinal growth. TGF-β was previously shown to be sufficient to inhibit hypertrophic differentiation in chondrocyte cultures . In addition, expression of a dominant-negative form of the TGF-β type II receptor in transgenic mice resulted in increased hypertrophic differentiation , suggesting that TGF-β is necessary to prevent hypertrophy in vivo. Likewise, inhibition of matrix mineralization by TGF-β1 is most likely not a consequence of reduced hypertrophic differentiation. Previously, it was shown that treatment with TGF-β1 resulted in only a 40% inhibition in hypertrophy but a complete inhibition of matrix mineralization, suggesting that TGF-β inhibited matrix mineralization independently of cellular differentiation . In the present report, we demonstrate that TGF-β1 completely inhibited matrix mineralization in the absence of PTHrP even though these cultures were completely hypertrophic. Immunolocalization of TGF-β type I and type II receptors suggests that all cells in the cartilage rudiment are potentially able to respond to TGF-β1; however, the data also suggest that some cell types may be more sensitive to treatment. High levels of receptor protein were localized to perichondrial cells, a subset of resting chondrocytes at the ends of the skeletal element, cells in the proliferating zone closest to the hypertrophic zone, and the hypertrophic cells in the center of the long bone rudiment. High levels of the TGF-β receptors in the perichondrium suggest that these cells are able to respond to TGF-β directly. Treatment with TGF-β was shown to increase the number of BrdU-labeled cells in the perichondrium, and TGF-β1 was able to stimulate perichondrial growth in the absence of PTHrP. Localization of the TGF-β receptors in chondrocytes suggests that TGF-β could act directly on chondrocytes to regulate growth or hypertrophic differentiation; alternatively, TGF-β1 could act indirectly through the perichondrium. Recently it was demonstrated that growth and differentiation of long bone cartilage are mediated by factors from the perichondrium . Removal of the perichondrium from chick long bone rudiments resulted in an increase in the length of the explants, an increase in DNA synthesis in the zone of proliferation, and increased hypertrophic differentiation . Long growth continued in the perichondrium-free chick cultures in the presence of exogenously added PTHrP even though hypertrophic differentiation was inhibited . This is in agreement with our results in mouse metatarsal organ cultures where PTHrP inhibited hypertrophic differentiation but did not affect incorporation of BrdU. In addition, we observed that inhibition of longitudinal growth by TGF-β1 occurred independently of PTHrP, suggesting that, if the effect of TGF-β1 on growth is mediated through the perichondrium, another factor is involved. Fibroblast growth factor receptor 3 (FGFR3) is expressed in chondrocytes in the proliferating zone , and FGFR3-null mice demonstrate enhanced and prolonged longitudinal bone growth . The ligand for the FGFR3 that regulates endochondral bone formation is not known. TGF-β may modulate the expression or activity of FGFs from the perichondrium and thereby indirectly regulate longitudinal growth. This suggests that TGF-β1 mediates hypertrophic differentiation indirectly through PTHrP which is most likely secreted from the perichondrium. TGF-β1 stimulated expression of PTHrP mRNA in the cartilage rudiment cultures, and expression was localized to the perichondrium and periarticular area. TGF-β1 has been shown to stimulate expression of PTHrP mRNA and protein in several cultured cell types, including carcinoma cell lines, uterine and ovarian epithelial cells, and articular chondrocytes . Additional support of an indirect effect of TGF-β mediated through the perichondrium comes from transgenic mice that express a truncated, dominant-negative TGF-β type II receptor in the perichondrium. These mice demonstrate increased hypertrophic differentiation in the growth plate and suggest that TGF-β signaling in the perichondrium is required to regulate hypertrophy . Development of skeletal elements requires the coordination of signals from several sources. It was recently shown that Ihh and PTHrP form a negative feedback loop that provides a mechanism for chondrocytes to sense and downregulate their rate of differentiation . Misexpression of Ihh in chick limb cartilage rudiments resulted in inhibition of chondrocyte differentiation and increased expression of Ptc and Gli in the perichondrium, suggesting that the perichondrium contained the Ihh responding cells . Misexpression of Ihh also resulted in increased expression of PTHrP, and it was shown that PTHrP was required for the inhibitory activities of Hh on chondrocyte differentiation . Expression of a dominant-negative form of the TGF-β type II receptor in mouse perichondrium results in increased terminal chondrocyte differentiation and increased and persistent expression of Ihh . Since Ihh normally acts as a negative regulator of differentiation, it was proposed that TGF-β signaling is required for Ihh-mediated inhibition of chondrocyte differentiation. There is precedent for such a model. The Drosophila protein, Dpp, a member of the TGF-β superfamily, can act as a secondary signal downstream of Hh to regulate the patterning of the imaginal disks , and BMP-2 and BMP-4 are induced by sonic Hh in the chick limb and hind gut . Furthermore, Hh- and TGF-β–related genes are coexpressed at many sites of cell–cell communication in the mouse embryo . The present study suggests that TGF-β acts upstream of PTHrP to negatively regulate chondrocyte differentiation; however, whether or not TGF-β mediates the effects of Ihh on PTHrP has not yet been addressed. Future experiments will provide further evidence of how signaling from several factors is coordinated to build and maintain a functional skeletal system. | Study | biomedical | en | 0.999995 |
10330407 | All chemicals were obtained from Sigma Chemical Co. unless stated otherwise. All tissue plastics and tissue culture media were obtained from GIBCO BRL . All fluorescence indicators were from Molecular Probes Inc. The procedure is essentially as described by Peuchen et al. with slight modifications. A sucrose-based separation step was added to the procedure to aid in the removal of cell debris and dense myelin, thereby promoting cell attachment and growth. Earle's balanced salt solution (EBSS) was used throughout the isolation protocol for incubations and washes. Cerebra were taken from 5- to 8-wk-old Sprague-Dawley rats. The cortex was separated from the other brain structures, the meninges and blood vessels were removed. The tissue, kept at 4°C, was chopped into small pieces, resuspended in EBSS, and forced through a 280-μm metal mesh (tissue pan; Sigma Chemical s Co). The filtered suspension was washed by centrifugation at 400 g for 4 min at room temperature. The pellet was resuspended in a Ca 2+ , Mg 2+ –free EBSS solution containing 50,000 U/ml trypsin (EC 3.4.21.4, isolated from porcine pancreas), 1.033 U/ml collagenase (EC 3.4.24.3, type XI, isolated from Clostridium histolyticum ), 336 U/ml deoxyribonucleate 5′-oligonucleotidohydrolase (EC 3.1.21.1, type IV, isolated from bovine pancreas). The enzymatic digestion was stopped after 15 min at 36°C by the addition of 10% heat-inactivated FBS. The suspension was gravity-filtered through a 140-μm metal mesh and centrifuged at 400 g for 6 min. The pellet, resuspended in EBSS, was layered over a 0.4-M sucrose solution (molecular biology grade; BDH Chemicals Ltd.) and centrifuged at 400 g for 10 min. The resulting pellet was washed twice in EBSS, followed by one wash in d- valine–based minimum essential medium (MEM) with Earle's salts. The pellet was resuspended in d -valine MEM, which inhibits the growth of fibroblasts and endothelial cells, supplemented with 5% FBS, 2 mM glutamine, and 1 mM malate, and transferred to tissue culture flasks precoated with 0.01% poly- d -lysine. The flasks were placed in an incubator (95% air, 5% CO 2 , at 36°C) for 20–40 h, after which the medium was refreshed. After 6 d in culture, the supplemented d -valine MEM was replaced with MEM supplemented with l -valine. The cells reached confluency at 12–14 d in vitro and were harvested and reseeded onto 24-mm-diam glass coverslips (BDH) precoated with 0.01% poly- d -lysine for fluorescence measurements. Three or more days before experiment, astrocytes were plated onto glass coverslips with an initial plating density of ∼7,000 cells/cm 2 . Cells were washed with a physiological saline containing 156 mM NaCl, 3 mM KCl, 2 mM MgSO 4 , 1.25 mM KH 2 PO 4 , 2 mM CaCl 2 , 10 mM glucose, and 7.5 mM Hepes at pH 7.35. For the qualitative measurement of [Ca 2+ ] m and [Ca 2+ ] n the cells were loaded with 4.5 μM rhod-2/AM in the presence of 0.005% Pluronics 127 for 30 min at room temperature and washed thoroughly. Rhod-2 fluorescence was excited at 560 nm and emitted fluorescence was collected through a 590-nm-long pass barrier filter. High resolution imaging enabled identification of individual mitochondria for analysis. To demonstrate that rhod-2 could be used to monitor changes in nuclear Ca 2+ , the cells were loaded with 4.5 μM rhod-2/AM and 4.5 μM fura-2/AM in the presence of 0.005% Pluronics 127 for 30 min at room temperature. The dyes were excited sequentially at 490 (for rhod-2), 340, and 380 nm (for fura-2) using specific filters housed in a computer-controlled filter wheel (Cairn Research Ltd.) and fluorescence at wavelengths longer than 520 nm was measured. For analysis of [Ca 2+ ] cyt waves in single astrocytes, the cells were loaded with 4.5 μM fluo-3/AM in the presence of 0.005% Pluronics 127 for 30 min at room temperature. Fluo-3 was excited at 490 nm and emitted fluorescence ≥ 530 nm was collected. For analysis of changes in mitochondrial membrane potential (Δψ m ), cells were loaded with 3 μM of tetramethylrhodamine ethyl ester (TMRE) for 15 min at room temperature. TMRE fluorescence was excited at 546 nm and emitted fluorescence was collected through a 590-nm-long pass barrier filter. Fluorescence measurements were obtained via an epifluorescence inverted microscope (Nikon Diaphot) equipped with an oil immersion 63× fluorite objective (NA 1.3). Fluorescence was excited using a 75 W xenon arc lamp and emitted light projected onto the face of a slow scan, frame transfer 800 × 600 pixel, 12 bit cooled CCD camera (Digital Pixel Ltd.), acquisition rate ∼2 frames per second full frame, binning pixels 3 × 3. To resolve the rising phases of both nuclear and [Ca 2+ ] m signals and to monitor wave propagation better, fast imaging was carried out using a fast readout, cooled interline transfer CCD camera allowing image acquisition rates up to 50 images per second with pixels binning at 4 × 4. All imaging data were acquired and analyzed using software from Kinetic Imaging. Because rhod-2, fluo-3, and TMRE are single wavelength fluorescent indicators, it was not possible to apply the ratiometric method for quantitative determination of [Ca 2+ ] m , [Ca 2+ ] cyt , and Δψ m , respectively. Therefore, the information derived from the measurements of rhod-2, fluo-3, and TMRE fluorescence was normalized as a function of the first image. Nuclear Ca 2+ ([Ca 2+ ] n ) monitored by fura-2 was expressed as the ratio of fura-2 fluorescence after excitation at 340 and 380 nm. All imaging experiments were carried out at room temperature. Greater care was taken for the wave propagation experiments with an average temperature of 21 ± 1°C to avoid any artifact due to the temperature-dependence of Ca 2+ wave kinetics . Excitatory light was kept to a minimum with neutral density filters and a computer-controlled shutter to minimize photobleaching and photodynamic injury to cells. Confocal microscopy was used for colocalization studies and for high spatial resolution of mitochondrial depolarization waves. In brief, astrocytes were dual-loaded with rhod-2/AM as described above and with 20 nM MitoFluor Green/AM for 30 min at room temperature. Cells were imaged using a confocal laser scanning microscope (model 510; Carl Zeiss, Inc. ). Cells were illuminated using the 488-nm emission line of an argon laser and fluorescence was collected simultaneously between 505 and 530 nm for MitoFluor Green and at λ ≥ 585 nm for rhod-2. To spatially resolve waves of mitochondrial depolarization, astrocytes were loaded with TMRE as described above and were illuminated with the 488-nm emission line. Emitted fluorescence was collected at λ ≥ 585 nm. After testing for normality and variance homogeneity, the data were subjected to the unpaired t test. When the variance was heterogeneous, the Mann-Whitney U test was used. The 0.05 level was selected as the point of minimal statistical significance. All the curves shown in figures are representative of at least three measurements performed on four separate cell preparations. Adult rat cortical astrocytes were loaded with rhod-2/AM, a Ca 2+ -sensitive fluorescent indicator that accumulates in mitochondria in response to Δψ m . Fig. 1 a illustrates a typical image of a rhod-2–loaded adult rat cortical astrocyte showing the localization of rhod-2 within rod-shaped, brightly fluorescent structures of variable organization, reminiscent of mitochondria. However, a diffuse small cytosolic signal and an accumulation of the dye in the nucleus (with rhod-2 also labeling nucleoli) were usually noticed . To confirm that the main rhod-2 fluorescence signal did originate from mitochondria, astrocytes were dual-loaded with rhod-2/AM and MitoFluor Green/AM, an indicator that labels mitochondria independently of mitochondrial function . The small overlap of the excitation spectra of rhod-2 and MitoFluor Green allows the simultaneous use of both the dyes . Colocalization of MitoFluor Green and rhod-2 within the cell was confirmed by merging the two images and by the analysis of colocalization shown in Fig. 1 d. It should be stated that the relative compartmental loading varied substantially between batches of rhod-2 and between preparations, but the difference was quantitative, not qualitative (i.e., some batches of dye gave much clearer localization into mitochondria, others gave a brighter relative cytosolic [and nuclear] signal, but the partitioning was still between cytosol/nucleus and mitochondria). Physiological activation of the P 2U purinoceptor by ATP in adult rat cortical astrocytes has been shown previously to raise [Ca 2+ ] cyt via an IP3-mediated pathway . We took advantage of the concomitant localization of rhod-2 within the nucleus (a mitochondrion-free volume of the cell) to confirm and monitor changes in [Ca 2+ ] cyt in response to ATP challenge, as [Ca 2+ ] n follows the changes in [Ca 2+ ] cyt by diffusion of Ca 2+ from one compartment into the other . The temporal relationship between [Ca 2+ ] n and [Ca 2+ ] m after a brief pulse of 100 μM ATP is shown in the time sequence of Fig. 2 a. The sequential quantitative changes in [Ca 2+ ] n and [Ca 2+ ] m , measured along the longitudinal axis of the cell as indicated by the line in the first image of Fig. 2 a, are shown in Fig. 2 , b and c. Before agonist application, the rhod-2 fluorescence was relatively dim and the mitochondrial signal was not obviously brighter than much of the cytosol . Immediately after the mobilization of intracellular Ca 2+ stores by ATP, an overall increase over both the nucleus and the cytosol was observed, followed by a delayed increase in signal over mitochondria (selected close to nucleus) that remained elevated long after [Ca 2+ ] n recovery. The rise in [Ca 2+ ] m presented a wavelike pattern in that mitochondria at one end of the cell tended to respond before those at the other . [Ca 2+ ] n rose rapidly (time to reach half the peak amplitude, t 1/2 = 1.95 ± 1.1 s, n = 26), reaching a normalized fluorescence peak of 1.75 ± 0.36 ( n = 57), and returned to its resting level within a minute . A quantitative analysis showed that [Ca 2+ ] n returned to resting values with a decay time constant of 4.84 ± 1.75 s ( n = 41). In contrast, [Ca 2+ ] m rose slowly ( t 1/2 = 3.17 ± 1.3 s, n = 26) and reached a fluorescence [Ca 2+ ] m peak corresponding to a 2 ± 0.57-fold increase ( n = 63). It remained at a high plateau within the experimental time scale . Long-term recording of the ATP-induced changes in [Ca 2+ ] m indicated that full recovery to the resting [Ca 2+ ] m took 28.6 ± 10.5 min . Plotting changes in [Ca 2+ ] m as a function of the changes in [Ca 2+ ] n revealed four phases of intracellular Ca 2+ handling triggered by the IP3-induced mobilization of intracellular Ca 2+ stores . Immediately after ATP application (phase 1), [Ca 2+ ] n rose very rapidly without any important change in [Ca 2+ ] m . [Ca 2+ ] m started increasing together with [Ca 2+ ] n (phase 2). In the following 70 s (phase 3), [Ca 2+ ] m fell slowly despite the rapid return of [Ca 2+ ] n to its resting value. Finally, [Ca 2+ ] m progressively and slowly decreased (phase 4) to its resting levels. We cannot exclude that the measured rise in the extranuclear fluorescence probably reflects a combination of cytosolic and mitochondrial Ca 2+ signals. However, neither the peak nor the recovery of the [Ca 2+ ] m signal appeared to be contaminated by the [Ca 2+ ] cyt signal that peaked and returned to basal level far more quickly than [Ca 2+ ] m (see below for time courses of [Ca 2+ ] cyt and [Ca 2+ ] n ). In addition, the flat morphology of astrocytes in culture allowed the resolution and selection of individual mitochondria for image analysis. Mitochondrial calcium uptake depends on Δψ m and dissipation of the potential provides the easiest way to examine the consequences of [Ca 2+ ] m accumulation for cell Ca 2+ signaling. To this end, we chose to use the mitochondrial protonophore p -trifluoromethoxy-phenylhydrazone (FCCP), a mitochondrial uncoupler that dissipates the proton gradient across the inner mitochondrial membrane, hence abolishing the Δψ m . Under such conditions, activation of the mitochondrial ATPase in reverse mode may lead to the rapid consumption of ATP . Therefore, FCCP (1 μM) was always applied in combination with 2.5 μg/ml oligomycin, to inhibit mitochondrial consumption of cellular ATP. The time series shown in Fig. 4 a, and more particularly the corresponding surface plot and line image , illustrate the changes in [Ca 2+ ] cyt and [Ca 2+ ] n upon FCCP application, followed by 100 μM ATP. FCCP initially induced a transient rise in [Ca 2+ ] cyt localized to the perinuclear and the cytosolic regions, most likely reflecting Ca 2+ release from mitochondria that was only followed later by a rise in [Ca 2+ ] n . In the experiment illustrated in Fig. 4 b, the FCCP-induced change in rhod-2 fluorescence was ∼1.2-fold. The mean increase in rhod-2 signal in response to FCCP application was 1.76 ± 0.43 ( n = 29) fold (see also below). Subsequent application of ATP elicited a diffuse, transient rise in nuclear and cytosolic rhod-2 fluorescence , without any apparent mitochondrial rhod-2 signal, demonstrating the effective inhibition of Ca 2+ uptake in deenergized mitochondria. The relative change in the nuclear Ca 2+ signal appeared far bigger than that in the cytosol . This apparent nuclear amplification is likely to occur because of an underestimation of [Ca 2+ ] cyt changes resulting from the creation of a pool of indicator sequestered in mitochondria that was insensitive to changes in [Ca 2+ ] cyt after the dissipation of Δψ m by FCCP . Collapse of Δψ m by FCCP did not significantly alter the peak amplitude of the ATP-induced [Ca 2+ ] n transient or the time required to reach half the peak amplitude of [Ca 2+ ] n (control cells, t 1/2 = 1.95 ± 1.1 s, n = 26; FCCP-treated cells, t 1/2 = 1.93 ± 0.95 s, n = 38; P = 0.93, t test). However, the decay phase of the [Ca 2+ ] n was significantly longer in the presence of FCCP . This result strongly suggests that mitochondrial Ca 2+ buffering plays a substantial role in hastening the restoration of [Ca 2+ ] n and, hence, [Ca 2+ ] cyt to basal levels. The [Ca 2+ ] cyt transient had a very similar time course to that of the [Ca 2+ ] n , strengthening the assumption that changes in [Ca 2+ ] n are a true index of changes in [Ca 2+ ] cyt . Dissipation of Δψ m by FCCP reverses the activity of the Ca 2+ uniporter, which becomes a Ca 2+ efflux pathway . We applied 1 μM FCCP to resting astrocytes dual-loaded with rhod-2 and fura-2, in the presence of 2.5 μg/ml oligomycin. Data are illustrated in Fig. 5 . Short application of FCCP induced a steep rise in [Ca 2+ ] n monitored by rhod-2 (solid circle) that returned to basal level within a minute (mean peak amplitude = 1.76 ± 0.43, n = 29). We were concerned that the change in the rhod-2 signal might reflect some form of redistribution of dye and dequench (i.e., as seen with TMRE, although this is not expected on theoretical grounds; see below) rather than a true reflection of changing [Ca 2+ ] n . We have recorded rhod-2 fluorescence simultaneously with a second widely used Ca 2+ -sensitive fluorescent indicator, fura-2. FCCP caused a rise in fura-2 ratio (open circle, mean peak amplitude = 2 ± 0.6, n = 21) sharing kinetic characteristics that could be superimposed on rhod-2. In principle, only the nonfluorescent AM ester form of rhod-2 exhibits a net positive charge, enabling its sequestration into mitochondria in response to Δψ m . Once the AM ester is cleaved within mitochondria, the resulting rhod-2 is hydrophilic and negatively charged and is unlikely to cross (mitochondrial) membranes or redistribute upon mitochondrial depolarization. As FCCP interferes with nonmitochondrial stores in some preparations, we repeated these experiments using antimycin A1 (an inhibitor of mitochondrial respiratory complex III) in the presence of oligomycin. Qualitatively, similar results were observed under these conditions (data not shown). Therefore, these data suggest that the rise in rhod-2 signal upon mitochondrial depolarization is an accurate reflection of changes in [Ca 2+ ] cyt and that Ca 2+ is released from mitochondria in these resting astrocytes. To investigate the spatio-temporal organization of the [Ca 2+ ] cyt changes in astrocytes, we examined Ca 2+ waves in fluo-3–loaded astrocytes at higher time resolution (∼7–14 Hz). Single-cell stimulation was performed either chemically by pressure application from a micropipette filled with ATP (20 μM) or mechanically with a micromanipulator-driven glass micropipette filled with the recording saline. We chose an ATP concentration of 20 μM to temporally resolve the Ca 2+ wave as stimulation with 100 μM induced a rapid wave that could not be resolved (data not shown). After either chemical or mechanical stimulation, a wave of [Ca 2+ ] cyt was routinely observed throughout the stimulated cell, usually spreading in directions away from the point of stimulation. Fig. 6 a shows some sample images from a sequence to illustrate the propagation of an intracellular [Ca 2+ ] cyt wave after ATP application. The time course of propagation was defined by selecting a line along the wave path on the image series. The intensity profile for the line for each frame was plotted as a two-dimensional line image (the post hoc equivalent of a line scan on a confocal system) as shown in Fig. 6 c. The wave propagation is seen as a gradual increase of the fluorescent signal across the cell from one side to the opposite side as a function of time. The wave velocity was readily measured simply as the slope of distance with time across the cytosol . The mean value for the velocity of the [Ca 2+ ] cyt wave was 21.6 ± 11.5 μm/s (Table I , n = 133) and 24.4 ± 10.7 μm/s (Table I , n = 129) under chemical and mechanical stimulation, respectively. These velocities were not significantly different, suggesting that once initiated, the waves were propagated by the same mechanism. The mean rate given by all these data was 22.9 ± 11.2 μm/s (Table I , n = 262). The time course of the amplitude changes in [Ca 2+ ] cyt signal for six regions of the cell during the propagation of the wave as a function of time is shown in Fig. 6 b. The peak amplitude of the wave was sustained throughout the regions of propagation although the rate of rise tended to decrease with propagation of the wave. Similar results were found for mechanically induced Ca 2+ waves (data not shown). The [Ca 2+ ] cyt at this sustained level (∼1.8-fold increase from resting values) remained within the measurable range. Indeed, saturation of the fluo-3 signal by application of 20 μM ionomycin showed a fluorescence enhancement of 5.7 ± 3.3-fold ( n = 77) in comparison to resting [Ca 2+ ] cyt . Determination of the diffusion rate of Ca 2+ across the nucleus of ATP-stimulated cells showed a significantly higher rate than the Ca 2+ wave velocity calculated over the cytosol (Table I , 34.6 ± 26.7 μm/s, n = 27, Mann-Whitney U test, P < 0.05) corresponding to a 60% increase. We asked about the consequences of mitochondrial calcium uptake, for mitochondrial function and, as described below, for calcium signaling. Upon chemical and mechanical stimulation, waves of [Ca 2+ ] m propagating across single astrocytes could be observed . However, the features of a [Ca 2+ ] m wave front were hard to characterize given the spatially irregular punctate rhod-2 signal. In addition, the rhod-2 signal arising from the cytosol obscured the initial rising phase of the [Ca 2+ ] m wave characteristics. To overcome this problem, we followed changes in Δψ m , as influx of Ca 2+ into mitochondria induces a small transient mitochondrial depolarization . Indeed, monitoring changes in Δψ m tends to amplify the response to mitochondrial calcium uptake and provides a sensitive index of a small calcium flux during mitochondrial Ca 2+ uptake . To follow changes in Δψ m , astrocytes were loaded with the potentiometric dye TMRE that selectively accumulates in mitochondria. The concentration of TMRE into polarized mitochondria causes autoquenching of TMRE fluorescence that is relieved by mitochondrial depolarization. Therefore, TMRE dequench is associated with a rise in fluorescence. Induction of a Ca 2+ wave with a brief deformation of the plasma membrane initiated a wave of mitochondrial depolarization spreading from the stimulation point across the cell seen clearly in the confocal image series shown in Fig. 7 a. A line image constructed from a similar image series but obtained using the fast readout CCD camera clearly showed the initiation sites of the propagating wave as mitochondria (the bright lines on the line image), whereas TMRE diffused slowly into the nucleus (asterisk). Determination of the wave velocity gave a mean value of 9.7 ± 4.2 μm/s ( n = 89). Fig. 7 b illustrates the changes in Δψ m recorded in six regions of the cell as a function of time. The peak amplitude of the transient mitochondrial depolarization was sustained over long distances throughout the regions of wave propagation . Indeed, roughly the same peak amplitude of Δψ m signals was seen at the cytosolic stimulation point 1 and the more distal point 6, suggesting again the regenerative nature of the propagating wave. In addition, the rate of rise tended to decrease with propagation of the wave, strikingly mirroring the gradual decrease of the rate of rise of the [Ca 2+ ] cyt wavefont. Thus, the propagation of the Ca 2+ wave causes a wave of mitochondrial depolarization in its wake. To determine the impact of mitochondrial Ca 2+ uptake on the spatio-temporal characteristics of calcium signaling, fluo-3–loaded astrocytes were treated either with 2.5 μg/ml antimycin A1 or with 10 μM rotenone (an inhibitor of mitochondrial respiratory complex I), both in association with 2.5 μg/ml oligomycin. This protocol induced a complete collapse of Δψ m (assessed with TMRE, data not shown), preventing any subsequent Ca 2+ uptake into mitochondria. Whatever the mitochondrial inhibitor used, mechanical or chemical stimulation elicited a typical [Ca 2+ ] cyt wave spreading away from the stimulation point, very similar to control cells in terms of pattern and regenerative nature of the propagation. Fig. 8 shows a typical example of Ca 2+ waves spreading across an astrocyte upon ATP challenge either in control conditions (a) or after treatment with antimycin A1/oligomycin (b). The differentiated line image (corresponding to a time series of iteratively subtracted images, only showing changes occurring between consecutive frames) given in the lower part of each frame showed the wavefront propagating through the cell. It is clear that the Ca 2+ wave traveled faster in the antimycin-treated cells . Determination of the wave velocity in antimycin-treated cells showed a mean value of 35.6 ± 23 μm/s ( n = 121) in response to ATP application and 31.1 ± 10.5 μm/s ( n = 66) to mechanical stimulation (Table I ). These values were not significantly different and the pooled data gave an average Ca 2+ wave rate in antimycin-treated cells of 34 ± 19.6 μm/s ( n = 187). Rotenone-treated cells exhibited a Ca 2+ wave rate of 40.4 ± 28.6 μm/s ( n = 50) to ATP challenge and of 30.6 ± 12.4 μm/s ( n = 35) to mechanical stimulation (Table I ). The averaged Ca 2+ wave rate was 36.4 ± 23.7 μm/s ( n = 85), remarkably similar to the diffusion rate of Ca 2+ through the mitochondrion-free nucleus (see above). This corresponds to a significant 48% and 58% increase of the [Ca 2+ ] cyt wave velocity in antimycin- and rotenone-treated astrocytes, respectively. Statistical analysis of the Ca 2+ wave velocities from antimycin- and rotenone-treated cells revealed no significant difference. These results clearly demonstrate that mitochondrial calcium uptake exerts a negative feedback action upon the kinetics of Ca 2+ wave propagation in astrocytes. In the present study, we have addressed several related questions: (a) Do mitochondria take up Ca 2+ during physiological Ca 2+ signaling in adult rat cortical astrocytes? (b) If so, what are the temporal characteristics of the mitochondrial response? (c) What are the consequences for mitochondrial potential and (d) what are the consequences for the spatio-temporal characteristics of the Ca 2+ signal? Our data show that mitochondria in these cells take up Ca 2+ during physiological Ca 2+ signaling, that they tend to retain that Ca 2+ for prolonged periods of time, and that the uptake not only shapes [Ca 2+ ] cyt signals but also exerts a negative feedback on the rate of propagation of Ca 2+ waves, by acting as high capacity fixed Ca 2+ buffers. Little information is available about the functional consequences of [Ca 2+ ] m accumulation with respect to [Ca 2+ ] cyt handling in glial cells. Stimulation of IP3-mediated ER Ca 2+ release by ATP in astrocytes routinely induced a simultaneous rise in [Ca 2+ ] n and in [Ca 2+ ] m . However, these signals exhibited quite distinct temporal features: the [Ca 2+ ] n transient recovered within 1 min. In contrast, the mitochondrial response rose 1.6-fold more slowly than the nuclear signal, and full recovery took several minutes. A longer time of recovery to basal level for [Ca 2+ ] m than for [Ca 2+ ] cyt also has been described in the glial cell line RBA-1 , in oligodendrocytes , in hepatocytes , in myocytes , and in adrenal chromaffin cells , although the specific rates varied widely between these different cell types. These observations differ strikingly from results obtained with aequorin targeted to mitochondria in HeLa cells . This discrepancy could result from the use of different cell types, different methodologies, different temperatures, the relative inaccuracy of aequorin at [Ca 2+ ] values below 300–400 nM ( K d for Ca 2+ = 1 μM), or alteration of the apparent kinetics of the [Ca 2+ ] m signal by rhod-2 given its relatively high affinity to Ca 2+ compared with the low affinity aequorin. The mitochondrial model elaborated by Magnus and Keizer also predicts a delay between increases in [Ca 2+ ] cyt and [Ca 2+ ] m . This delay may result from the positive cooperativity of uniporter activation by extramitochondrial Ca 2+ (Hill coefficient ∼2) . Mitochondria start accumulating cytosolic Ca 2+ from [Ca 2+ ] cyt ∼500 nM but do not saturate readily . Rates of [Ca 2+ ] m increase after intracellular Ca 2+ mobilization have been demonstrated to be more than one order of magnitude faster than that predicted on the basis of the amplitude of the mean rise in [Ca 2+ ] cyt . This has been explained by proposing that mitochondria closely apposed to Ca 2+ release sites would be exposed to local concentrations of Ca 2+ much higher than those measured in the bulk of the cytoplasm . Such a close localization of mitochondria to IP3 receptors recently has been observed in rat astrocytes in culture , and is also suggested by the appearance of transient mitochondrial depolarizations due to focal calcium release from the sarcoplasmic reticulum in cardiac myocytes . Depolarization of the mitochondrial potential with FCCP completely prevented [Ca 2+ ] m loading after a challenge with ATP. In addition, the rate of decay of the [Ca 2+ ] n signal was significantly slowed, suggesting that mitochondrial Ca 2+ uptake plays a significant role in the clearance of [Ca 2+ ] cyt loads in these cells. The effects of the mitochondrial uncoupler on Ca 2+ clearance were almost certainly not attributable to ATP depletion, as the mitochondrial ATPase was inhibited throughout by oligomycin to minimize mitochondrial consumption of cellular ATP. In contrast to our results, FCCP exposure did not influence the kinetic parameters of the depolarization-triggered [Ca 2+ ] cyt transient in oligodendrocytes , suggesting that [Ca 2+ ] m accumulation does not play an important role in shaping Ca 2+ signals in oligodendroglia. It seems likely that differences in such contributions of mitochondria to calcium signaling may vary simply as a function of the relative mitochondrial density or intracellular location in different cell types. The [Ca 2+ ] n signal in the astrocytes after ATP stimulation recovered with a monoexponential time course. In a number of cell types, [Ca 2+ ] cyt decay characteristically follows a biphasic sequence in which a fast initial decay is followed by a much slower secondary decay that appears to be maintained by the reequilibration of [Ca 2+ ] m by the mitochondrial Na + /Ca 2+ exchange . The absence of such a plateau phase in the astrocyte model seems consistent with the very prolonged retention of Ca 2+ in the mitochondria of these cells. This may reflect a low [Na + ] content of astrocytes and the absence of Na + influx during this form of stimulation (in contrast to depolarization-induced transients in the excitable sensory neurons, gonadotropes, and chromaffin cells). Indeed, while the accumulated [Ca 2+ ] m is clearly released back into the cytosol, this occurs at such a low rate that it does not cause a significant change in [Ca 2+ ] cyt . Therefore, the [Ca 2+ ] m efflux pathway does not significantly shape the [Ca 2+ ] cyt signal in these cells. We noted that depolarization of the mitochondrial potential routinely released Ca 2+ into the cytosol within quiescent cells, suggesting that mitochondria in this preparation were often Ca 2+ -loaded at rest. Given the very prolonged time course of [Ca 2+ ] m reequilibration, perhaps this is not very surprising as only occasional spontaneous activity would keep the mitochondria primed with a significant Ca 2+ content. A substantial Ca 2+ pool has been found in mitochondria of oligodendrocytes under resting conditions , in contrast to the very modest [Ca 2+ ] m content in neurons , T lymphocytes , rat gonadotropes , and rat chromaffin cells . In astrocytes, mitochondria seem to contribute to cytosolic Ca 2+ clearance during the [Ca 2+ ] n decay only but not in the fast rising phase of the [Ca 2+ ] n transient. In addition, dissipation of Δψ m by FCCP did not appreciably affect the peak amplitude of the ATP-induced [Ca 2+ ] n response. These results are similar to those described in adrenal chromaffin cells , T lymphocytes , rat gonadotropes , and HeLa cells . Simpson et al. found that FCCP pretreatment caused variable effects on the norepinephrine-evoked [Ca 2+ ] cyt responses in astrocytes, ranging from complete block of the response to minor effects. In contrast, we consistently found no significant alteration in the amplitude of the Ca 2+ peak induced by ATP in FCCP (+ oligomycin)-treated astrocytes. This disparity could be partly explained by the absence of oligomycin in most of their experiments, leading to ATP run-down in the course of the norepinephrine stimulation. Stimulation of astrocytes triggers an increase in [Ca 2+ ] cyt that can propagate as a wave through the cytoplasm of individual cells and through astrocytic networks . Thus, we were interested in determining the role of mitochondria upon the spatio-temporal characteristics of these Ca 2+ waves. Both mechanical and chemical stimuli were used to trigger Ca 2+ waves. In addition to triggering Ca 2+ influx, mechanical stimulation recently has been shown to activate phospholipase C in astrocytes . The propagation velocities of these Ca 2+ waves were nearly identical regardless of the initiating stimulus (pooled control rate = 22.9 ± 11.2 μm/s, n = 262), suggesting that once initiated, the waves were propagated by the same mechanism. This range of Ca 2+ wave velocities correlates well with those found in hippocampal astrocytes and retinal astrocytes . The amplitude of the Ca 2+ signal from the wave initiation site could be maintained or could even increase during spread across the cell, strongly suggesting that the wave propagates as an actively regenerative process and not simple long-range passive diffusion following release from a point source. The propagating [Ca 2+ ] cyt wave was associated with a propagating wave of mitochondrial depolarization that traveled at around 10 μm/s following mechanical stimulation. Thus, during the propagation of an intracellular [Ca 2+ ] cyt wave, mitochondria take up Ca 2+ as the wave travels across the cell. The discrepancy between the velocity of the [Ca 2+ ] cyt wave and of the wave of mitochondrial depolarization probably reflects fundamental differences in the behavior of the fluorescent dyes fluo-3 and TMRE. Whereas fluo-3 fluorescence rises immediately upon Ca 2+ binding, the increase in TMRE fluorescence after mitochondrial depolarization results from dye egress from mitochondria, dequench of fluorescence and then diffusion of dye through the cytosol, inevitably underestimating the rate of progression of the wave of mitochondrial depolarization. Furthermore, the change in Δψ m reflects the rate of Ca 2+ flux, which tends to be slower at the distal end of the cell compared with the origin of the wave front. Nevertheless, the appearance of a wave of mitochondrial depolarization clearly demonstrates that mitochondria take up Ca 2+ as a [Ca 2+ ] cyt wave travels across the cell. To determine whether mitochondria play a functional role in the propagation of the Ca 2+ wave in astrocytes, mitochondrial Ca 2+ uptake was prevented by dissipation of the mitochondrial potential using antimycin A1 or rotenone (each in association with oligomycin). The Ca 2+ waves presented the same regenerative pattern of propagation as in the control cells, but the wave velocity increased substantially. Thus, mitochondrial Ca 2+ uptake appears to slow the rate of propagation of the cytosolic signal. Taken together, these results suggest that functional mitochondria exert a negative feedback on the propagation of intracellular Ca 2+ waves in adult rat cortical astrocytes. What could be the underlying mechanism of the negative feedback control exerted by mitochondria upon the propagating Ca 2+ wave? Current models to account for the propagation of Ca 2+ signals suggest that IP3 induces a wave by diffusing through the cell, priming IP3 receptors, and releasing Ca 2+ as puffs from ER release sites . The rise in [Ca 2+ ] cyt generates additional IP3 through the Ca 2+ -dependent activation of phospholipase C providing a positive feedback step, whereas the diffusion of Ca 2+ to neighboring IP3 receptors activates increasing numbers of IP3-primed IP3 receptors , as type 1 IP3 receptors display a bell-shaped sensitivity to Ca 2+ that functions as a coagonist with IP3 to release stored Ca 2+ . The peak sensitivity to Ca 2+ lies at ∼300 nM, whereas at higher concentrations Ca 2+ becomes inhibitory. However, type 2 IP3 receptors, lack the Ca 2+ -dependent inactivation at high [Ca 2+ ] cyt . Thus, depending on the type of receptors expressed by a cell, mitochondrial control of local [Ca 2+ ] cyt through Ca 2+ uptake could result in either a positive or a negative control on the Ca 2+ wave propagation. The importance of mitochondria in the regulation of propagating [Ca 2+ ] cyt waves was first demonstrated in oocytes of the amphibian Xenopus laevis that express type 1 IP3 receptors . These authors found that IP3-mediated Ca 2+ waves were synchronized and increased in both amplitude and velocity by addition of substrates for mitochondrial respiration. The additional substrate hyperpolarizes the Δψ m , promoting [Ca 2+ ] m uptake and slowing IP3 receptor inactivation by reducing the Ca 2+ concentration in the immediate vicinity of active IP3 receptors . In the present study, we found that mitochondria exert a negative control on Ca 2+ wave propagation. Rat astrocytes in culture express predominantly type 2 IP3 receptors . In addition, mitochondria in astrocytes are located near IP3 receptors, which correspond to the amplification sites for Ca 2+ waves . One possible explanation would be that mitochondria in close proximity to IP3 receptors participate in a local Ca 2+ buffering that alters gating kinetics of type 2 IP3 receptor channels. The fall in local [Ca 2+ ] cyt would decrease the affinity of the IP3 receptors to IP3, decreasing further IP3-mediated Ca 2+ release. Less Ca 2+ will be available to feedback onto the Ca 2+ releasing IP3 receptors and to diffuse away to stimulate nearby IP3 receptors. This mechanism would completely account for the slowing of the intracellular Ca 2+ wave . In this model, type 2 IP3 receptors will not be inherently self-limiting, because Ca 2+ passing through an active type 2 channel cannot negatively feedback and turn the channel off . This hypothesis is further supported by the observation that the diffusion rate of Ca 2+ across the (mitochondrion-free) nucleus was of the same order of magnitude as the Ca 2+ wave velocities in cells in which mitochondrial uptake had been abolished (∼34 μm/s). Such differences in the wave rates between nucleus and cytosol have been observed in other cell types . This probably reflects the paucity of Ca 2+ buffers in the nucleus, and strengthens the concept that mitochondria are predominant cytosolic Ca 2+ buffers in astrocytes. Studying the impact of cytoplasmic Ca 2+ buffering on the spatial and temporal characteristics of intercellular Ca 2+ signals in astrocytes, Wang et al. found that pretreatment with an exogenous Ca 2+ chelator such as BAPTA dramatically decreased the Ca 2+ wave velocity within individual astrocytes. According to these authors, the positive feedback role of Ca 2+ in wave propagation is primarily local, acting at the Ca 2+ release sites, while Ca 2+ diffusion constitutes an important factor as a rate-limiting step during the initiation and propagation of Ca 2+ waves . One major consequence of the presence of an exogenous Ca 2+ buffer might be to reduce the peak-free Ca 2+ concentration at a puff site, thereby attenuating the regenerative potential of the wave. These authors suggested that the presence of endogenous cytosolic Ca 2+ buffers with a relatively low affinity for Ca 2+ would permit wave propagation but still restrict this mode of intracellular Ca 2+ signaling to a very localized range . Ca 2+ uptake into mitochondria is a low affinity, high capacity process . In addition, a rapid mode of Ca 2+ uptake recently has been discovered in liver mitochondria allowing [Ca 2+ ] m accumulation for low [Ca 2+ ] cyt . Taken together, the data indicate that Ca 2+ wave kinetic parameters are controlled tightly by active Ca 2+ buffering by mitochondria. Working on the same cell model, Simpson et al. drew completely opposite conclusions , suggesting that mitochondria located close to IP3 receptors promote Ca 2+ wave progression by preventing Ca 2+ -dependent inactivation of the IP3 receptor at high [Ca 2+ ] cyt . This hypothesis relies on the bell-shaped response of the IP3 receptor to Ca 2+ , characteristic of type 1 IP3 receptor . However, they showed that astrocytes essentially possess type 2 IP3 receptors . The fact that mitochondria are located near IP3 receptors does not necessarily mean that they play a positive role in Ca 2+ wave propagation. On the contrary, such a close location could represent a highly plastic mechanism whereby the cell could finely tune intracellular Ca 2+ wave characteristics, thus modulating this newly discovered form of nonsynaptic long-range signaling in the brain . Under physiological conditions, mitochondrial status could contribute to the modulation of the intercellular propagation of Ca 2+ waves within the astrocyte network. Indeed, modulation could simply rely upon supply of mitochondrial respiratory substrates to mitochondria or transient inhibition of mitochondrial respiration, regulating Δψ m , and modulating mitochondrial Ca 2+ uptake. In the astrocyte, functional active mitochondria would permit wave propagation but could restrict the extent of spread of this mode of intercellular Ca 2+ signaling. Modulation of such a mitochondrial contribution would allow a differential activation of specific neurons in the brain and may directly participate in information processing in the CNS. Brain mitochondria have been found to be impaired in some pathologies such as ischemia/reperfusion injury and Parkinson's disease . In such conditions, [Ca 2+ ] m uptake is impaired and mitochondria will no longer be able to regulate Ca 2+ wave spread. One possible consequence might be an increased spread of intercellular Ca 2+ waves in the astrocytic networks that might eventually lead to Ca 2+ deregulation in the surrounding nervous system. The astrocytic Ca 2+ wave may be important for the phenomenon known as Leao's spreading depression (SD) : a slow wave of depression of neuronal activity . It is accompanied generally by an increase in intracellular Ca 2+ concentration . More recently, a similar, if not identical, phenomenon has been shown to be induced by hypoxia . A glial role was suggested in SD since this depression propagates through diverse heterogeneous brain regions with similar velocity (33–100 μm/s). The Ca 2+ waves in the astrocytes studied therein, more particularly in those where mitochondrial Ca 2+ uptake was prevented, bear similarities to SD such as their high rates of propagation. The fact that hypoxia seems to mimic such a depression may support our hypothesis of negative mitochondrial feedback on Ca 2+ wave propagation. In such a condition, mitochondrial function would be disrupted, Δψ m dissipated and finally mitochondrial Ca 2+ uptake prevented. This would inexorably lead to further spreading of the glial Ca 2+ wave, stimulating in passing neuronal networks that normally are not activated by these astrocytes, and may contribute to brain activity disorganization. In conclusion, we have characterized an important role of mitochondria in glial Ca 2+ signaling, i.e., a strong negative feedback control on intracellular Ca 2+ wave propagation via their high Ca 2+ buffering capacities. Physiological alteration of mitochondrial function would allow the fine tuning of the Ca 2+ wave spreading through the astrocyte network, hence modulating their information processing functions . Besides, in pathological conditions, a mitochondrial dysfunction could contribute to the pathogenesis of numerous CNS disorders such as ischemia and epilepsy. | Other | biomedical | en | 0.999999 |
10330408 | Percoll SPBs were pelleted twice to remove Percoll , then diluted ∼50-fold into bis-tris (bt) buffer containing 1 mg/ml heparin, 0.1 mM DTT, and left overnight in ice. Extracted SPBs were separated on gradients of 1.75, 2.0, 2.25, and 2.5 M sucrose-bt in a Beckman SW40 rotor at 40,000 rpm for 4 h. SPB cores (no band was visible) were taken from the 2.0 M layer and the 2–2.25 M interface, then diluted with 1:1 bt-DMSO , pelleted (330,000 g , 15 min), and treated with either alkaline phosphatase or λ phosphatase ( New England Biolabs ; 90 U/μl, 3 h, 37°C) before SDS gel electrophoresis. For thin section EM the very thin pellet of SPB cores was located with a hand magnifier and encapsulated in 2% agarose after fixation to prevent breakup during processing. SDS gel bands were digested with trypsin and the tryptic peptide masses determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in a PerSeptive Biosystems Voyager-DE STR mass spectrometer using matrix peaks and trypsin peptides as internal standards. The NCBI nonredundant database of >300,000 proteins was searched using MS-fit ( http://prospector.ucsf.edu ) set at 50 parts per million, 0–300 kD and with all modifications and extra cleavages excluded. This decreased the number of peptides matched but increased the specificity of the match. All the proteins identified in the SPB core gel were the top match with MOWSE scores (P factor 0.4) of between 2 × 10 5 and 3 × 10 9 apart from Spc29p which had a score of 1 × 10 4 . A second search allowing methionine oxidation, protein NH 2 -terminal acetylation, and two missed tryptic cleavages was then carried out to match further peptides. For the identifications in this paper the first number in brackets is the number of tryptic peptides identified followed by the percentage sequence covered. These were: Spc110p dimer (18, 21%), Pom152p (13, 10%), Spc110p (42, 33%), Nud1p (12, 21%), Cnm67p (23, 33%), Spc42p (22, 36%), Spc29p (9, 42%), Sec53p (14, 38%), and Cmd1p (9, 61%). All yeast strains were prepared in Nasmyth's (IMP, Vienna) K699 background or the isogenic diploid K842, all vectors used were the pRS series . Most strains were made using the direct PCR method in K842 using as markers either the Schizosaccharomyces pombe HIS5 or the Kluyveromyces lactis URA3 genes followed by sporulation at 23°C. Standard yeast genetic methods were used . All strains were checked by colony PCR. All base pair numbers start from the A of the presumed initiator methionine. All tags were placed either at the NH 2 terminus (written as for example green fluorescent protein GFP-Spc110p) or at the COOH terminus (written as Spc42p-GFP). All tagged strains had the wild-type gene removed and grew at the same rate as untagged strains. The construct for Spc42p-GFP was prepared by PCR amplification of GFP (S65T) and insertion into the ClaI site of SPC42 at the 3′ end. This was designed to insert a short polypeptide linker (GA) 5 between the COOH terminus of Spc42p and the NH 2 terminus of GFP. The construct was integrated into the TRP1 locus of the covered knockout strain AY4 as either a single or about three copies (IAY18) and the wild-type gene removed by plasmid shuffle. Strains containing a deletion of CNM67 marked with HIS5 and using the K . lactis URA3 marker to tag either NUD1 or SPC72 with GFP at the 3′ end were prepared, together with the same strains rescued with CNM67 on a LEU2 CEN vector. A strain containing a deletion of CNM67 and SPC42-GFP was prepared by mating with IAY18. Temperature-sensitive (ts) mutants in NUD1 and SPC29 were prepared first by cloning the wild-type genes by gap repair to give bases −1501 to 4054 for NUD1 and bases −666 to 1382 for SPC29 and preparing the covered knockout strains from the heterozygous knockout diploid strains . The two genes were amplified under error prone conditions using primers at −5 to 16 and 2726 to 2705 for NUD1 and −666 to −645 and 1382 to 1371 for SPC29 (the 3.1-kb open reading frame at the 5′ end of SPC29 terminates at −269). These were transformed into the covered knockout strains together with the gapped plasmids, cut at PflMI (165) and BsiWI for NUD1 and BsmI (−495) and ClaI (684) for SPC29 . Plasmids were recovered from ts strains, and integrated at the TRPI locus as single copies to give nud1-44 and spc29-20 after removal of the wild-type gene. Both alleles were recessive, grew at normal rates on plates at 23°C, did not grow above 30°C, and were fully rescued by the wild-type genes on CEN plasmids at 37°C. Both alleles were sequenced between the PCR primers and the changes found for nud1-44 were Q96L, E127V, I418V, S419P, V504G, S534C, N600I, N613D, and I633F. For spc29-20 they were A-70G, T-15C (these two changes are in the 5′ noncoding region), E166D, and I220T. Two additional alleles were also examined nud1-52 , I353F and Y696N, and spc29-10 , A-389Δ, T-143Δ, A-121G, and L165Q. The changes in the promoter region in the two spc29 alleles were not responsible for the ts phenotype since the phenotype remained when this region was replaced by the wild-type promoter. The constructs for GFP-Spc110p and GFP-Spc29p were prepared by PCR amplification of GFP between BspLU11 I sites and ligation of this into NcoI sites at the initiator methionine of SPC110 in pPY133 and SPC29 (the NcoI site was made by PCR). The initiator methionines were replaced with a short polypeptide linker (GAGA) between it and the COOH terminus of GFP. These constructs replaced the wild-type gene after integration at the TRP1 locus as multiple copies for GFP-SPC110 and as either single (for immunoEM) or multiple copies for GFP-SPC29 . Versions of the earlier ts alleles of SPC42 and SPC110 integrated at the SPC42 or SPC110 loci without markers were prepared either by the pop-in/pop-out procedure to give spc42-10i , or by replacement of the disrupted SPC110 with the PCR-amplified ts allele to give spc110-1i and spc110-2i . A tetraploid version of K699 homozygous at the MAT a locus was prepared by transformation of K842 with GAL-HO to make two diploid strains homozygous at either the MAT a or MAT α locus. These strains were mated and GAL-HO used again to prepare the tetraploid strain homozygous for MAT a . The ploidy of this strain was confirmed by flow cytometry. Strains containing GFP-labeled Spc42p, Spc110p, and Spc29p in mps2-1 were prepared by backcrossing mps2-1 with K699 once, then crossing in IAY18, JKY1143, and a strain containing multiple integrated copies of GFP-SPC29 . These particular strains would not be completely in K699 background. A version of Spc42p with all 34 serines changed to alanine (S:A Spc42p) was prepared by a combination of site-directed mutagenesis, directed mutagenesis by PCR, and reconstruction of the gene by overlapping 80-mer oligos. This sequence is available from the authors. The S:A Spc42p gene was integrated as a single copy at the TRP1 locus of AY4 and the wild-type gene removed by plasmid shuffle. The integrated gene was amplified by PCR and sequenced to confirm all serines were replaced by alanine. The copy number in this transformant was further increased by integration of multiple copies of the S:A Spc42p gene at the URA3 locus. A version of this gene under the control of the GAL promoter and with a hemagglutinin tag at the NH 2 terminus was prepared and integrated as a single copy at the URA3 locus in K699. We removed a potential bipartite nuclear localization sequence (NLS) in Spc110p situated between residues 24–59 by replacing K24, 26, 54, and 55, and R27, 56, 58, and 59 with alanine using overlapping oligos in a construct designed to overexpress SPC110 under the control of the GAL promoter and with a myc tag at the NH 2 terminus. This construct was integrated at the URA3 locus of K699 and IAY18 as a single copy. As a control to test the function of ΔNLS-Spc110p we inserted the SV-40 T-antigen NLS (MARPKKKRKVA) between the myc tag and the NH 2 terminus and used the wild-type promoter. Strains containing only this compensated version of Spc110p grew normally. Yeast two-hybrid assays were performed essentially as described by Clontech . Two-hybrid constructs were made by PCR with a (GA) 5 polypeptide linker at the NH 2 -terminal end, cloned into plasmids pGAD424 and pGBT9 (full-length Spc42p in the higher expression vectors pACT2 and pAS-1 was toxic in yeast), transformed into Y190 and Y187, respectively, and lacZ assays performed after mating. The fragments used were: Cnm67p-Cterm (amino acids 442–581), Spc42p-coil-Cterm (49–363), Spc42p-Nterm-coil (1–141), and Spc110p-Cterm (781–944). Constructs containing only the coiled-coil domains of Spc42p (49–141) or Spc110p (161–800) were not detected by Western blotting, and constructs containing the NH 2 terminus of Cnm67p (1–180) in pGBT9 showed nonspecific activation of the reporter gene. The immunoEM protocol described in this paper was developed specifically to detect transient SPB duplication intermediates which might be rarely present even in synchronized cells. We used a preembedding staining method because of its greater sensitivity; however, a major problem with SPB antigens is that even short periods of formaldehyde fixation (1 min in the case of Spc42p) can abolish their reactivity with both polyclonal and monoclonal antibodies. In the case of Spc42p we tried a number of different epitope tags, including single and multiple myc and hemagglutinin tags at the NH 2 and COOH termini, but found GFP to be far superior in retaining reactivity after formaldehyde fixation. We found that during processing for immunoEM the cells fractured open; this was very advantageous because we could avoid detergent permeabilization and thus preserve the nuclear membrane which was necessary for the proper identification of the half-bridge, satellite, and duplication plaque. Cells were harvested by centrifugation, washed once with H 2 O, then fixed in 3.7% formaldehyde solution (BDH Chemicals), 0.1 M potassium phosphate, pH 6.5, for 20 min at 22°C. After three washes with 0.1 M potassium phosphate, pH 6.5, and one with 0.1 M phosphate citrate buffer, pH 5.8 (PC), the cells were incubated with 10% vol/vol glusulase ( DuPont NEN ) and 0.1 mg/ml zymolyase 20T (ICN Biomedicals) in PC at 30°C for 1 h. The samples were then washed once with PC, incubated with 50 mM glycine in PBS at 4°C for 5 min, washed twice with 0.5 ml PBS-BSA, and incubated with either affinity-purified rabbit anti-GFP antibody (a gift from K. Sawin, ICRF, London, United Kingdom) diluted 1:150 in PBS-BSA, 1% solution P (90 mg PMSF, 2 mg pepstatin in 5 ml absolute ethanol), 30–100 μg/ml of purified 9E10 or 12CA5, or with affinity-purified rabbit anti-Tub4p diluted 1:3,000 for 1 h at 22°C. After washing three times with PBS-BSA, the samples were incubated with a 1:50 dilution of Nanogold-labeled goat anti–rabbit Fab′ fragment or 1:20 dilution of goat anti–mouse Fab′ fragment (Nanoprobes Inc.) in PBS-BSA, 1% solution P at 22°C for 1 h. After one wash with PBS-BSA, and three with PBS, the samples were fixed with 2.5% glutaraldehyde (biological grade; Polysciences Inc.) in 40 mM potassium phosphate, pH 6.5, 0.5 mM MgCl 2 for 2 h at 22°C. The glutaraldehyde was removed by washing once with buffer, then incubating the samples three times with 50 mM MES, 200 mM sucrose, pH 6.0, at 4°C for 5 min each. The Nanogold particles were then silver enhanced in the dark for 3–5 min at 22°C with NPG silver enhancement solution . After enhancement the samples were washed three times with cold 200 mM MES, pH 6.15, in the dark over 10 min, removed from the dark room, and washed twice with 0.1 M sodium acetate, pH 6.1. After postfixing with 2% osmium tetroxide the samples were processed for serial thin section EM. EM, flow cytometry, elutriation, and GAL induction were as before , as was immunofluorescence with anti-GFP and anti-Tub4p . The anti-GFP antibodies used were affinity-purified rabbit polyclonals kindly given by K. Sawin (ICRF) and P. Silver (Dana-Farber Cancer Institute, Boston, MA), and mouse monoclonal 3E6 (Quantum Biotechnology). Cells (10 7 /ml) were treated with α-factor (10 μg/ml, 20 μg/ml for tetraploid cells) for 105 min (or 3 h for the prolonged treatment) at 30°C, or with DAPI (5 μg/ml) for 20 min. At present little is known about the process of SPB duplication because intermediates have not been observed by EM of vegetatively growing cells due presumably to the rapidity of the process. It has been known for some time that the process probably starts with the assembly of the satellite structure at the distal end of the half-bridge . Assembly of the satellite occurs between the end of anaphase and Start . There are some characteristic features of the half-bridge evident from these and other micrographs and those in Fig. 1 : the lipid bilayers are more densely stained and are continuous with the bilayers outside of the half-bridge . The bilayer on the inner nuclear side has a very thin layer of electron dense material closely associated with it whereas the cytoplasmic outer bilayer has a thicker layer which we call the half-bridge outer layer, it runs parallel to the bilayer in the cytoplasm with a gap of ∼15–20 nm and appears to intersect with the central plaque and the satellite . We looked for intermediate structures after the satellite stage by examining cells synchronized with α-factor and released for 30 min to increase the fraction of cells undergoing SPB duplication. In some cells at this time point the half-bridge structure appeared elongated and the satellite was replaced by a large plaque-like structure on the cytoplasmic side of the half-bridge , which we call the duplication plaque. This plaque, which was similar in diameter to the existing central plaque, appeared to have a more direct association with the cytoplasmic lipid bilayer of the half-bridge than the half-bridge outer layer. The duplication plaque is very similar in structure to the partly assembled SPB attached to the cytoplasmic side of the half-bridge observed at the arrest phenotype of mps2-1 , ndc1-1 , and mps1-737 . There was a characteristic bend in the half-bridge associated with the side of the duplication plaque proximal to the existing SPB. At this time point the distal end of the half-bridge was often fused with usually a pore structure immediately adjacent to the fused half-bridge . This pore, which was not morphologically distinguishable from a nuclear pore complex, may aid in fusion of the half-bridge. A proposed further stage in the duplication process was found in some cells where the duplication plaque was partly inserted into the nuclear membrane so that it appeared to be in direct contact with the nucleoplasm. This insertion was often at an angle to the existing SPB, and the plaque did not appear to have nuclear microtubules attached, though material, presumably outer plaque, was attached to the cytoplasmic side . Finally at 45 min after α-factor release at 30°C all cells examined had side-by-side or separated SPBs. A simple interpretation of these images is that the duplication plaque is a precursor of the central plaque and its assembly is initiated from the satellite. Towards the end of this assembly the distal end of the half-bridge fuses to allow insertion of the duplication plaque into the nuclear envelope, followed by the formation of nuclear microtubules. These results suggest that SPB duplication is initially a cytoplasmic event with the later stages taking place in the nucleus. We now wished to examine the role of the main structural components of the SPB in the cytological events of SPB duplication described above. Since the initial events in SPB duplication mainly involve the central part of the SPB, we analyzed an SPB core fraction by MALDI mass spectrometry analysis to identify the main components concerned. These heparin-extracted SPB cores appeared to contain a partly depleted outer plaque, intermediate layer 1 (IL1), and the central plaque, which in conventional EM thin sections includes IL2 ; the inner plaque appeared to be absent . A thin section through the whole pellet of this material shows only profiles of SPB cores sectioned at various angles, suggesting that they are highly enriched . Coomassie-stained SDS gels of SPB cores show only a few main bands when compared with intact spindle poles. Alkaline phosphatase was used to sharpen the bands of the phosphoproteins , and gels from two preparations are presented to show the bands consistent between different preparations . In addition we show λ phosphatase–digested material to show that there are no main bands concealed by the alkaline phosphatase . The main SPB core bands were identified by MALDI mass spectrometry as Pom152p, Spc110p, Nud1p, Cnm67p, Spc42p, Spc29p, Sec53p (phosphomannomutase), and Cmd1p (calmodulin). All of these proteins had been identified previously in our preparation of spindle poles . It seems likely that Pom152p and Sec53p are contaminants as they were in the earlier preparation . Spc110p, Nud1p, Cnm67p, Spc42p, and Cmd1p are all SPB components and have been shown to localize to the central and outer plaques , but Spc29p has not yet been localized within the SPB. We have now localized GFP-Spc29p by immunoEM to the nuclear side of the central plaque in 90% of the SPBs examined . The other 10% showed additional cytoplasmic staining of the central plaque. However in ∼25% of the SPBs, which serial sectioning showed were single SPBs, there was an additional focus of GFP-Spc29p staining to one side. When the half-bridge was correctly oriented in the section, this staining was clearly located at the distal cytoplasmic end of the half-bridge . Thus, GFP-Spc29p has both a nuclear and cytoplasmic localization like some other SPB components such as Spc98p . When synchronized cells were examined, staining of GFP-Spc29p at the end of the half-bridge was absent in cells with small buds and detected again when large-budded and unbudded cells were present. This suggests an association between the staining and the presence of the satellite, and indeed later we show localization of GFP-Spc29p to the satellite in synchronized cells . We also examined Nud1p-GFP and Spc42p-GFP log phase cells and found a similar proportion of SPBs showing cytoplasmic staining at the distal end of the half-bridge (data not shown). The proportion of single SPBs showing distal half-bridge staining (20–25%) is higher than the proportion of satellite-bearing SPBs in a log phase culture in rich medium (∼8% in the diploid K842, data not shown). This suggests that in addition to satellite staining, a precursor to the satellite might also be stained. Some of these SPBs did appear to lack satellites; however, these could have been obscured by silver deposition. In addition, during processing for immunoEM satellite-bearing SPBs could have been enriched and the satellite partly removed. Therefore, a more definite conclusion as to whether a satellite precursor is being stained should come from immunoEM of GFP-tagged SPB components in some of the cell cycle mutants. In summary we have characterized an SPB core fraction and shown that it is composed mainly of six SPB components all of which localize to the central and outer plaques. Since the initial stages of SPB duplication are cytoplasmic (see above) we were particularly interested in the main SPB components which had a cytoplasmic location in the SPB and how they were arranged, since components close to the central plaque are more likely to be involved in the initial events of SPB duplication. First we considered the outer plaque components Cnm67p, Nud1p , and Spc72p . Spc72p is depleted from the SPB core preparation suggesting it has a peripheral location, consistent with the finding that it interacts with the Tub4p complex . Cells containing a deletion of CNM67 have depleted outer plaques but intact central plaques ; thus, we have used this strain to establish which of these outer plaque components are still present at the SPB. We present most of the data as the GFP and DAPI staining given by unfixed cells as this is probably free from fixation artifacts such as the absence of staining caused by over-fixation . In unfixed anaphase, B cells' SPB position can be predicted without the need for tubulin staining as close to the two front edges of the separating chromatin . For fixed cells, we mostly used immunoEM instead of immunofluorescence because of its greater resolution. As expected, we found that Spc42p-GFP localizes to the SPB region in a strain containing a deletion of Cnm67p which has a depleted outer plaque. However, to our surprise we found that the outer plaque component Spc72p-GFP also localized to the SPB region in the same background. The reason for this became clear on immunoEM where, in contrast to Spc42p-GFP which localized as normal to the central plaque (data not shown), Spc72p-GFP localized to the half-bridge in all the SPBs examined , including those in short spindles. Cytoplasmic microtubules are attached to the half-bridge throughout the cell cycle in cells deleted for Cnm67p , and Spc72p is presumably associated with the Tub4p complex at this site. When this strain was rescued by transformation with CNM67 , Spc72p-GFP now localized normally to the outer plaque . When Nud1p-GFP was localized in the strain deleted for Cnm67p we found two types of staining pattern. Most cells with buds had a prominent dot which was probably not associated with the SPB since it was not coincident with DAPI staining . However, in unbudded cells and cells with small buds we found a weakly staining dot which was DAPI-associated (data not shown) and immunofluorescence showed this was SPB-associated . ImmunoEM of these cells showed that most of the SPBs were unstained, but ∼10% had Nud1p-GFP localized to the half-bridge , or with an unstructured particle associated with the half-bridge (data not shown). Most of the stained SPBs appeared to have a satellite or were side-by-side SPBs showing that they had come from unbudded or small-budded cells. When these cells are rescued with CNM67 we found that in all cells Nud1p-GFP now localized to the SPB region and immunoEM showed it had returned to the outer plaque in all the SPBs examined . In conclusion these results show that while the localization of Spc42p-GFP to the cytoplasmic side of the central plaque is independent of Cnm67p, the localization of Spc72p-GFP and Nud1p-GFP to the outer plaque requires Cnm67p. This, coupled with the depletion of Spc72p in the SPB cores, suggests that the approximate order of cytoplasmic SPB components from the nuclear membrane out is Spc42p, Cnm67p, Nud1p, and Spc72p. The finding that Spc42p is the closest cytoplasmic SPB component to the nucleus suggests that it should interact in some way with the nuclear SPB components Spc110p, Cmd1p, and Spc29p. We tested for interactions between Spc42p and Spc110p since both have been detected in an enriched complex , and we also sought to confirm possible interactions between Spc42p and Cnm67p using both a genetic and a two-hybrid approach. We found synthetic lethal effects between SPC110 and SPC42 . An enhanced ts phenotype (by 5°C) was found in the double spc110-2i spc42-10i mutant. Synthetic lethal effects were also detected between CNM67 and SPC42 but not SPC110 . Here cells containing a deletion of CNM67 maintained by CNM67 on a CEN URA3 plasmid were spotted at various dilutions onto 5-fluoroorotic acid media at 23°C which selects against the URA3 plasmid. When the spc42-10i mutation was present, the plasmid loss rate was reduced 1,000-fold as judged by the absence of colony growth at high dilutions, but was unaffected by the presence of the spc110-1i or spc110-2i mutations. We also looked at the interaction between Spc42p, Spc110p, and Cnm67p by two-hybrid analysis . Here we found an interaction between the NH 2 terminus of Spc42p and the COOH terminus of Spc110p and between the COOH terminus of Spc42p and the COOH terminus of Cnm67p. These interactions may not be direct and may also involve other SPB components, particularly the nuclear components Cmd1p (calmodulin) and Spc29p (we were able to detect a very weak two-hybrid interaction between full-length Spc29p and the NH 2 terminus of Spc42p, data not shown). Both Spc29p and Cmd1p localize close to the nuclear edge of the central plaque ; this is where the COOH terminus of Spc110p probably resides since Cmd1p has been shown to interact with this part of Spc110p . The localizations of these SPB components are shown schematically in Fig. 9 . The finding that Spc42p has different interactions with its NH 2 and COOH termini suggests that it exists as a single layer in the SPB rather than the double layer which is its probable arrangement in the overexpressed polymer . Of the six core SPB components described in the above section, five are essential and one, Cnm67p , is non-essential. The cytoplasmic peripheral component Spc72p is also non-essential , although this depends on the genetic background . Of the five essential genes, EM phenotypes have been described for CMD1 , SPC42 , and SPC110 . spc42 mutants have a defect in SPB duplication, whereas spc110 and cmd1-101 mutants appear to duplicate most of the SPB normally but have a defect in nuclear microtubule attachment; thus, the phenotype of these alleles is compatible with the location of the gene product in the SPB. We wished to examine the phenotypes of the two remaining essential SPB core components to see whether they were also compatible with their location in the SPB. Elutriated G 1 nud1-44 cells released at 37°C passed through mitosis at a slightly slower rate than wild-type cells . Of 20 cells examined by EM at 2 h, 3 showed satellite-bearing SPBs, 4 side-by-side SPBs, 12 short spindles, and 1 a postanaphase spindle. At 4 h cells (>80% had large buds) contained postanaphase spindles . Eight of these cells were examined by EM and SPBs were found at both ends of the spindle . This phenotype suggests that the cells are unable to exit mitosis. The SPBs appeared to have a depleted outer plaque and cytoplasmic microtubules were often found to terminate at the half-bridge . This part of the phenotype is similar to that of cells lacking Cnm67p which also has a depleted outer plaque . spc29-20 cells synchronized with α-factor and released at 37°C completed DNA replication at 1 h, and immunofluorescence at 1.5 h suggested that ∼25% of the cells contained spindles (data not shown). However, EM analysis of nine spindles (and three side-by-side SPBs) between 1 and 2 h showed an abnormality: one apparently normal-sized SPB and one small SPB . The small SPB, which is presumably the new one, remains inserted in the nuclear envelope at these time points and appeared to have a normal half-bridge . Thus, although SPB duplication can occur in spc29-10 cells, SPB assembly is apparently defective. At 4 h nearly 80% of the cells had large or multiple buds, and immunofluorescence showed that two-thirds had one focus of microtubule staining while the other third showed an additional much weaker focus of microtubule staining which was not apparently associated with nuclear DNA . Complete serial sectioning of whole nuclei from seven cells showed one apparently normal SPB , but the nuclear microtubules from these SPBs were not apparently connected to another structure. We presume that the weaker focus of microtubule staining seen by immunofluorescence represents the smaller SPB which has become separated from the main body of the nucleus. We did not find these SPBs by EM, possibly due to partial breakdown of the structure, as was found in some spc110 alleles . Flow cytometry of both nud1-44 and spc29-20 showed they progressed normally through the first round of DNA replication, but did not appear to arrest with a G 2 DNA content. However, we could not reach a definite conclusion on this because the peaks at later time points were often not distinct and could be caused by increased background due to the increased cell size (data not shown). Two other alleles, nud1-52 and spc29-10 , were examined by immunofluorescence after an asynchronous block and showed similar phenotypes to nud1-44 and spc29-20 , respectively (data not shown). The apparent depleted outer plaque and consequent attachment of cytoplasmic microtubules to the half-bridge in the nud1-44 spindles is in agreement with the localization of Nud1p to the outer plaque in wild-type cells. However, Nud1p must have an additional essential function as shown by the ts phenotype: it is required for cells to exit mitosis. This function is not dependent on its localization to the outer plaque since Nud1p remains an essential gene product in the absence of Cnm67p (data not shown) where it can localize to the half-bridge . Possibly the requirement for cells to exit mitosis might depend on a localization of Nud1p to the half-bridge which we have not yet been able to detect in wild-type cells. Incidentally, while most SPB components are coiled-coil proteins , Nud1p is a leucine-rich repeat protein , it has at least six repeats with a consensus sequence of XLX 2 LNLSXNXaX 2 aX 2 aX 2 a where the second asparagine is at positions 510, 553, 576, 597, 630, and 652 (X is any amino acid and a is normally aliphatic). It is not clear what this homology means. Leucine-rich repeats are found in functionally diverse proteins and are involved in interactions with other proteins . The phenotype of spc29-20 shows a defect in spindle structure, probably caused by one SPB being smaller than the other. This could be due to partial dissociation of the new SPB as was found in some spc110 alleles . Alternatively, if preassembled Spc29p were stable at 37°C in the mutant, then a partial SPB might assemble from the satellite but not grow to full size due to defective soluble Spc29p. The primary defect in this mutant may be caused by the smaller SPB having insufficient microtubules to carry all the chromosomes. In conclusion we show that the phenotypes of ts mutants in Nud1p and Spc29p are in part compatible with their localization to the SPB. In the scheme for SPB duplication presented earlier the simplest interpretation of the order of events is that the satellite is the precursor of the duplication plaque, although there are other possibilities such as the satellite dissociating from the half-bridge during duplication and production of a new SPB from fission of the parent . It is difficult to distinguish between these alternatives, particularly if the events are very fast and thus only rarely represented even in synchronized cells. The precursor–product relationship between the satellite and duplication plaque would be more clearly established, though not proven, if these structures shared common components and had a similar morphology. We looked for the presence of SPB components in both the satellite and the duplication plaque, starting with the cytoplasmic components since the initial steps in duplication occur in the cytoplasm. We consistently detected Spc42p-GFP and Nud1p-GFP in both the satellite and duplication plaque . For Spc42p-GFP it was essential to have multiple copies of SPC42-GFP integrated; this increased the size of the SPB and made it easier to distinguish the duplication plaque from the satellite. GFP-Cnm67p was also detected in both but less consistently in the satellite; whether this is due to a lower concentration or greater susceptibility to fixation is not clear. Spc72p-GFP localized to the half-bridge in early G 1 cells . Tub4p stained the inner plaque of the existing SPB and the cytoplasmic surface of the half-bridge , consistent with the growth of cytoplasmic microtubules from the half-bridge at this stage of the cell cycle . There was also some possible staining of the duplication plaque , indicating that part of the periphery of the outer plaque has been assembled. We detected GFP-Spc29p in the satellite , but have not yet found a clear duplication plaque in this strain. Duplication plaques were seen infrequently in all the strains investigated because of their transient nature, but we presume that since GFP-Spc29p is present in both the satellite and the existing SPB, then it will also be present in the duplication plaque. We were concerned that the satellite may have an altered composition in α-factor–treated cells, so we repeated some of the results on G 1 elutriated cells and were able to detect Spc42p-GFP and Nud1p-GFP in the satellite and Spc72p-GFP in the half-bridge . We also looked at the localization of a nuclear SPB component, GFP-Spc110p, and could detect it in the inner plaque but not in the satellite or duplication plaque . This version of Spc110p had been tagged with GFP on the NH 2 terminus which localizes to the inner plaque . However, in case we were unable to detect Spc110p elsewhere because fixation reduced antibody accessibility we did an indirect experiment on unfixed cells. The mutant mps2-1 arrests during SPB duplication just before insertion with a structure on the cytoplasmic surface of the half-bridge very similar in morphology to the duplication plaque ; the arrest phenotype looks very similar to Fig. 1 C. At later stages of the arrest the partly assembled SPB detaches from the existing SPB and migrates some distance away while remaining attached to the cytoplasmic face of the bridge or nuclear membrane, eventually coming to rest above an area of the nucleus containing little nuclear DNA . The two SPBs are thus cytologically distinguishable: the existing SPB is associated with nuclear DNA staining while the partly assembled SPB which resembles the duplication plaque is not. We tagged Spc110p, Spc29p, and Spc42p with GFP in this mutant to see whether these were detectable in the partly assembled SPB. Spc42p-GFP has been detected already in both SPBs in mps1-737 which has a similar phenotype and we found a similar result (data not shown). GFP-Spc29p gave the same result: two dots were detected and only one was associated with the DAPI staining . This is in agreement with the nuclear and cytoplasmic localization of Spc29p and indicative of its presence in the duplication plaque. However, with GFP-Spc110p we found only one dot, which was always associated with the nuclear DNA, suggesting that it corresponded to the existing SPB . The partly assembled SPB could be detected with anti-Tub4p in this strain . Thus, these results indicate that Spc110p, and presumably calmodulin since it is bound to Spc110p , is assembled into the SPB after insertion into the nuclear membrane. Since the satellite and duplication plaque contain common components, we might expect them to have a similar morphology. The morphology of the satellite cannot be readily observed by EM in haploid cells due to its small size. Since the diameter of the SPB increases with increasing ploidy , we thought the morphology of the satellite might be clearer in a tetraploid strain. When this strain was arrested in G 1 with α-factor so that all the SPBs would be satellite-bearing, the satellites now appeared plaque-like with a more similar morphology to the duplication plaque. In summary these results show that both the satellite and duplication plaque contain the same cytoplasmic SPB components and can have a similar morphology, all suggestive of a precursor–product relationship. The immunoEM results in the last section suggest that the satellite and duplication plaque contain common cytoplasmic SPB components. We wished to confirm this proposal by changing the structure of the cytoplasmic part of the existing SPB, since similar changes might also occur in the satellite and duplication plaque. We made use of polymorphic structures produced by Spc42p, these were found unexpectedly while attempting to investigate the function of the phosphorylation of Spc42p , which occurs predominantly on serines (data not shown). To investigate the function of this phosphorylation we prepared a version of Spc42p in which all 34 serines were changed to alanine (S:A Spc42p). To our surprise S:A Spc42p was fully functional, although bilateral mating was lethal (data not shown). Although SPBs appear to have a normal morphology, there is an unusual structural polymorphism associated with S:A Spc42p. On GAL overexpression, instead of the normal dome-shaped structure , a large electron-dense ball was associated with each central plaque . ImmunoEM showed that this structure contained overexpressed S:A Spc42p . In some cells, and in all cells treated with α-factor, the balls were associated with the central plaque and with the distal end of the cytoplasmic side of the half-bridge, where it presumably initiated from the satellite . Thus, perturbations introduced into the existing SPB are also evident in the satellite. A similar structural transition was found on prolonged α-factor treatment (3 h) of the S:A Spc42p strain. We noticed that in wild-type cells prolonged α-factor treatment appears to deplete the outer plaque as shown by SPBs which are about to fuse during mating . Furthermore, we found that Cnm67p-GFP is not detectable at the SPB under these conditions in either unfixed (data not shown) or fixed cells, although it returns to the SPB on release from α-factor . When we examined the morphology of SPBs in the S:A Spc42p strain after 3 h in α-factor, all the central plaques had been transformed into balls . This structural transition may be due in part to the removal of a stabilizing interaction with Cnm67p and may also be responsible for the bilateral mating defect found in this strain. Cells remained viable on release from α-factor and all SPBs had reverted to the normal plaque-like morphology by the time of spindle formation . It appeared that daughter SPBs formed with a normal plaque-like morphology . We attempted to see if the duplication plaque could be made to form a ball in a strain carrying S:A Spc42p and a deletion of Cnm67p. This was not successful and the central plaque remains as a plaque in this strain (data not shown). Thus, it seems that both the absence of Cnm67p and some environmental effect of the α-factor–treated cell are necessary for ball formation. In conclusion, we show that perturbations introduced into the central plaque are also evident in the satellite, suggesting they have similar structural features. In addition we show that changing all the serines in Spc42p to alanine can produce major reversible structural transitions in the central plaque. This shows the dominant role of Spc42p in central plaque structure and is consistent with the relatively small number of components found in this structure. A simple interpretation of the structure of the duplication plaque, given that it contains Spc42p which localizes to the cytoplasmic side of the central plaque , but apparently not Spc110p which localizes to the nuclear side of the central plaque , is that it consists of the cytoplasmic part of the central plaque attached to the half-bridge. Its orientation would be the same as the existing SPB since outer plaque components are able to assemble onto its cytoplasmic side. This model predicts that central plaque components such as Spc42p or Spc29p, instead of interacting with Spc110p as in the fully assembled SPB, would interact now either directly or indirectly with half-bridge proteins. This suggests that overexpressing Spc110p in the cytoplasm could compete with the interaction between the duplication plaque and the half-bridge, disrupt it, and inhibit SPB duplication. Since Spc110p is a nuclear protein when overexpressed , we altered a potential NLS so that the protein remained in the cytoplasm on overexpression. This version of Spc110p (ΔNLS-Spc110p) could not rescue a deletion of SPC110 unless the SV-40 T-antigen NLS was added to the NH 2 terminus. This suggests that the lack of function of ΔNLS-Spc110p is due just to the removal of the potential NLS. When ΔNLS-Spc110p was overexpressed, immunofluorescent labeling was detected at the SPB but not in the nucleus (data not shown). EM showed that cells arrested during SPB duplication with a duplication plaque-like structure partly detached from the half-bridge , although one end remained attached to the distal end of the half-bridge at the site formerly occupied by the satellite. This suggested that ΔNLS-Spc110p had inhibited attachment of the plaque to the half-bridge possibly by forming a large polyhedral structure . This would start to assemble between the half-bridge and the plaque and, if the plaque remained attached to the distal end of the half-bridge, cause it to swing up to beyond a vertical position. Thus, in Fig. 8 A2, the outer plaque side of the displaced plaque (arrowhead) is on the right. ImmunoEM confirmed this possible arrangement, with labeling of ΔNLS-Spc110p on the side of the displaced plaque proximal to the SPB , the silver particles presumably staining the margins of the large polyhedral polymer, with internal staining of the polymer probably blocked by extensive cross-linking due to fixation. Spc42p-GFP was detected as expected on the distal side of the plaque . This result is consistent with the model that the duplication plaque contains Spc42p and probably Spc29p, that these proteins are probably involved in the attachment of the plaque to the half-bridge, and that this attachment is inhibited by inappropriate expression of Spc110p. In this paper we have started to characterize the molecular events which occur during SPB duplication. EM of this process suggested that it starts with the assembly of the central part of the SPB, so to identify the more abundant proteins involved we prepared a highly enriched SPB core fraction. We found six main components by MALDI mass spectrometry, all of them previously identified , which is a remarkably small number of main components for such a large organelle. We can estimate the mass of the SPB cores assuming each has ∼1,000 copies of Spc110p and Spc42p and assuming the other components (except for Nud1p) are in equivalent copy number as indicated from the Coomassie-stained gels . This gives a mass of ∼0.3 GDa, which is at the lower end but compatible with the STEM mass analysis figure of 0.54 ± 0.23 GDa . We have presented an approximate model for the arrangement of some of these components in the SPB . This has Spc110p, Cmd1p, and Spc29p on the nuclear side of the central plaque and Spc42p in the IL2 on the cytoplasmic side of the central plaque. Note that in thin section EM of glutaraldehyde-fixed yeast cells the IL2 and central plaque layers appear merged, and we have called this merged structure the central plaque . On the cytoplasmic side of the central plaque Cnm67p and Nud1p localize to the vicinity of IL1 and the outer plaque, and finally Spc72p is localized to the outer plaque itself since it is absent in SPB cores which have depleted outer plaques. However, it is clear that rearrangements of these components can occur in some circumstances, suggesting that the SPB is a dynamic organelle. Thus, Spc72p and Tub4p relocate from the outer plaque to the half-bridge in G 1 , presumably reflecting in part the assembly of cytoplasmic microtubules from the half-bridge in G 1 . Nud1p, normally in the outer plaque , can relocate to the half-bridge in G 1 /S in the absence of Cnm67p, although we do not know the function of this localization or whether this can happen in wild-type cells. In addition, Cnm67p and also possibly the entire outer plaque dissociates during prolonged α-factor treatment, perhaps facilitating SPB fusion during mating . The combination of EM and immunoEM data presented in this paper suggests a number of stages in SPB duplication which are summarized in Fig. 9 . One of the most interesting findings is that SPB components are present in the satellite, suggesting that duplication is already partly achieved at this stage. This finding indicates that there may be analogies between the satellite and the prereplicative complex for DNA replication ; both are assembled at the end of the preceding cell cycle and contain components which are primed to act once cells have passed through Start. The presence of Spc42p in the satellite and the apparent disruption of duplication plaque formation in spc42-10 mutants are consistent with the proposal that an early stage in SPB duplication is the crystalline lateral growth of Spc42p . We presume that there are analogies between SPB duplication and centrosome duplication, since most processes in yeast, even the more specialized ones such as mating, use similar pathways to those found in higher eukaryotes. There are some common structural features between centrosome and SPB duplication (see Introduction), in particular the assembly of the new organelle at a set distance from the existing one. There may also be analogies between this process and the templated assembly of the new cytoskeleton in trypanosomes . Here new microtubules are nucleated by lateral microtubule-associated proteins in the preexisting microtubule cytoskeleton. This duplicates the exact pattern of the two dimensional microtubule array and can explain the earlier results showing cytoplasmic inheritance of the organization of the cell cortex in Paramecium . How might this templated assembly work in the case of the SPB? The bridge between side-by-side SPBs is a thin striated rectangular structure and is schematically shown in Fig. 9 with the two short edges adjacent to the central plaques of the two SPBs. One might envisage a protein complex localized specifically along the SPB-binding edges, or bridge components exposed at these two edges, which bind the central plaque components Spc29p and/or Spc42p. This binding always occurs at the cytoplasmic edge of the bridge and usually to the edge of the central plaque. Thus, the main function of the bridge is to provide two separate potential points of assembly along its SPB-binding edges. One of these is normally occupied by the existing SPB, the other is activated at some point in the preceding mitosis to bind cytoplasmic SPB components with the eventual formation of the satellite. There is a specialized attachment site for the distal end of the duplication plaque, and probably also for the satellite, at the cytoplasmic distal edge of the half-bridge. This site is different from the attachment of the nuclear face of the duplication plaque to the half-bridge, since overexpression of ΔNLS-Spc110p displaces the plaque from the half-bridge without detaching it from the distal cytoplasmic edge . What happens during insertion is not clear, but a possible clue comes from images such as Fig. 1 D and Fig. 8 B1 here, and Figure 3 c in Byers and Goetsch . These images suggest that the cytoplasmic edge of the half-bridge can bind to the nuclear face of the duplication or central plaque, in addition to the edge as described above. Thus, during insertion the half-bridge could retract with the cytoplasmic edge, presumably containing the satellite-binding site, withdrawing across the nuclear face of the duplication plaque . The length of the half-bridge outer layer, or at least the distance between the near edges of the two plaques, appears to remain constant throughout duplication , suggesting the retraction is confined to that part of the half-bridge immediately under the duplication plaque. The half-bridge outer layer may act as a rigid connecting strut between the existing SPB and the duplication plaque, perhaps stabilizing the latter during insertion. The retraction would expose the duplication plaque to nucleoplasm and thus allow assembly of nuclear components. This retraction may involve the half-bridge component Cdc31p , a member of the centrin family of proteins implicated in Ca 2+ -dependent contraction of basal body-associated fibers . Once SPB assembly is complete, the bridge would be severed in some way before spindle formation. This provides a new free SPB-binding edge associated with the half-bridge of each SPB for the assembly of the satellite during the next round of SPB duplication. A requirement for this model is that the self-nucleation rate for the assembly of SPB and half-bridge components is low, thereby directing assembly to preexisting templates or structures. This is certainly the case for structures assembled by Spc42p , Spc110p , and Spc72p . Thus, in this model the crucial element ensuring only two separate SPBs is the bridge and the specific binding of SPB components to either end of this structure. A problem for the future will be to characterize this binding further and also the role of the bridge in the other steps in SPB duplication, and the cell cycle controls which mediate them. | Study | biomedical | en | 0.999996 |
10330409 | A 20-kb genomic fragment containing full-length kif3A was selected from a murine genomic library of embryonic stem (ES) cell line J1 and probed using a mouse cDNA fragment encoding the P-loop exon of kif3A . The two independent phage clones obtained were cloned into pBS (Stratagene) vector. These clones were subsequently subcloned into pBS and mapped by a combination of several restriction enzymes and partial sequencing. We applied the conventional positive-negative selection together with the poly(A) trap strategy. As shown in Fig. 1 A, we applied a 1-kb-long HindIII-SalI fragment as a 5′-homologous arm and a 5-kb-long fragment as a 3′-homologous arm. Then poly(A)-less pGKneo positive selection cassette was flanked by these arms and cloned into a modified multiple cloning site in the DT-A cassette B vector ( GIBCO BRL ). Finally, this construct was prepared on a large scale with a QIAGEN miniprep kit. This plasmid was linearized by NotI and an appropriate concentration of vector (25 μg/ml) was prepared for the subsequent transfection into ES cells. We have performed the electroporation of targeting vector to J1 cell line basically according to Harada et al. with a slight modification in G418 concentration. After electroporation, the transfected cells were selected in the presence of G418 ( GIBCO BRL ) at a concentration of 175 μg/ml. About 10 d after electroporation, the growing positive selected colonies of ES cells were picked up in duplicated 96-well plates, one for cell stock and the other for genomic DNA preparation. Screening of recombinants was performed by genomic Southern blotting with 32 P-radiolabeled external probe (generated by PCR ∼0.4 kb) as indicated in Fig. 1 . Then the integrity of candidate clones was further confirmed with an internal and neo probes (data not shown). Three independent recombinants out of ∼450 ES colonies were obtained by this poly(A) trap targeting strategy. These recombinant clones were injected into the blastocysts recovered from superovulated C57BL/6N line and raised chimeric mice from two independent cell lines. We identified chimeric mice by contribution of agouti coat color. Male chimeras were bred with C57BL/6J to obtain heterozygous offspring, from the mating of which we could obtain kif3A null mutants. The generated mice were routinely probed by PCR for the identification of their genotype as described previously . In brief, a small portion of mouse tail was incubated in a tail lysis buffer (10 mM Tris-HCl, 25 mM EDTA, 1% SDS, and 75 mM NaCl, pH 8.0) supplemented with 100 μg/ml proteinase K (Merck). The Eppendorf tubes containing these samples were shaken in an air incubator maintained at 55°C overnight. Phenol-ethanol extraction and ethanol precipitation were performed before the PCR reaction. About a hundredth amount of each purified DNA was routinely used for PCR. The following two pairs of primer sets were used for genotyping offsprings: (a) neo primers, neoF 5′-TGG GCA CAA CAG ACA ATC GG-3′, neoR, 5′-ACT TCG CCC AAT AGC AGC CAG-3′; (2) internal primers, kif3A-F4, (346–369) 5′-TGT TCC ATA TAG CCC AGG ATA CCC-3′, kif3A-B1, (545–525) 5′-GAT GGT CCC TGA AAA TGG TGC-3′. To determine the embryonal genotype, we collected amniotic membranes. They were dissolved in aqua destillata supplemented with 100 μg/ ml proteinase K under shaking at 55°C for 1 h. A small amount (2 μl) of the lysate was used for PCR amplification. The whole embryos ranging from 9.5 to 10 dpc were killed and minced by using a pair of ophthalmological scissors in PBS supplemented with protease inhibitor cocktails (5 mM PMSF, 10 ng/ml leupeptin, pepstatin, benzamidine, and 100 mM DTT; Wako Pure Chemicals Co.) on ice. By using a Potter's homogenizer, small pieces of embryos were completely destroyed. The resultant homogenate was centrifuged at 15,000 rpm by using a Beckman TL-100 for 30 min and the supernatant was collected. The protein concentration of each sample was adjusted to 10 μg/ml and used for standard Western blotting procedure as described previously . Here we used the following antibodies to ascertain the expression level of KIF3A and KIF3B proteins in the mutant mice: both monoclonal and polyclonal anti-KIF3A antibodies , and polyclonal anti-KIF3B antibody . The embryos dissected from the pregnant mice were processed for the light microscopic observation by the following method. As soon as the embryos were extirpated from the uterus, a small portion of extraembryonic membranes was retained for the determination of genotype by PCR and the embryos proper were soaked into Bouin's fixative (75% pycric acid, 5% glacial acetic acid, and 25% formaldehyde; Wako Pure Chemicals Co.). After fixing embryos for 2 h at room temperature (RT), the samples were dehydrated by a graded series of ethanol solutions, followed by substitution of xylene. Finally, the whole mount embryos were placed in melted Paraplast (Oxford Labware) at 65°C for ∼90 min with two changes of this embedding material. The samples were embedded in fresh Paraplast after complete penetration of the samples by the embedding material. The blocks were cut by using a rotatory microtome (HM355; Carl Zeiss ) into 7-μm-thick serial sections, then the sections were mounted onto glass slides, which were in turn deparaffinized and stained with hematoxylin and eosin according to Kaufman with slight modifications. These sections were observed and photographed by a Nikon Optiphot-2 microscope. The dissected embryos were fixed by using half Karnovsky solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 2 h at RT. Then we soaked these embryos into 10% sucrose solution buffered by 0.1 M cacodylate for 20 min, followed by postfixation with 1% osmium tetraoxide in cacodylate buffer on ice for 15 min. After washing the embryos extensively with aqua destillata, dehydration with a graded series of ethanol solutions was performed. After completing the dehydration process, the media was substituted with isoamyl acetate and the embryos were left to stand in this solution at RT for over 30 min. They were then prepared for scanning electron microscopy by critical point freeze-dry procedure as described previously . In brief, they were soaked into liquid carbon dioxide under high pressure (JCPD-5; JEOL). Then the chamber was gradually heated to the critical point, where the embryos could be dried without being destroyed. The samples obtained were surface-coated using a gold spattering device under the optimal conditions for 10 min. The samples were viewed under a JEOL scanning electron microscope at 100 kV and photographed by a Polaroid. Embryos at 7.5 dpc were dissected out from the uterus and were fixed by the method of Mizuhira and Futaesaku to obtain good preservation of axonemal proteins. Then the samples were processed and observed routinely as described previously . Whole mount in situ hybridization of the mouse embryos other than lefty-2 was carried out principally according to Conlon and Rossant with several modifications. In brief, the embryos were dissected in sterilized RNase-free PBS and fixed with 4% paraformaldehyde not equilibrated with NaOH at 4°C. After incubation for 2 h, they were washed twice with PBT (PBS supplemented with 0.1% Tween 20), followed by bleaching with PBT and 30% hydrogen peroxide. They were then treated with proteinase K (10 μg/ml; Merck) for several minutes at RT. Embryos were further fixed with 0.2% glutaraldehyde containing 4% paraformaldehyde, prehybridized, and then hybridized with appropriate markers (overnight, 70°C). On the following day, the sample embryos were washed with buffers and treated with RNase to remove unbound or nonspecific binding probes. After that, alkaline phosphatase (AP)–conjugated anti-DIG antibody (Roche Diagnostics) was applied and incubated overnight at 4°C. On the next day, the embryos were washed with TBST (Tris-buffered saline containing 0.2% Tween 20) with medium changes every 1 h. The washing process continued into the following day when the final AP color reaction was performed. The reaction was carried out according to the standard method (NBT and BCIP; Roche Diagnostics) and the color reaction was stopped whenever the localization of markers became clear and distinct. We have performed a series of dehydration/rehydration processes to darken the color of the AP reaction precipitate products. The embryos were then soaked in a glycerol/TBST solution to make the body transparent for easier identification of staining at a three dimensional level. For staining the embryos by using the lefty-2 probe, we followed our previous method . Nodal cilia can be observed during the nodal formation and bulging at 7.5 dpc . The lower halves of the embryos dissected from the uterus were eliminated by using a pair of electrolytically sharpened tungsten needles. The half-egg like structure was embedded into a small hole of silicon-rubber membrane (thickness ∼300 μm) attached onto the surface of a glass slide. Another silicon membrane having a hole at its center (diameter ∼5 mm) was overlaid onto it. We filled the hole with motility solution (50% heat-inactivated rat serum, 49% DMEM and 1% 1 mM sodium pyruvate) and sealed it by simply placing a coverslip on it. Fluorescent beads (Fluosheres, carboxylate-modified microspheres; Molecular Probes) with a diameter of ∼220 nm were added to the assay solution to ∼5% for visualization of flow generated by the motile cilia. The preparations were viewed under VEC-DIC/FL microscopy ( Olympus Inc. ) and a series of motility assay was videotaped simultaneously. The embryos under investigation were genotyped after observation. Embryos were fixed with 4% paraformaldehyde and processed by a standard cryosectioning method. The blocks were sectioned with a Leica cryomicrotome, and extended on the surface of glass slides. The samples were permeabilized with 0.1% Triton X-100 and blocked with 5% skim milk followed by a standard immunocytochemical procedure as described elsewhere . For the staining by anti-axonemal dynein antibody, we generally followed the method of Umeda et al. with slight modifications. The stained preparations were examined under a confocal laser scanning microscope (Bio-Rad). Initially, we tried to obtain homologous recombinants by using a standard positive-negative selection vector and screened ∼800 ES cell colonies. However, we could not successfully obtain any recombinant clones, which led us to adopt the poly (A) less pGK-neo cassette with an A-T rich/pausing signal . Under this condition, three independent clones out of ∼450 colonies were isolated, all of which have been used for blastocyst injection. As shown in Fig. 1 B, wild-type embryos displayed a single 3.2-kb fragment when digested with EcoRI, while an additional 2.1-kb band was observed in heterozygotes by genomic Southern blotting. Another restriction enzyme (XbaI) being diagnostic for targeting events, also verified successful homologous recombination (data not shown). Genotypes were also identified by PCR where primer pairs explicitly detect the targeted events. As represented in Fig. 1 D, almost all littermates of heterozygous mothers intercrossed with male heterozygotes revealed proper separation of genotype according to Mendel's law. We made a necropsy of 20 pregnant heterozygotes expected to have homozygote offsprings during 7.5–11.5 dpc, which revealed that no homozygotes were observed after 10.5 dpc , suggesting the midgestational lethality of homozygous embryos. Western blotting analysis of all littermates revealed a complete absence of KIF3A protein from homozygous mice. Interestingly, KIF3B protein, being a counterpart in the KIF3 complex, was expressed at a normal level . kif3A homozygotes were generally smaller compared with heterozygous and wild-type littermates at 9.5 dpc. Furthermore, no living embryos were obtained when cesarean section of pregnant mice was carried out after 11 dpc (Table I ). At 9.5 dpc, the homozygotes displayed severe malformation characterized by swelling and degeneration of the neural tube mimicking the phenotype of hydrocephalus. Furthermore, extreme distension of the pericardium due to pericardial effusion was noted. A regular array of somites as seen in wild-type embryos could not be observed in homozygote embryos , simply showing degenerated bulk in the lower truncal part and abnormal somitegenesis, i.e., sirenomelia. In most cases, embryonic turning was not completed at 9.5 dpc, suggesting growth retardation/inhibition of the homozygotes. Staggering of the neural tube was prominent at this incomplete turning region. These phenotypes are suggestive of disturbed mesodermal development resulting in failure of neural tube induction. In addition, the severity of the phenotype observed at 9.5 dpc was almost correlated with the expression level of the KIF3A molecule, which revealed relatively strong signal intensity in the neural tube and the heart (idem, Cor). Moreover, we encountered abnormal heart looping. In wild-type cases , almost 100% of embryos displayed a D-loop pattern while a few atypical phenotype also existed. On the contrary, in homozygotes, ∼40% of the embryos revealed a L-loop type heart . In some cases (∼20%), incomplete cardiac looping, i.e., a tuba rectae, was observed. These results suggested that randomization of the heart looping occurred in kif3A homozygous mice. To address this point more precisely, we scrutinized these embryos with lefty-2 RNA probe, to see whether it involves abnormal lefty-2 expression, as lefty-2 product is considered to be one of the earliest left-right determinant in early murine development . As has been observed in our previous report for kif3B −/− , the expression of lefty-2 was bilateral or downregulated in kif3A −/− embryos . In some cases, the expression was extended to the contralateral paraxial mesoderm while normal pattern still existed . On the contrary, almost all wild-type embryos and heterozygotes displayed normal left-side restricted pattern . Regarding a cardiogenesis, hypoplasia of the myocardium was another striking feature of kif3A homozygous embryos. A sagittal section of the heart wall indicated fairly underdeveloped myocardium compared with normal embryos , which might be a cause of circulatory insufficiency in utero. The node is normally formed at 7.5 dpc and is essential for further induction of mesodermal anlage and in turn for neural tube formation. As Chlamydomonas orthologues of the KIF3 complex have been reported to engage in IFT , we focused on the ciliated mesodermal cells which normally accommodate monocilia . As shown in Fig. 7 , A and B, the nodal region forms a small dimple at the base of the dorsal part of embryo where the cell density is quite high. Since the cells constituting the node are smaller than surrounding endodermal cells at this developmental stage, this region could readily be discriminated from other parts of the embryonic body. At a glance, the wild-type/heterozygous cells normally exhibited cilia ranging from 2 to 4 μm in length and having pinpoint-like ends . However, in the case of kif3A −/− mice , the surface structure displayed quite striking differences. There were almost no mature cilia, although there existed some very short rudimentary processes. This phenotype was invariably observed among homozygous mouse littermates, suggesting the possible role of the KIF3A molecule in constructing and maintaining the monocilia structure. To decipher the degree of dysfunction of nodal pit cell cilia in kif3A −/− embryos, we have carried out a motility assay. An embryonic body harboring a node was placed under the VEC-DIC/FL microscope and fluorescent beads were added to the solution surrounding the embryos. By using this method, we can simultaneously observe both ciliary movement and nodal flow. In wild-type and heterozygous embryos, distinct flow generated by the rotating cilia was recorded and this flow was directed leftward with reference to the presumed body A-P axis . This regular flow was especially evident in the posterior part of the node. On the contrary, in KIF3A-deficient embryos, we could identify no such a regular flow, while rather Brownian movement of beads was observed . This result clearly indicates that the cilia of nodal pit cells is involved in gradient formation within the nodal groove, implying some important roles of the KIF3A molecule in the determination of early body planning. We have carried out immunostaining of nodal cilia to determine whether KIF3A molecules are really installed in the motile cilia. Cryosection of the node stained with anti–α-tubulin (DM1A) indicates the presence of cilia containing MT cytoskeleton . Double immunostaining of the same embryo with an anti-KIF3A antibody revealed the localization of KIF3A molecule within the cilia , suggesting that the KIF3A molecule might be engaged in the construction and maintenance of the ciliary structure. These results are almost the same as those obtained from the KIF3B-deficient embryos , strongly suggesting that KIF3A is really a counterpart of KIF3B in vivo. Collectively, KIF3A molecules may be essential for ciliary formation, determining the subsequent developmental cascades. Moreover, staining with an anti-outer axonemal dynein antibody and anti-axonemal dynein intermediate chain also revealed the presence of this molecule within the monocilia . Because the nodal area is composed of several optical sections by confocal laser scanning microscopy while that for cilia is made of a few sections, the background fluorescence in the nodal area seems to be higher. However, as there are no positive staining of cilia in the negative control panel , we could state that the localization of ciliary axonemal dynein is specific. We could also identify both inner and outer arm dynein localizing on the surface of doublet MTs with in the monocilia by a conventional electron microscopy. These results collectively imply that axonemal dynein is a candidate cargo of KIF3A that may in turn be engaged in the movement of monocilia. As both development of the mesodermal structure and the determination of L-R asymmetry were highly affected in the kif3A −/− mice, we also examined the expression pattern of some developmental markers for delineating the role of the KIF3A molecule in this process. Pax6 expression in 9.5 dpc embryos did not exhibit any apparent changes in the eyes except for slightly reduced expression in the forebrain . Brachyury is a mesodermal marker that is normally expressed in the midline structures with a strong accent at the tail bud region. At 8.5 dpc, its expression patterns in both embryos were very similar, but midline staining corresponding to the notochord was fainter in the case of homozygous embryos. Of noteworthiness, another midline mesodermal marker, sonic hedgehog , exhibited the most dramatic change in homozygote embryos even at early developmental stage . Normally, this marker is expressed in the node, notochord and subsequently in the floor plate. Furthermore, the ventral half of the mesencephalon also expresses it resulting from induction by the underlying floor plate at this region . In the case of homozygous embryos , the expression of shh at the thoracic notochord was significantly reduced and the staining per se became staggered to a certain extent. Moreover, the expression in the mesencephalon was completely absent in homozygous mice while abundant expression could be encountered in wild-type embryos . This absence of shh expression in brain structures might result in abnormal development of the neural tube, leading to the hydrocephalus-like phenotype and neural tube hypoplasia. Actually, exencephaly was relatively frequently observed in our kif3A null mutants under the genetic background with lesser contribution of C57BL/6J, suggesting the possibility that some modifier loci of this phenotype are present in the 129/Sv strain. In addition, reduced expression of shh in the rostral region together with reduced T expression in the truncal part collectively suggest the influence of the KIF3A molecule in the formation of A-P axis possibly through transporting inductive signals or their receptors. In summary, this is one of the first reports describing the general function of the KIF3 complex in vivo. The null mutant embryos displayed smaller body size and early developmental failure characterized by mesodermal hypoplasia and degeneration followed by the development of neural tube defects. Furthermore, determination of L-R asymmetry was randomized, exhibiting the phenotype represented by L-loop formation of the cardiac tube (situs inversus). This phenotype was presumably due to defective nodal cilia as identified in our previous kif3B mutant . These results reinforce our biochemical data that KIF3A is a counterpart of KIF3B . Another new idea on the function of KIF3A is that it is involved in mesodermal formation, which then regulates the development of neural tissues. Here we should like to discuss the possible role of the KIF3A molecule with special reference to (a) the early developmental events and (b) the determination of laterality. KIF3A is reported to be already expressed in ES cells and precedes that of KIF3B. Considering the biochemical data that KIF3A could remain soluble in the absence of its counterpart , the earlier death of kif3A −/− embryos than that of kif3B homozygous embryos is quite reasonable. Although the macroscopic phenotype was not so evident until 8.5 dpc in kif3A null mutants, microscopical changes in kif3A homozygotes were even evident early in the node formation stage (7.5 dpc). The node is generally believed to be responsible for the induction of further midline mesodermal structure. The absence of cilia on the surface of nodal pit cells in kif3A null mutants and the normal distribution of KIF3A molecules within these cilia in wild-type embryos strongly suggest that the nodal cilia are to some extent constructed and maintained with the aid of KIF3A molecules. Although a previous report suggested the presence of axonemal outer arm dynein in primary cilia of mammalian sperm and lung ciliated epithelium, and it might be responsible for actual motility, our data presented here and in a previous study indicated KIF3A and KIF3B are essential for transporting these ciliary components. Axonemal outer arm dynein per se is probably transported down within the cilia as a transporting complex for the following two reasons. On the first line, our immunocytochemical staining clearly demonstrated the colocalization of KIF3A and outer axonemal dynein within monocilia . Secondarily, mutations in a putative axonemal dynein heavy chain ( lrd ), being abundantly expressed in these ciliated nodal cells , de facto resulted in situs inversus. These results are in good agreement with the case of Chlamydomonas FLA10, where large IFT complexes are transported by this KIF3 orthologue and depletion of this motor protein resulted in the absence of normal motile flagella . Then by what mechanism are the early developmental events disorganized, giving rise to multisystem failure? Although there is no direct evidence interfacing the function of KIF3A and early inductive events, it is supposable that the defective nodal cilia could not properly distribute some presumably soluble substances to their proper destinations. Alternatively, intracellular transport of some kinds of receptors to proper direction might be affected. Especially, the abnormal expression pattern of Brachyury ( T ) and sonic hedgehog ( shh ) could partly explain the hypoplasia of midline structure and degeneration of posterior body structure. The generally accepted idea holds that shh is secreted as a complex form bound to cholesterol . Considering the size of this complex and cilia, it is reasonable that the lack of cilia resulted in disturbance of orthotopic shh expression in kif3A −/− homozygote mice. In addition, spontaneous mutation of shh , cyclopia , and targeted disruption of shh partly share the phenotypes observed in this kif3A null mutant. These ideas collectively imply the relationship between the KIF3 molecule and transport of the Shh molecule. From another standpoint, as neural induction depends on the mesoderm, abnormal neural tube formation could be attributed to this general failure of anterior development. Indeed, the lack of shh staining at the ventral half of the mesencephalon implies that the neural phenotype is partly secondary to mesodermal abnormality. In fact, expression of shh at 7.5 dpc in homozygotes is already more altered than that of wild-type embryo, displaying very faint trace amount with no nodal accentuation. However, considering that neuronal morphogenesis also involves cellular movement and assortment by themselves, the neural phenotype might be explained independently by genuine neuronal failure brought about by kif3A disruption. In addition, as some reports suggested the presence of cilia on the neuron , the neural phenotype may partially be attributable to the lack of neuronal cilia. As has been discussed in the previous section, KIF3A might transport materials required for the cilia assembly and maintenance, so that the absence of KIF3A molecules results in ciliary disorganization, leading to several developmental defects. One of the most remarkable phenotypes in these mutant mice was randomization of L-R asymmetry. As we have previously reported in kif3B null mutant mice , these null mutant mice exhibited randomized heart loop formation. Importantly, failure of cardiac looping to occur in neither direction was also encountered in kif3A null mutant embryos (∼20%). Then what mechanism is responsible for left-right determination? Our experiment by using fluorescent beads clearly indicated that the nodal cilia propelled certain kinds of substances in the extraembryonic fluid in the vicinity of the node from right to left. A concentration gradient that reaches the threshold for switching on lefty expression is probably formed and it might further destine the L-R asymmetry. This conclusion is further supported by our recent unpublished work showing randomness of left-right asymmetry occurs in mutant whose nodal pit cells harbor primary cilia, but they are immotile (Okada, Y., S. Nonaka, and N. Hirokawa, unpublished data). As a candidate of determinants, we expect that N-Shh protein might be involved since the cholesterol-conjugated form of N-Shh could not diffuse out a significant distance from a source of secretion and be concentrated to where it should be localized. There is no direct evidence for the relationship between lefty and shh , but it is now generally accepted that shh expression in chick is restricted to the left side before that of lefty-2 . Furthermore, although the expression of shh is bilateral in mammals, it is most probable that the leftward flow of the extraembryonic fluid generated by nodal cilia produce the concentration gradient of secreted morphogens including Shh which are upstream of the L-R determinants such as lefty-1 , -2 . The absence of this flow in kif3A null mutant mice might result in disturbance of the correct topology of determinants. As a next step in delineating the total function of KIF3 complex in vivo, conditional gene targeting is now under way. Furthermore, we could also examine the genetic interaction between these molecules by making double knockout mice for KIF3A/B. From another standpoint, by using the cell biological disciplinary, now we are also conducting experiments to unveil the function of KIF3 complex in mesodermal induction and neural development. | Study | biomedical | en | 0.999998 |
10330410 | Monoclonal anti-hemagglutinin (HA) antibody (12CA5) was obtained from BabCo, polyclonal anti-Myc was obtained from Santa Cruz Biotechnology, Inc. , and monoclonal anti-Myc antibody (9E10) was a gift from Phil Tsichlis (Fox Chase Cancer Center, Philadelphia, PA). Antibodies against activated Erk and Jnk were obtained from Promega Corp. , anti-vinculin was from Sigma Chemical Co. , and anti–phospho-MLC was a kind gift from Fumio Matsumura (Rutgers University, New Brunswick, NJ). Fluorochrome-conjugated goat anti–mouse and anti–rabbit antibodies were from Jackson ImmunoResearch Laboratories, Inc. Wild-type and mutant forms of Pak1 were subcloned from pJ3H as SalI/(EcoRI → blunt) fragments into SalI/EcoRV-cut pTet-Splice . These plasmids express NH 2 -terminal HA-tagged Pak1 when cells are grown in the absence of tetracycline. The tetracycline-regulated expression vectors pUM-Rac1 V12 and pUM-Rac1 N17 were kindly donated by Mark Symons (Onyx Corp., Richmond, CA). NIH-3T3 cells and derivatives were maintained in DME plus 10% calf serum (CS). The NIH-3T3 variant S2-6 , bearing a tetracycline-regulated transactivator, was used to construct cell lines that inducibly express various forms of Pak1 or Rac1. To prepare stable, regulated clonal cell lines, S2-6 cells were cotransfected with either pTet-Splice-Pak1 or pUM-Rac1 plasmids plus a plasmid encoding a puromycin resistance gene using a calcium phosphate precipitation method . 48 h after transfection, the cells were selected in media containing 2.5 mM histidinol (to retain the tetracycline-VP16 transactivator), 2 μg/ml puromycin (to select for the tetracycline-regulated expression vector), and 1 μg/ml tetracycline (to repress transgene expression during selection). Clonal cell lines were isolated and expanded and at least 24 lines for each form of Pak1 or Rac1 were examined for inducible transgene expression by anti-HA or anti-Myc immunoblot. Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 50 mM NaF, 10 mM β-glycerol-phosphate) containing 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 μg per ml aprotinin. 10 μg extract was fractionated by 10% SDS/PAGE and transferred to polyvinyl difluoride (Immobilon) membranes. Membranes were blocked using fat-free milk, probed with antibodies, and developed using an alkaline-phosphatase–based chemiluminescent system ( Dupont / New England Nuclear ). HA-tagged Pak1 was immunoprecipitated using mAb 12CA5 (BabCo) from 35-mm wells of S2-6 cells grown in tetracycline-free DME. The immunoprecipitates were washed three times in NP-40 lysis buffer, once in 0.5 M LiCl, and then once in protein kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 1 mM DTT). The immunoprecipitates were then incubated at 30° C for 20 min in 25 μl protein kinase buffer containing 25 μM ATP plus 5 μCi γ-[ 32 P]ATP. The reactions were stopped by the addition of 5 μl 6× SDS/PAGE sample buffer, boiled for 5 min, then separated by 10% SDS/PAGE. The dried gel was then autoradiographed. Cells were plated at 3.2 × 10 5 cells/35-mm culture dish in DME plus 10% CS and transfected with expression plasmids using a calcium phosphate precipitation method . Cells cultured on coverslips were fixed in 3.5% formaldehyde, permeabilized in 0.1% Tween-20 and blocked with 3% BSA in PBS. After incubation with primary antibodies, the cells were stained with rhodamine X-conjugated goat anti–rabbit antibodies along with AMCA-conjugated and Cy5-conjugated anti–mouse antibodies. For staining of filamentous actin, 0.1 μM Oregon Green or FITC-conjugated phalloidin was included during incubation with the secondary antibodies. Confocal microscopy was performed using a Bio-Rad MRC 600 laser scanning confocal microscope. Cells from one inducibly expressing clonal line for each of two constitutive active mutant forms of Pak were plated at 30% confluence into 35-mm wells with and without coverslips. 24 h later the cells were washed one time in PBS and refed with DME plus 10% CS alone or with one of the following: 1, 0.2, 0.1, 0.04, or 0.02 μg/ml tetracycline and incubated for another 8 h. At that time, the cells were again washed and refed with DME plus 0.2% CS, retaining the same tetracycline concentrations as 8 h earlier. 16 h later, HA-tagged Pak was detected in the cells on coverslips by indirect immunofluorescent staining, while the remaining cells were lysed in NP-40 lysis buffer and immunoblotted as described above. Pak1 expression was induced by growth in tetracycline-free DME plus 10% CS for 16 h. After protein induction, the cells were treated with trypsin, washed in the presence of soybean trypsin inhibitor and replated in tetracycline-free DME plus 0.1% BSA onto plates coated with 1% BSA for 30 min. Uninduced cells were treated in the same manner. The cells were then transferred to a plate coated with 50 μg/ml fibronectin and allowed to attach for 40 min. Phase contrast images were captured thereafter at 30-s intervals for calculation of cell speed and at 2-min intervals for long term analysis (4 h sampling). The cells were tracked with the aid of Isee™ imaging software and the results were tabulated using Excel™. For measurements of haptotaxis, Pak1 expression was induced by growth in tetracycline-free DME plus 10% CS for 16 h. Uninduced cells were treated in the same manner in the presence of tetracycline. After this pretreatment, the cells were detached from the plate with trypsin, washed in the presence of soybean trypsin inhibitor, resuspended in DME plus 0.1% BSA in the presence or absence of tetracycline, and then loaded into the top wells of a Boyden chamber at a concentration of 15,000 cells/well. The bottom wells contained DME plus 0.1% BSA, with or without tetracycline. The separating filter was coated on the underside only with 200 μg/ml collagen, type I. The number of cells that successfully migrated through the filter in three to six wells were counted after staining with hematoxylin. To assess the effects of Pak1 on motility, we established stable cell lines in which Pak1 expression is regulated by tetracycline. These cell lines included HA-tagged wild-type, constitutive-active, and kinase-dead mutants of Pak1, as well as Myc-tagged activated and dominant-negative forms of Rac1 . Two constitutive active forms of Pak1, one that binds Cdc42/Rac normally (Pak1 E423 ), and one that does not (Pak1 L83,L86 ), were included in order to assess the role of p21 binding in any phenotypes induced by Pak1 expression . Transgene expression was tightly controlled by tetracycline, displaying 5–10-fold induction, as assessed by densitometry of anti-HA immunoblots . Importantly, transgene expression was barely detectable in cells grown in tetracycline-containing media, even on prolonged exposure of immunoblots. As expected, immunoprecipitated Pak1 E423 displayed the most protein kinase activity, followed by Pak1 L83,L86 . Neither wild-type nor kinase-dead forms of Pak1 displayed detectable kinase activity, consistent with previous results . We and others have previously shown that transient expression of Pak1 affects the structure of the actin cytoskeleton in Swiss 3T3 , HeLa , and PC12 cells . However, the effects of stable Pak1 expression on populations of cells have not been reported. Our tetracycline-regulatable cell lines permit large numbers of cells to be examined, each expressing similar levels of transgenic Pak1. In addition, we can titrate the level of Pak1 expression by varying the concentration of tetracycline. In induced NIH-3T3 cultures, wild-type Pak1 has only modest effects on F-actin distribution as we previously reported in transiently transfected Swiss 3T3 cells , although dorsal ruffles develop in a few (<20%) cells . Expression of an activated Pak1 mutant, Pak1 L83,L86 , resulted in formation of not only dorsal ruffles but also of large, asymmetric lamellipodial structures characteristic of the leading edge of the cells . These latter structures resemble those found in motile fibroblasts. Interesting, asymmetric lamellipodia were seen in >50% of cells expressing another activated mutant, Pak1 E423 ; the remaining cells have a morphology characterized by a marked loss of stress fibers and few lamellipodia similar to those observed upon transient transfection . That these differences are due to the different cell types used for these studies is unlikely, given that transient transfection of S2-6 cells with pCMV6M-Pak1 E423 , results in a flattened, stress fiber-free cell phenotype (data not shown). A more likely explanation for differences in cell morphology may relate to the very high expression levels achieved in transient transfection versus the more moderate, but stable levels attained in the tetracycline-regulated cells. In support of this latter hypothesis, when a small amount of tetracycline (0.04 μg/ml) was added to the medium, the population of cells as a whole produced lower levels of Pak1 E423, with concomitantly higher percentage of the cells having asymmetric lamellipodia as Pak1 production decreased (50.4% lamellipodia in tetracycline-free medium; 76.1% lamellipodia in medium containing 0.04 μg/ml tetracycline; 800 cells counted in total). Expression of a kinase-dead form of Pak1 also induced formation of lamellipodia, but, compared with cells expressing activated Pak1, these cells tended to have increased numbers of such structures dispersed throughout the cell body, giving the cells a multi-lobed appearance . Often, lamellipodia accumulated on multiple sides of the cell, a phenotype that is virtually never observed in cells expressing activated forms of Pak1. To ensure that the NIH-3T3 derivatives used in these studies respond to Rho-family GTPases like other commonly studied fibroblasts, we examined the cytoskeletal effects of Rac1 in the parental S2-6 NIH-3T3 cells. As in Swiss 3T3 and REF52 cells , activated Rac1 induced cell flattening and circumferential lamellipodia, formation of cortical focal complexes, and a loss of central stress fibers , while the dominant-negative version induced compact but elongated cells (data not shown). Thus, Rac1 expression gave rise to phenotypes similar to those described previously in Swiss 3T3 and REF52 cells, indicating that the S2-6 cells behave in a similar manner as these well-described cell systems. We have previously argued that, in Swiss 3T3 cells, the effects of Pak1 L83,L86 on F actin organization are independent of Rac1 . However, Obermeier et al. showed that Pak1 L83,L86 induces neurite outgrowth in PC12 cells, and that ∼40% of these effects are inhibited by coexpression of a dominant-negative form of Rac1, implying that Pak1 may operate upstream of Rac1. To reexamine this issue in fibroblasts, we transiently transfected Rac1 N17 into the tetracycline regulated cell line expressing Pak1 L83,L86 . Cell expressing Pak1 L83,L86 alone have, as expected, large asymmetric lamellipodia . Coexpression of Rac1 N17 has no apparent effect on this phenotype . In cells that lack Pak1 L83,L86 expression (i.e., those grown in the presence of tetracycline), Rac1 N17 induces cell shrinkage, indicating that this protein is biologically active (data not shown). To quantitate these effects, we scored the phenotypes of 400 cells per group grown in the presence or absence of tetracycline with or without coexpressed Rac1 N17 . As expected, when grown in the presence of tetracycline, few cells display lamellipodia. Upon induction, ∼91% of cells expressing Pak1 L83,L86 alone, and 90% of cells coexpressing Rac1 N17 display characteristic lamellipodial structures . A second activated Pak1 mutant, Pak1 E423 , was less potent in inducing lamellipodia (56% of cells), but, as with Pak1 L83,L86 , coexpression of Rac1 N17 did not inhibit formation of these structures. The results of these studies show that Rac1 N17 has very little effect on the phenotype elicited either by Pak1 L83,L86 or Pak1 E423 , and therefore, in this cell type, the effects of these activated Pak1 mutants on F actin organization are independent of Rac1. Because the morphology induced by activated Pak1 mimics that seen in motile fibroblasts, we investigated the role of this enzyme in regulating cell movement. Although the control of cell motility at the molecular level is poorly understood, it has been shown in a number of systems that Ras, and related GTPases of the Rho family clearly play important roles in this process . We assessed the effect of Pak1 expression on cell motility by tracking the movement of individual cells at 30-s intervals over a 1-h time course after their attachment to fibronectin-coated dishes . In the presence of tetracycline, the cell lines showed minor differences in basal movement rate . These differences probably reflect intrinsic clonal variation. However, in the absence of tetracycline, the average control cell speed increased by only 0.24 nm/s, never attaining the speed of Pak1-expressing cells, even though these latter cells have a lower basal speed. In fact, the average speed of cells expressing wild-type Pak1, Pak1 L83,L86 , Pak1 E423 , and Pak1 R299 increased to 17.03 ± 0.91, 17.58 ± 0.83, 21.06 ± 0.88, and 23.25 ± 1.07, respectively, when assayed in the absence of tetracycline. These data show that increased Pak1 levels cause an increase in cell movement, and that this increase is independent of protein kinase activity. Interestingly, although random cell motility is independent of Pak1's catalytic function, directional movement strongly correlates with kinase activity. 4-h cell tracking experiments indicate that cells expressing kinase-dead Pak1, though highly motile, display poor persistence of movement compared with wild-type or activated forms of Pak1 . In contrast to cells expressing activated Pak1, cells expressing kinase-dead Pak1 frequently and abruptly change course. These effects are readily apparent in cell path tracings . Here, plots derived from the paths of 10 randomly selected cells show that the overall motility of cells expressing either activated or kinase-dead Pak1 is increased, but that the complexity of the paths is strongly related to protein kinase activity. Cells expressing activated Pak1 move in relatively straight paths , whereas those expressing kinase-dead Pak1 move randomly . We speculated that these different types of movement, which are related to Pak1's protein kinase activity, might be reflected in other measurements of directional motility. In agreement with this prediction, cells expressing activated forms of Pak1 display increased haptotactic movement in response to an immobilized collagen gradient, whereas cells expressing kinase-inactive Pak1 show slightly decreased haptotaxis . As a control, we also assessed haptotaxis in Rac1-expressing cells. Activated Rac1 enhances haptotactic movement, whereas a dominant-negative form inhibits this directional movement (data not shown). Interestingly, neither Pak1 nor Rac1 affect chemotaxis towards soluble fibronectin , suggesting that this signaling pathway is regulated by a different set of proteins. The activation of MLC by phosphorylation has been correlated with cell motility , perhaps due to its role in retracting nondominant pseudopodia . Pak1 has been reported to affect myosin function in a variety of organisms , and mammalian Paks have been shown to phosphorylate MLC at serine 19 in vitro . We therefore asked whether Pak1 expression in fibroblasts results in increased MLC phosphorylation. For these studies we used an antibody that specifically recognizes the serine-19 phosphorylated form of MLC . Lysates from control cells, or cells expressing WT, activated, or kinase-dead forms of Pak1 were examined by immunoblot for protein expression, MAPK and SAPK activity, and phospho-MLC content. These studies show that activated forms of Pak1, like Rac1, induce activation of a SAPK (Jnk) but not a MAPK (Erk1) pathway . These results are in agreement with most published studies, which show modest Jnk and p38 activation by activated forms of Pak . As expected, control cells treated with growth-stimuli such as PDGF or LPA show Erk1 activation, while cells exposed to a hyperosmotic shock show Jnk activation, demonstrating that signaling pathways in the S2-6 cell lines are generally similar to those observed in most other fibroblasts. Phospho-MLC is apparent in growth-factor treated cells, as well as in cells expressing activated forms of Rac1 or Pak1, but not in control cells or cells expressing a kinase-dead form of Pak1. These results indicate that activated Pak1 induces MLC phosphorylation and that this phosphorylation is not mediated via downstream activation of Erk1. We also used the phospho-MLC antibody to localize activated MLC by immunofluorescence. Cells inducibly expressing an activated form of Pak1 were grown in the presence or absence of tetracycline for 16 h before fixation. These experiments show that cells expressing activated forms of Pak1 induce an accumulation and localization of phospho-MLC to the lamellipodia . In nonexpressing cells, phospho-MLC levels are low and are found mainly in a perinuclear location . In Pak1-expressing cells, the amount of total phospho-MLC is apparently significantly increased and a substantial fraction of this protein now localizes just beneath the leading edge of lamellipodia . These results are consistent with a previous study which showed that phospho-MLC accumulates in lamellipodia in locomoting cells . The p21-activated kinases comprise a family of 62–68-kD serine/threonine kinases. These enzymes are catalytically activated by binding specifically to activated forms of Cdc42 and Rac1, two GTPases of the Rho subfamily, thus making Paks good candidate effectors for these signaling molecules . In fact, a number of the Paks have been implicated as the downstream effectors for several Cdc42 and Rac1 regulated signaling pathways including modulation of the actin cytoskeleton and establishment of cell polarity , processes that are required for cell motility. Although it has been shown recently that both Cdc42 and Rac1 regulate motility and invasiveness of epithelial cells , and that Rac1 modulates growth factor–driven chemotaxis in fibroblasts and macrophages whether any of the Paks play a role in regulating these processes has not been established. Pak1 has two types of effects on cell morphology, one related to its protein kinase activity and one that is kinase independent . We and others have previously shown that at least some of Pak's kinase-independent effects on the actin cytoskeleton are mediated by one or more SH3-containing proteins that associate with proline-rich regions located in the NH 2 -terminal half of Pak1 . These SH3-containing proteins include, but are not necessarily limited to, the adaptor Nck and the guanine-nucleotide exchange factor PIX and its relatives . Because PIX is an activator of Rac1, Obermeier et al., have proposed that the kinase-independent activity of Paks on actin polymerization is mediated by Rac1 . In support of this model, they have shown that the kinase-independent effects of Pak3 on neurite extension in PC12 cells are partially inhibited by a dominant-negative from of Rac1. In contrast, we have argued that, in microinjected Swiss 3T3 cells, the kinase-independent effects of Pak1 are not inhibited by dominant-negative Rac1 . Possibly, cell-type differences, or the isoforms of Paks studied, account for these apparent discrepancies. However, it should be noted that less than half of Pak3's effects in PC12 cells are blocked by dominant-negative Rac1, that a nearly identical degree of inhibition is seen in control cells, and that dominant-negative Cdc42 has an even more inhibitory effect . Therefore, it is not clear if or how Pak3 functionally links Cdc42 to Rac1. Whatever the situation in PC12 cells, our present data show that dominant-negative Rac1 has no measurable effect on Pak1-driven actin reorganization in NIH-3T3 cells. Therefore, at least in cells of mesenchymal origin, the kinase-independent effects of Pak1 do not require Rac1. As with cell morphology, the effects of Pak1 on cell movement also have a kinase-dependent and independent component. Overexpression of wild-type or mutant forms of Pak1 all increase cell movement, but protein kinase activity affects the persistence of these movements. Cells expressing kinase-dead Pak1 are characterized by multiple, randomly arrayed, lamellipodia, that continuously form and recycle. These cells move more quickly than controls, but do so in a haphazard manner, frequently and abruptly switching direction. In contrast, cells expressing wild-type or activated forms of Pak1 are characterized by a single lamellipodium at the leading edge. These cells move more quickly than controls, and also display increased persistence. As with activated Cdc42 and Rac1 , such cells also display increased haptotaxis towards a collagen gradient. These differences are likely to reflect the failure of cells expressing kinase-dead Pak1 to polarize F-actin appropriately. It is also possible that the increased motility of these cells is related to weakened adhesion to the substratum, as activated Pak1 induces aberrant formation of focal adhesion complexes . The formation of membrane extensions such as pseudopodia is thought to be driven mainly by actin polymerization . This process is evident in cells expressing either kinase-dead or kinase-active versions of Pak1. As mentioned above, these effects are likely to be mediated by interactions between the NH 2 -terminal, non-catalytic, domain of Pak1 and other proteins that affect actin polymerization. Although our data argue against the possibility that Rac1 acts downstream of Pak1, it is possible that other GTPases that affect actin organization, such as Arf6, might be activated by Pak1 . Activation of this or a similar GTPase might account for the kinase-independent ruffling induced by Pak1. Actin polymerization by itself, however, is insufficient for directed cell movement. In gradient-directed cell movement, one or more pseudopodia become dominant, while the others are suppressed, and these dominant pseudopodia are stabilized as leading edge lamellipodia, whose firm attachment to the substratum are thought to provide an anchorage point for contractile forces. The process of selection, generation, and propagation of a leading edge lamellipodium from a group of random pseudopodia is poorly understood, but may involve stabilization of filaments through the formation of strong focal contacts. In addition, nonadherent extensions may be actively suppressed once a leading edge lamellipodium is established . In NIH-3T3 cells, expression of activated, but not kinase-dead, Pak1 results in large, polarized lamellipodia and persistent cell movement. Phospho-MLC is increased, both in the trailing edge and at the leading edge of the motile cells. A similar distribution has been noted previously in other motile cells . Phosphorylation at the tail is thought to be responsible for pushing the cell body forward by tail contraction. The function of phosphorylated MLC at the leading edge is less clear, but may contribute to the membrane extensions at the leading edges, either by translocating actin filaments to this location or by suppressing lamellipodia formation in the absence of the reinforcement provided by stable focal contact . According to this scenario, active Pak1 may concentrate at the incipient leading edge in resting cells, perhaps directed there by the exchange factor PIX, where it acts upon myosin II, augmenting the polarity of the cell. This might explain the effects of active Pak1 on directional cell movement. Whatever the exact function of myosin II in locomoting cells, our data are consistent with the notion that Pak1 has two types of effects on cell morphology and motility; a kinase-independent activity that affects actin polymerization, and a kinase-dependent function that increases phospho-MLC levels and stabilizes leading edge lamellipodia. How does Pak1 affect the phosphorylation state of the MLC? The effect of Pak1 on MLC may be direct, as Pak1 has been shown to phosphorylate serine 19 of the MLC in vitro . Alternatively, Pak1 might induce MLC phosphorylation indirectly, via activation of MAPK or Rho kinase, both of which affect MLC kinase activity . Although Paks are usually considered to be activators of SAPKs such as Jnk and p38, rather than of MAPKs such as Erk1 and -2 , Pak2 has recently been shown to phosphorylate Raf-1 . This phosphorylation is required for efficient Raf-1 activation. In addition, Pak1 phosphorylates MEK on serine 298, increasing its affinity for its activator, Raf-1 . These data suggest that Pak1 activity positively regulates the MAPK pathway. As activation of MAPK is associated with increased MLC phosphorylation and cell movement , this pathway might explain Pak1's effects on motility. However, in our inducible cell lines, neither wild-type nor mutant forms of Pak1 appreciably stimulate Erk1 or -2, while activated forms do modestly activate Jnk. Therefore, activation of MLC via MAPKs cannot explain our findings. Alternatively, since Pak1 is associated with PIX, a guanine-nucleotide exchange factor for Rac1 , Pak1 might activate Rho kinase through a GTPases cascade (Cdc42 to Rac1 to RhoA) . However, coexpression of a dominant-negative form of Rac1 did not interfere with Pak1-induced cell polarity changes, making it unlikely that its motility effects are mediated through a known GTPase cascade. Therefore, if Pak2 activates Rok, it is not likely via downstream activation of Rac1 and RhoA. In this regard, it is interesting to note that fission yeast Pak1p has recently been shown to operate upstream of the Rok homologue Orb6p . Whether fission yeast Rho1p is interposed between Pak1p and Orb6p is not known, but these data do reinforce the notion that some of Pak's effects on myosin phosphorylation might be mediated by Rok. Therefore, at present we are uncertain if Pak1 directly phosphorylates MLC or does so indirectly through Rho, Rok, and/or MLCK. These possibilities may be distinguishable through the use of C3 toxin or dominant-negative forms of RhoA or Rok. | Other | biomedical | en | 0.999997 |
10330411 | PAK antibody was purchased from Santa Cruz, vinculin (Vin 11-5) from Sigma Chemical Co. , and Nck, p130 Cas , and clathrin heavy chain antibodies were obtained from Transduction Labs. FAK antibody (BC3) was a generous gift of Dr. J.T. Parsons (University of Virginia, Charlottesville, VA), antitalin (8d4) was provided by Dr. K. Burridge (University of North Carolina, Chapel Hill, NC). p50-COOL-1 antibody, which recognizes p85/p75PIX/COOL, was as described . Individual glutathione S-transferase (GST) fusion proteins spanning paxillin amino acid (aa) 54–313, or encompassing LD1 (aa 1–20), LD2 (aa 141–160), LD3 (aa 212–231), LD4 (aa 263–282), and LD5 (aa 299–317) were expressed in Escherichia coli (DH5α) and purified on glutathione agarose beads as previously described . Tissue lysates (newborn rat brain, a gift of Qin He, State University of New York, Syracuse, NY, or embryonic day 17 chicken gizzard smooth muscle) were prepared by Dounce homogenization in 10 vol lysis buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1 mM EGTA, 2 mM MgCl 2 , 0.1% β-mercaptoethanol, 0.5% TX-100) containing protease inhibitors (Complete™ EDTA-free; Boehringer Mannheim ), followed by clarification for 15 min at 15,000 g . Alternatively, cultured cells were washed twice with ice-cold Hanks' buffer and processed as for tissue lysates. Lysates (0.5–1 mg protein) were incubated with the individual fusion proteins (50 μg) in fusion protein-binding buffer (same as lysis buffer, but with the Triton X-100 adjusted to 0.1%) for 120 min at 4°C and then washed four times in binding buffer. The washed precipitates were boiled in SDS-PAGE sample buffer, followed by SDS-PAGE and Western blot analysis, or processed for in vitro kinase assay. For in vivo coprecipitation experiments, rat brain was homogenized in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% NP-40, 10% glycerol. Paxillin was precipitated with antipaxillin antibody (Transduction Labs) and the blots probed with antibodies to PIX, PAK, paxillin, and p130 Cas . Mouse Ig was used as a control. To demonstrate an association in vivo between paxillin and p95PKL, green fluorescent protein– (GFP–) p95PKL was transfected into COS 7 cells. 72 h after transfection, the cells were lysed in 10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1% NP-40, 10% glycerol, and clarified by centrifugation at 15,000 g for 15 min. Paxillin was precipitated with antipaxillin antibody and the blots probed with antibodies to paxillin, GFP ( CLONTECH Laboratories, Inc.), and p130 Cas . Mouse IgG was used in a control precipitation. Fusion protein precipitates were washed four times in fusion protein lysis buffer and once in kinase buffer (20 mM Tris-HCl, pH 7.6, 20 mM MgCl 2 , 10 mM MnCl 2 , 1 mM EDTA, 1mM EGTA, 40 μM ATP). The pellets were resuspended in 20 μl kinase buffer and incubated for 20 min at room temperature in the presence of 5 μCi [ 32 P]γ-ATP (>4,000 Ci/mmol; ICN Biomedicals). The effect of activated p21 GTPases on precipitated kinase activity was determined by the addition of GTPγS- or GDP-loaded GST p21 GTPases (Rho, Rac, or Cdc42) and 2.5 μg myelin basic protein, as previously described . The reactions were terminated by boiling in SDS-PAGE sample buffer. To evaluate if phosphorylation of precipitated proteins regulated binding to the paxillin fusion protein, the kinase reaction was followed by several washes of the immobilized GST fusion protein complex with kinase buffer before boiling in SDS-PAGE sample buffer. Results were visualized by SDS-PAGE on 10 or 15% gels followed by autoradiography. Asynchronously growing CHO.K1 fibroblasts were washed in serum-free DME and then incubated for 24 h in labeling buffer (8 ml methionine- and cysteine-free DME, 1 ml complete DME, 1 ml FBS [Summit Biotechnology], 2 mM glutamine, 200 μCi Trans 35 S-label™ [>1,000 Ci/mmol; ICN Biomedicals]). The cells were washed in Hanks' buffered saline and lysed in lysis buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1 mM EGTA, 2 mM MgCl 2 , 0.1% β-mercaptoethanol, 0.5% Triton X-100) containing protease inhibitors. The lysate was clarified in a microfuge for 15 min at 14,500 g , and the supernatant incubated with the appropriate fusion protein (50 μg) in binding buffer (lysis buffer containing 0.1% Triton X-100) for 120 min at 4°C. GST–LD-binding proteins and associated proteins were washed four times with binding buffer and analyzed on SDS-PAGE gels followed by enhanced fluorography using Amplify™ (Nycomed Amersham, Inc. ). Alternatively, metabolic labeling of individual proteins was performed by coupled transcription/translation reaction in a cell-free reticulocyte lysate system (TNT; Promega Corp. ) as directed by the manufacturer. The reaction mixture containing the [ 35 S]methionine- or [ 35 S]cysteine- (in vitro translation grade; ICN) labeled proteins was clarified by centrifugation before incubation with GST fusion proteins in fusion protein binding buffer for 120 min at 4°C as described above. LD4-binding proteins from chicken smooth muscle tissue lysate were separated by SDS-PAGE and transferred to PVDF membrane. Protein bands were visualized by Ponceau S staining and excised. Proteolytic digestion, HPLC fractionation, and internal peptide sequencing was performed by Dr. John Leszyk (Worcester Foundation for Biomedical Research, Worcester, MA). Several of the peptide sequences generated from the p95 protein (p95PKL) precipitated by LD4 of paxillin were identical to the product of the KIAA0148 gene, which encodes a 50-kD protein . The equivalent cDNA was isolated by reverse transcriptase-PCR from human U937 cells and a probe spanning nucleotides 527 to 999 of the KIAA0148 cDNA was used to screen a Molt-4 human leukemia cDNA library (Stratagene). Sequencing of a 2.5-kb clone revealed sequence in the 3′ end that coded for the additional peptide derived from the microsequencing of p95. A full-length clone of p95 was isolated and sequenced on both strands at the BioResource Center of Cornell University (Ithaca, NY). The sequence for p95PKL has been deposited in GenBank/EMBL/ DDBJ . Several additional p95PKL clones, representing alternative splice variants, were isolated and will be described elsewhere. The cDNA for p85 PIX was generated by reverse transcriptase-PCR using RNA prepared from NIH3T3 cells. Forward and reverse primers were derived from the mouse PIX cDNA sequence . p95PKL, p85PIX, and mPAK3 were subcloned into pcDNA3 for coupled transcription/translation analyses. SH-SY5Y human neuroblastoma cells were grown in medium consisting of 1:1 mixture of DME and Ham's F-12 ( GIBCO BRL ) containing 15% vol/vol FBS (Summit Biotechnologies), 100 IU/ml penicillin, and 50 μg/ml streptomycin and maintained at 37°C in an atmosphere of 5% CO 2 . 18 h before treatment, the cells were removed to serum-free medium. IGF-1 was added to a final concentration of 10 nM for the indicated time. SH-SY5Y cells were transfected using SuperFect (QIAGEN, Inc.) according to the manufacturer's protocol. Stable transfectants were obtained by growing in media containing 200 μg/ml G418. NIH3T3 and COS 7 cells were maintained in DME (Mediatech) supplemented with 10% vol/vol FBS, 1 mM glutamine, and 1% penicillin/streptomycin. CHO.K1 cells were cultured in modified Ham's F-12 (Mediatech) supplemented with 10% vol/vol heat-inactivated, certified FBS, and 1% penicillin/streptomycin ( Sigma Chemical Co. ) at 37°C in a humidified chamber with 5% CO 2 . Lipofectamine ( GIBCO BRL )-mediated transfection of CHO.K1 and COS 7 cells was as described elsewhere . Indirect immunofluorescence analysis was performed as previously described in Brown et al. . Photographs were taken on a Zeiss Axiophot photomicroscope equipped with epifluorescence illumination using Kodak Tmax 400 film. Images were scanned using Kodak Coolscan II™ and processed using Adobe™ Photoshop 3.0.5. GST fusion proteins of paxillin LD4 were purified from bacterial lysates as described previously and dialyzed against microinjection buffer (10 mM KPO 4 , pH 7.5, 5 mM KCl, 0.1% β-mercaptoethanol). Aliquots of dialyzed fusion protein (1 mg/ml) were stored at −70°C. Microinjection was performed using standard protocols using a Narishige micromanipulator and borosilicate glass capillary needles (World Precision Instruments) pulled on a Brown-Flaming micropipette puller ( Sutter Instrument ). To measure migration rates of injected cells, confluent NIH3T3 cells were wounded by clearing the cells from one side of the coverslip with a Teflon spatula. The first line of cells immediately adjacent to the wound were injected with either GST or GST–LD4. Cells were allowed to migrate for 24 h and then fixed and processed for immunofluorescence microscopy. Slides were coded, and microinjected cells scored blind for position in one of three categories: baseline, cells remaining at the edge of the original wound, i.e., at the site of injection; pack, cells which had migrated, but were not at the leading edge; and leading edge, cells at the front of the migrating pack. Four replicates were examined for each injected protein (>200 cells per treatment) and the data analyzed by t test. Paxillin comprises an NH 2 terminus containing five LD motifs and a COOH terminus made up of four LIM domains . Previously, we have localized the vinculin and FAK binding sites on the NH 2 terminus of paxillin to extended regions encompassing LD motifs 2 and 4 . To determine the ability of vinculin and FAK to bind independently to the discrete paxillin LD motifs, each LD motif of paxillin was synthesized as a GST fusion protein and incubated with unlabeled smooth muscle cell lysate in precipitation-binding assays followed by Western blotting. Consistent with our previous findings , FAK was precipitated by LD4, and to a lesser extent, LD2 . Vinculin exhibited strong binding to LD2, the domain previously identified as the principal binding site for this protein, in addition to a low binding to LD4. In contrast to FAK, vinculin also bound to LD1. Both vinculin and FAK binding to the aa 54–313 paxillin fusion protein was observed as previously shown . Thus, FAK and vinculin differentially associate with the individual paxillin LD motifs . A reprobe of the blot confirmed that smooth muscle talin is unable to bind to the individual LD motifs or to aa 54–313 of paxillin . Having established that the individual LD motifs can support specific and differential binding of known focal adhesion binding partners for paxillin, we determined if the individual LD fusion proteins associate with additional cellular proteins. GST–LD fusion proteins were incubated with [ 35 S]methionine/cysteine metabolically labeled CHO.K1 lysates. GST–LD-binding proteins were visualized by SDS-PAGE followed by fluorography . Each LD motif precipitated a discrete profile of cellular proteins. LD1 precipitated a protein of 120 kD (vinculin) and a prominent band of 50 kD. LD2 precipitated protein(s) of 120 kD (vinculin and FAK). LD3 and 5 precipitated no obvious 35 S-labeled binding partners in this assay. In contrast, LD4 precipitated several prominent proteins ranging from 80 to 150 kD. Importantly, a paxillin fusion protein spanning aa 54–313, and therefore containing LD2–5, precipitated a complement of proteins similar to the individual LD motifs . This suggests that the binding of proteins to the individual paxillin LD motifs was comparable to their binding when presented in the context of an intact NH 2 terminus. Similar results were obtained using lysates from NIH3T3 and Swiss 3T3 fibroblasts (data not shown).The 68-kD protein precipitated in approximately equal amounts by all fusion proteins also is precipitated to the same extent by GST alone (not shown), and is likely a heat shock protein. To identify LD4-binding proteins, GST–LD4 fusion protein was incubated with a smooth muscle tissue lysate. The bound proteins were subjected to SDS-PAGE, transferred to PVDF membrane, and visualized with Ponceau S staining. Individual protein bands, absent in a GST control precipitation, were excised and subjected to microsequencing. Proteins of ∼150, 97, 95, and 80 kD precipitated by LD4 were analyzed. The peptide sequencing data is summarized in Table I . Two peptide sequences from the 150-kD protein were identical to clathrin. One peptide sequence from each of the 97- and 80-kD bands matched with two forms of the recently identified guanine nucleotide exchange factor (GEF), PAK-interacting exchange factor , also called COOL , or p85SPR . The selective binding of PIX and clathrin to paxillin LD4 was confirmed by Western blotting . Multiple immunoreactive bands in the PIX immunoblot are consistent with the existence of multiple isoforms . Importantly, deletion (dl) of LD4 from a GST–paxillin fusion protein spanning the NH 2 terminus (GST–54-313 dl LD4) resulted in a complete loss of PIX binding . It should be noted that this fusion protein retains the ability to bind FAK via LD2 . Four peptides derived from the p95 protein (p95PKL) were sequenced. Peptides one through three were 100% identical to the previously reported KIAA0148 gene . However, the cDNA for KIAA0148 encodes a 50-kD protein. This cDNA was used to probe a human leukemia library to isolate a full-length p95PKL clone. The full-length p95PKL cDNA contained coding sequence for the fourth peptide derived from the original microsequencing analysis. Analysis of other cDNA clones isolated in the screen indicates that there are multiple splice variants of p95PKL. These will be described elsewhere. The PKL gene encodes a 757-aa protein with a calculated molecular weight of ∼85 kD. It is closely related to CAT , the 53-kD protein KIAA0148 , and G protein-coupled receptor kinase-interacting protein (GIT-1), a β-adrenergic receptor kinase-binding protein . An alignment of PKL, CAT, and KIAA0148 is presented in Fig. 4 . Although p95PKL, CAT, and KIAA0148 show significant identity within the first 471-aa, p95CAT contains a 50-aa deletion . PKL is more distantly related to the 130-kD ARF–GAP protein, ASAP . p95PKL contains several discrete structural and functional domains within the primary aa sequence. The NH 2 terminus (aa 3–111) exhibits homology to a family of ARF–GAP Golgi proteins, including ASAP (aa 461–563, 34% identity, 53% conservation), that have been implicated in the regulation of secretory pathways and mitosis . Located within aa residues 11–44 of this domain is a GCS family zinc-finger characterized by a C 2 C 2 H 2 zinc-coordinating residue scheme. This zinc-finger is contained within a consensus SAT motif (suppressor of Arf t s mutation) spanning aa residues 11–60 . The fact that the p95PKL related proteins GIT-1 and ASAP have been confirmed to exhibit ARF–GAP activity predicts PKL, CAT, and KIAA0148 will likely share this function. The p95 family contains three ankyrin repeats spanning aa 167–199, 200–232, and 134–166. The combined p95PKL, ARF–GAP, and ankyrin repeat region shows 26% identity to and 43% conservation with the tumor suppressor BRCA1-associated RING domain protein, BARD1 . A possible role for Ca 2+ calmodulin regulation of p95PKL is suggested by the presence of an EF hand (aa 417–430), and two partial IQ motifs on the COOH terminus (aa 654–671 and 710–718). The EF hand is present in p95PKL, KIAA0148, and GIT-1, but is absent from the PKL splice variant CAT and ASAP. The COOH terminus of p95PKL and p95CAT shares homology to a region of myosin maintained on clip and hook proteins such as restin/CLIP-170, giantin, EEA1, and hook-1, which are involved in vesicle and organelle linkage to microtubule networks (data not shown). Of particular relevance to this study is the identification of two potential paxillin-binding subdomains within p95 family members . PBS1 (aa 119–155) displays 32% identity and 68% conservation with the vinculin PBS . The second potential PBS sequence on p95PKL (PBS2, aa 643–679) is 19% identical and 57% conserved with the FAK PBS. The similarity of the p95 family PBS regions to those of vinculin and FAK indicate that these are candidate regions of p95PKL interaction with paxillin. The finding that both p95PKL and FAK bind to LD4, while vinculin binds to LD1 and LD2 , favors the p95PKL PBS2 sequence as the primary paxillin-binding region. PIX was previously identified through a high affinity association with PAK complexed with the p21 GTPase, Cdc42. Both PAK and Cdc42 are involved in reorganization of the actin cytoskeleton in response to soluble growth factors, and both PAK and PIX have been colocalized with paxillin at focal complex sites formed in response to activation of Cdc42 and Rac . Therefore, we sought to determine if PAK was associated with the LD4 motif of paxillin also. Lysates from rat brain were incubated with the individual GST–paxillin LD motifs and binding of PAK to the precipitates was evaluated by Western blotting. PAK was precipitated by GST–LD4 and the GST–54-313 paxillin fusion containing LD2–5 . PAK was not detected in individual LD1, 2, 3, or 5 precipitates. The filter was reprobed with an antibody to the focal adhesion protein p130 Cas . No binding of Cas to any of the paxillin fusion proteins was detected . The SH3-SH3-SH3-SH2 adaptor protein Nck interacts with PAK via the second SH3 domain . The ability of paxillin to coprecipitate Nck with PAK was confirmed by blotting the GST–54-313 paxillin precipitate with anti-Nck antibody . Importantly, deletion of LD4 from this GST– paxillin fusion protein (GST–54-313 dl LD4) resulted in complete loss of PAK and Nck binding , whereas the binding of FAK to this fusion protein was diminished but not eliminated, due to the presence of LD2, a second FAK binding site . No binding of the adaptor protein Crk was detected in any of the precipitates. A specific association of PIX and PAK with paxillin in vivo was confirmed by coimmunoprecipitation from brain lysate . The failure of the largest PIX isoform to precipitate efficiently with paxillin suggests that intact paxillin may discriminate between PIX family members for binding. No binding of p130 Cas to either the paxillin or control IgG precipitate was detected. To determine whether p95PKL, PIX, or PAK interact directly with paxillin, metabolically labeled proteins were synthesized using a cell-free coupled transcription/translation system. 35 S-labeled p95PKL, PIX, and PAK were incubated with GST fusion proteins of PIX, PAK, paxillin NH 2 terminus (aa 54–313), or GST. Bound proteins were detected by SDS-PAGE followed by fluorography. Previously reported interactions between PAK and PIX were confirmed using this cell-free system . However, neither of these proteins were precipitated by the paxillin NH 2 terminus . In contrast, p95PKL was enriched in both the GST paxillin NH 2 terminus and GST– PIX precipitates, but was not precipitated by either GST or GST–PAK . These data support a role for p95PKL in mediating the association of paxillin with PIX. p95PKL also bound to GST full-length paxillin but not to a GST fusion protein of the four LIM domains of paxillin (data not shown). The association of p95PKL with paxillin and PIX provides a potential mechanism for recruiting PAK to the NH 2 terminus of paxillin. To confirm that the paxillin LD4 motif supports p95PKL binding, a precipitation assay was performed using GST–LD4 and the NH 2 terminus of paxillin containing a deletion of LD4 (GST–54-313 dl LD4). p95PKL bound to the GST–LD4 fusion protein, but not the LD4 paxillin deletion mutant . To delineate further the PIX- and paxillin binding sites on p95PKL, two in vitro translation products of p95PKL were generated encoding aa 1–576 and 448–757, respectively, and tested for binding to GST–PIX and paxillin. PIX bound only to aa 1–576 of p95PKL, whereas paxillin bound to the 448–757-aa region of p95PKL containing the PBS2 region . To demonstrate an in vivo association between paxillin and p95PKL, GFP–p95PKL was transfected into COS 7 cells. Western blotting of a paxillin precipitate from lysates of these cells confirmed that GFP–p95PKL, but not p130 Cas , coprecipitated with paxillin . PIX has been suggested to mediate PAK activation by Cdc42 . To determine if the PAK associated with LD4 was enzymatically active, precipitation kinase assays using rat brain lysate were performed in the presence of the exogenous substrate for PAK, myelin basic protein. Phosphorylation of myelin basic protein was detected in coprecipitates of GST–LD4 in the absence of exogenously added GTPase, but not with GST–LD1 precipitates . The associated kinase activity was stimulated further by the addition of GTP-loaded GST– Cdc42. Enhanced phosphorylation of coprecipitating proteins of 100–120, and 70 kD was also noted . Although Rac stimulates PAK in vivo, GST–Rac, as well as GST–Rho, were unable to activate LD4 associated PAK in this in vitro assay, consistent with previous reports . Previously, we have demonstrated that the GST paxillin NH 2 terminus precipitates both tyrosine and serine kinase activity from tissue and cell lysates . The tyrosine kinase has been identified as FAK, while the serine kinase is currently unidentified. These kinases phosphorylate the paxillin fusion protein on residues that are also phosphorylated on paxillin in vivo in response to integrin-mediated cell adhesion , as well as a coprecipitating 95-kD protein that was heavily phosphorylated on both tyrosine and serine residues . To investigate the possibility that the p95 phosphoprotein was PKL and/or PIX, the precipitation kinase assay was repeated with GST–54-313 and GST–54-313 dl LD4 . Deletion of LD4 completely eliminated the 95- and 68-kD phosphorylated bands, consistent with the loss of binding of the p95PKL/PIX/PAK complex. In contrast, the 120-kD band that previously has been confirmed to be FAK was reduced, but not eliminated, consistent with the binding data presented in Fig. 5 B. To determine if phosphorylation of the coprecipitating proteins caused dissociation of any of the proteins, the complex was washed extensively after the kinase reaction, before electrophoresis. The profile of precipitating phosphoproteins was identical to the unwashed sample , indicating the paxillin/p95PKL/PIX/PAK complex is not perturbed by phosphorylation in this assay. Hic-5, a paxillin superfamily member , may act as a functional antagonist of paxillin in vivo . The highest level of homology between the two proteins occurs within the LD motifs and the LIM domains . Hic-5, like paxillin, is targeted to focal adhesions via the LIM domains, and also binds vinculin, FAK/FRNK (FAK-related nonkinase), and Pyk2 . To test whether Hic-5 also precipitates a p95PKL/PIX/PAK complex, the NH 2 terminus of Hic-5 was expressed as a GST fusion protein and used in precipitation experiments. Western blot analysis of proteins precipitated by the Hic-5 NH 2 terminus confirmed the binding of FAK, PAK, Nck, and PIX . p130 Cas served as a negative control. Direct binding of the Hic-5 NH 2 terminus to p95PKL was detected by using 35 S-labeled, in vitro translated p95PKL protein . To evaluate the subcellular distribution of p95PKL, the protein was expressed as a GFP fusion protein in CHO.K1 fibroblast cells. GFP–PKL localized to small focal adhesions associated with the ends of actin stress fibers , and to paxillin-containing focal adhesions . GFP–paxillin also localized to focal adhesions at the ends of actin stress fibers . Some labeling of both GFP–PKL and GFP–paxillin along the stress fibers was also noted. GFP alone demonstrated a diffuse cytoplasmic and nuclear staining with no enrichment at the ends of actin stress fibers . To examine a role for the LD4 motif of paxillin in p21 GTPase and PAK-dependent cytoskeletal reorganization such as lamellipodia formation , SH-SY5Y neuroblastoma cells were transfected with either wild-type chicken paxillin or an LD4 deletion paxillin mutant. Cells expressing wild-type paxillin were generally more spread and contained a much more organized actin stress fiber network than cells expressing the LD4 deletion mutant, although both constructs localized to focal adhesions as determined by immunofluorescence staining with a chicken paxillin-specific antiserum . Actin was visualized with rhodamine phalloidin . Serum-starved cells were stimulated with insulin-like growth factor-1 (IGF-1) to induce lamellipodia . Cells expressing wild-type chicken paxillin exhibited numerous, large lamellipodia in response to 5 min exposure to IGF-1 . In contrast, the cells expressing the LD4 deletion mutant formed fewer, substantially smaller lamellipodia . FAK and PAK have a role in stimulating cell migration . To test whether the interaction of FAK and PAK with paxillin through the LD4 motif is potentially important for FAK- and/or PAK-mediated cell motility, GST or GST–LD4 fusion protein was microinjected into NIH3T3 cells at the edge of a wounded monolayer of cells cultured on glass coverslips. A comparison of the rate of migration of injected cells into the wound indicated a statistically significant reduction in the rate of migration of cells injected with GST–LD4, as compared with control GST-injected cells . Taken together, these data support a role for the paxillin LD4 motif in coordinating actin cytoskeleton dynamics at the plasma membrane. We previously identified a novel leucine-rich repeating sequence within the NH 2 terminus of paxillin, termed paxillin LD motifs . These motifs are implicated in the binding of paxillin to vinculin, FAK, and the E6 oncoprotein from papillomavirus . In this study, we have demonstrated that individual LD motifs can function independently to support the differential binding of the focal adhesion proteins vinculin and FAK. Several proteins were identified that were precipitated by paxillin LD4. These include clathrin and a new 95-kD, ARF–GAP domain- and ankyrin repeat-containing protein. PAK, Nck, and members of the Cdc42/Rac1 guanine nucleotide-exchange factor family PIX/COOL also associated with LD4. In vitro binding experiments using isolated proteins demonstrated that p95PKL binds directly to paxillin and PIX. Since direct binding of PAK to paxillin was not observed, the precipitation of PAK by paxillin LD4 from cell and tissue lysates likely occurs as a result of its association with PIX . Similarly, it is expected that LD4 precipitation of Nck is a result of its stable association with PAK . The coprecipitation of endogenous PIX family members and PAK with paxillin from brain lysate , as well as previous reports detailing coprecipitation of PIX and PAK , provides evidence for the existence of these interactions in vivo. P95PKL is a member of a family of ARF–GAP proteins that include the closely related GIT-1, CAT, and KIAA0148, as well as the more distantly related ASAP1. Both GIT-1 and ASAP1 exhibit ARF–GAP activity and have been implicated in membrane remodeling events associated with Arf function. GIT-1 binds the β-adrenergic receptor kinase and is involved in coordinating receptor endocytosis , while ASAP, the largest family member, which also binds Src and Crk, has been localized to the plasma membrane where it is suggested to coordinate membrane trafficking associated with cytoskeletal remodeling or cell growth . KIAA0148 is a smaller (50 kD) protein, containing highly conserved ARF–GAP and ankyrin-repeat regions, but lacking the more diverse COOH terminus domain, which in the larger proteins may be important in conferring binding specificity for accessory proteins. In this regard, it is of note that the paxillin binding site on p95PKL is within aa 577–757, and therefore not shared with KIAA0148. In contrast, PIX, which binds to aa 1–576 of PKL but not 448–757, potentially binds both p95PKL and KIAA0148. Thus, KIAA0148 may be a functional antagonist for some, or all, of the larger ARF–GAP family members. Such a mechanism has been demonstrated with FAK and FRNK, which comprises only the COOH-terminal, noncatalytic domain of FAK , and more recently, with the regulation of PAK activity by the PIX family of GEFs . Paxillin itself is a member of a larger family that is characterized primarily by the conservation of NH 2 -terminal LD motifs and COOH-terminal LIM domains . One family member, Hic-5, shares several additional features with paxillin, including focal adhesion targeting via the LIM domains and binding to FAK, FRNK, vinculin, and CSK . The p95PKL/PIX/PAK/Nck protein complex also bound to the NH 2 terminus of Hic-5, suggesting conservation of function of the LD motifs of these two proteins. In contrast, unlike paxillin, Hic-5 is not tyrosine phosphorylated during cell adhesion and consequently is unable to bind the SH2 domain-containing adaptor, Crk . This suggests that Hic-5 and paxillin may act as functional antagonists with regards to Crk-dependent signaling events, such as cell migration and activation of the Ras–MAP kinase pathway via C3G. It will be important to determine the relative contribution of each of these multifamily members to particular cellular events and signal transduction pathways. What is the functional significance of a paxillin, p95PKL ARF–GAP protein association? A role for ARF1 and ARF6 proteins in cytoskeletal reorganization, including cortical actin reorganization during cell spreading is apparent . The capacity of paxillin to assemble a PAK-containing complex through the ARF–GAP proteins PKL and CAT provides a possibility for the integration of ARF and Rho family signal transduction at the cytoskeleton. In this regard, we and others recently have determined that paxillin cycles in an ARF-dependent manner, between the trans-Golgi/endosomal network and focal adhesions . One possible model would predict that regulation of different ARF family members by ARF–GAPs such as p95PKL determines the subcellular localization of paxillin and other focal complex/adhesion components, including PAK, PIX Cdc42, and vinculin . Thus, cell adhesion or growth factor stimulation would promote the release of paxillin and associated proteins from a perinuclear vesicle compartment, permitting targeting to sites of cell matrix interaction, and thereby contributing to the Cdc42/Rac/Rho paradigm. Interestingly, clathrin was also precipitated specifically by paxillin LD4. Although clathrin has not been observed in focal adhesions, α-actinin and the vitronectin receptor have been reported to bind clathrin . It is unknown at this time whether the binding of clathrin to paxillin is direct or indirect. However, clathrin does contain a well conserved PBS . The functional consequence of clathrin binding to paxillin remains to be determined, but it also may be associated with a role for p95PKL ARF–GAP activity and PAK in vesicular transport , endocytosis , and membrane trafficking to the leading edge during cell motility or in response to growth factors . It is also a potential factor in the redistribution of paxillin to the trans-Golgi/endosomal network in epithelial cells, where colocalization of paxillin and clathrin has been observed (Brown, M.C., and C.E. Turner, unpublished observations). Stimulation of p21 GTPase activity in response to integrin-mediated cell adhesion or growth factors results in reorganization of the actin cytoskeleton and the formation of distinct sites of cell attachment with the extracellular matrix. Rac and Cdc42 activation leads to the formation of small focal complexes at the base of lamellipodia and filopodia, respectively, while larger focal adhesions associated with robust actin stress fibers are the hallmark of Rho activity . Focal complexes and focal adhesions contain several common components including transmembrane integrin receptors and intracellular proteins, such as paxillin, vinculin, and FAK . PIX has been suggested as the protein that recruits PAK to focal complexes , but the protein responsible for PIX targeting has not been identified. It has been proposed that paxillin serves an adaptor function at these sites of adhesion, using the LIM domains for focal adhesion targeting and the NH 2 terminus in a scaffolding capacity, recruiting multiple signaling components such as the tyrosine kinases FAK, PYK2, Src, and Csk . In view of the binding of a p95PKL/ PIX/PAK complex to the NH 2 terminus of paxillin, and the localization of a pool of PKL to focal complexes/adhesions in CHO.K1 cells, future experiments will be directed towards evaluating a potential role for p95PKL in recruiting PIX and PAK to the paxillin-rich sites of cell adhesion. In support of a role for paxillin in p21 GTPase- and PAK-dependent cytoskeletal reorganization, we found that overexpression in neuroblastoma SH-SY5Y cells of a paxillin mutant lacking the LD4 motif caused an overall reduction in actin cytoskeletal organization in comparison to wild-type paxillin-expressing cells, and substantially reduced lamellipodia formation in these cells in response to IGF-1. Overexpression of PAK in neuronal PC12 cells also stimulates lamellipodia in the absence of IGF-1 . This response, which requires structural elements within both the NH 2 - and COOH-terminal region of PAK, but is not dependent on PAK kinase activity, is inhibited by coexpression of dominant-negative Rac or by interfering with the interaction between PAK and PIX . Thus, the ability of the paxillin LD4 deletion mutant to inhibit lamellipodia formation in response to IGF-1 likely is due in part to the failure of a PAK/PIX complex to target effectively to the plasma membrane through an association with paxillin. Similarly, the well-spread phenotype of SH-SY5Y cells overexpressing wild-type paxillin, as compared with the LD4 deletion mutant, provides further evidence for a role for paxillin in Cdc42 and Rac-dependent regulation of cell spreading . Finally, the absence of stress fibers in the paxillin LD4 deletion-expressing cells suggests that this mutant may also affect Rho-dependent signaling. Regulated attachment and detachment of integrin receptors to the extracellular matrix, as well as reorganization of the actomyosin cytoskeleton is required for cell movement . Recent reports have indicated a role for both PAK and FAK in cell migration . A paxillin/ FAK and/or a paxillin/PAK association at the plasma membrane may be required for the cytoskeletal changes necessary for efficient cell motility. Importantly, PAK has been shown to phosphorylate and inhibit MLCK , thereby providing a potential mechanism by which PAK can promote a motile Cdc42/Rac phenotype while inhibiting the less motile morphology characterized by robust stress fibers resulting from Rho-dependent activation of MLCK . Thus, microinjection of the nontargeting GST–LD4 fusion protein into NIH3T3 cells may be exerting an inhibitory effect on cell migration by sequestering sufficient FAK, PAK, and Cdc42 or Rac1 in the cytoplasm, preventing efficient lamellipodia and filopodia formation and extension at the leading edge of the cells. Cell spreading and cell migration also have been associated with increased kinase activity of FAK . We previously have shown that the NH 2 terminus of paxillin, when expressed as a GST fusion protein, precipitates FAK and serine kinase activities. The paxillin residues phosphorylated by these kinases in vitro are also major sites of paxillin phosphorylation during cell spreading . Conversely, inhibition of paxillin tyrosine phosphorylation blocks cell spreading . Since p95PKL is phosphorylated on both tyrosine and serine residues in in vitro paxillin precipitation kinase assays , it will be important to determine whether p95PKL is a target for FAK and/or PAK during cell adhesion/migration. These data are also consistent with the observation that p95CAT, (a PKL family member) is tyrosine phosphorylated during cell adhesion . In conclusion, we have demonstrated that the LD motifs of paxillin exhibit differential binding to several molecules important in the regulation of actin cytoskeleton dynamics. This includes the identification and cloning of a novel protein, p95PKL, that links paxillin indirectly to PAK and shares homology with ARF–GAP proteins involved in vesicular transport. Future experiments will examine the role of paxillin in coordinating the potential cross-talk between these paxillin-binding proteins | Other | biomedical | en | 0.999996 |
10330412 | Sequences encoding fragments of Ig-like molecules were amplified by PCR from embryonic day 11–13 (E11-E13) brain mRNA and subcloned into plasmid pSKII (Stratagene) as outlined previously . One clone, termed kb14, which contained a 290-bp insert with a novel Ig-like sequence was used to isolate clone pCMV/z14 by hybridization screening of an E16 λZAP library and in vivo excision as a pBK-CMV plasmid (Stratagene). As clone z14 did not contain the complete coding sequence of the novel molecule, a 5′-terminal EcoRI-PstI restriction fragment of 200 bp was used as a hybridization probe to isolate further phages from a chicken spinal cord cDNA library (λUNI-ZAP; Stratagene). Two of them, termed clones sc3 and sc4, which contain the L-form and the S-form, respectively, were subcloned into pSKII (Stratagene). Nucleotide sequences of z14, sc3, and sc4 were determined on both strands by the dideoxy chain termination method using the ALF (automated laser fluorescent) DNA sequencer ( Pharmacia ). The clones differed in two amino acid positions: V159 (sc4) versus G159 (z14/sc3) and S281 (sc4) versus P281 (z14/sc3). The complete insert of pSKII/sc4 was excised using XbaI and ApaI (partial digest) and ligated into pBK-CMV that had been linearized with NheI and ApaI to obtain pCMV/C11B3. In this step, the E . coli promoter of pBK-CMV which may interfere with eucaryotic expression was deleted. To express the L-form of neurotractin, the COOH-terminal part of pCMV/z14 was excised by digestion with NotI (partial) and MluI and subcloned into NotI-MluI–linearized pCMV/C11B3 to obtain pCMV/C11A1. Plasmids pCMV/C11B3 and pCMV/C11A1, which encode neurotractin-S and -L, respectively, were transfected into COS cells as described previously . To construct fusion proteins of human IgG Fc domains with neurotractin, the extracellular domains of neurotractin-L and -S were amplified from pCMV/C11A1 and pCMV/ C11B3, respectively, using the primers 5′-AAGAATTCCAGCGCGGAGCGGCGCGGAGAT-3′ and 5′-TTGAATTCTGATCCCATACTGGGCCGTACT-3′. Amplification products were subcloned via EcoRI into plasmid pIG2 , and sequences were confirmed by the dideoxy chain termination method. Expression in COS cells and purification of fusion proteins was done as described . To isolate telencephalic cells, E8 telencephali were incubated for 20 min at 37°C in HBSS with 1 mg/ml trypsin. Tissue was rinsed in HBSS, dissociated in DME and 10% FCS, and then cells were seeded at a density of 50,000 cells/cm 2 in tissue culture dishes (Petriperm™; Bachofer). The dishes had been precoated with 5 μl of test protein (12.5–100 μg/ml) for 4 h at 4°C, washed with HBSS, and blocked with DME and 10% FCS for 45 min at 37°C. Cultures were incubated for 40 or 72 h at 37°C, fixed, and stained essentially as described using mAb 5E directed to NCAM . To count attached cells, nuclei were labeled with the DNA-staining reagent H33258 ( Boehringer Mannheim ). Neurite outgrowth was quantified as follows: images containing nuclei of attached cells were captured separately from images with neurons using appropriate filter settings (images with neurons were edited manually because of a low signal-to-noise ratio). Images were then processed by an automated procedure to count cell nuclei and neurites and to determine neurite lengths essentially as detailed previously . In this study low cell densities and numbers of neurites allowed us to measure the exact lengths and numbers of neurites and to normalize with respect to the number of attached cells. Data were pooled from several independent experiments as follows. For quantification of neurite initiation, 80 images (450 × 450 μm) with a total of 2,800 attached cells were evaluated for 25 μg/ml neurotractin-L. For 50 μg/ml L-form, 109 images (5,500 cells) were evaluated, for 100 μg/ml, 125 images (6,700 cells), for the S-form, 31 images (680 cells), and for Fc control substrate, 28 images (890 cells). To quantify neurite elongation, 61 images with a total of 202 neurites were processed for 25 μg/ml neurotractin-L, 102 images (483 neurites) for 50 μg/ml, and 118 images (683 neurites) for 100 μg/ml neurotractin-L. Lower neurotractin-L concentrations in the coating solution (12.5 μg/ml) did not result in significant neurite extension. Statistical significance of differences was evaluated using the Mann-Whitney U Test implemented in the Statview program (Abacus Concepts, Inc.) Preparation of embryonic and adult chick brain plasma membranes, immunoblots, and release of GPI-linked proteins by phosphatidylinositol-specific phospholipase C (PI-PLC) was performed as outlined previously . Monoclonal antibodies directed against a 45–55-kD glycoprotein fraction of GPI-linked neural proteins from chicken brain were generated as described previously . To isolate both isoforms of neurotractin by immunoaffinity chromatography from PI-PLC supernatants mAb NTRA-1 was coupled to CNBr-activated Sepharose 4B ( Pharmacia Biotech AB ). NH 2 -terminal and internal peptide sequences of neurotractin have been determined essentially as reported previously . Polyclonal antibodies to neurotractin were generated in rabbits by subcutaneous injection of 10 μg neurotractin in 2-wk intervals. N-linked carbohydrates were cleaved from proteins with a mixture of endoglycosidase F and peptide- N -glycosidase F of Flavobacterium meningosepticum according to instructions of the manufacturer (Oxford GlycoSystems). Generation and characterization of function-blocking polyclonal Fab fragments directed to chicken neural IgSF members Ng-CAM, neurofascin, Nr-CAM, F11, axonin-1, NCAM, or gicerin , and of polyclonal antibodies that recognize cell surface–expressed CEPU-1 or LAMP has been reported in previous studies. Incubation of chicken eggs, preparation of tissue sections and immunofluorescence analysis were done as outlined in detail previously . To generate probes specific for the second and the third Ig-like domain of neurotractin, the corresponding sequences were amplified by PCR with the primer pairs 5′-AAGGATCCAACAATGCAGGTGCACCTCAC-3′/5′-TTGAATTCGGACTGAGACGTCGTTTTCTGC-3′ and 5′-AAGGATCCCACAATTCAGGAACTTAAATCC-3′/5′-TTGAATTCGAGGCAGGCTGGCATTGGTCA-3′, respectively, using plasmid pCMV/ C11A1 as a template and the products were subcloned via BamH1/EcoRI into plasmid pKSII (Stratagene). The plasmids were linearized and in vitro transcription was performed on 1 μg of template DNA/reaction using T3- (antisense strand) or T7- (sense strand) RNA polymerase (Fermentas) and a digoxigenin (DIG) nucleotide labeling mixture ( Boehringer Mannheim ). The resulting RNA was purified on Sephacryl columns ( Pharmacia ) and the concentration was estimated on an agarose gel. Brain tissue was dissected from different embryonic stages and either directly frozen and cut on a cryostat or fixed at first overnight in PBS/4% paraformaldehyde followed by PBS/30% sucrose . Sections were postfixed in PBS/4% paraformaldehyde for 10 min, washed twice in PBS/0.1% diethylpyrocarbonate for 15 min and equilibrated in 5× SSC for 15 min. Prehybridization was performed for 2 h in hybridization solution (50% formamide, 5× SSC, 40 μg/ml denatured salmon sperm DNA) at 58°C followed by overnight hybridization in the same buffer containing 0.4 μg/ml DIG-labeled RNA at 58°C. Sections were washed in 2× SSC at room temperature (RT) for 30 min, in 2× SSC at 65°C for 1 h, in 0.2× SSC for 1 h, in PBS/0.1% Tween 20 at 65°C for 10 min, and finally in PBS/0.1% Tween 20 at RT for 10 min. Blocking was performed in PBS/0.1% Tween 20/0.5% skimmed milk powder (containing 20% sheep serum) for 2 h at RT followed by anti-DIG alkaline phosphatase-conjugated antibody ( Boehringer Mannheim ; 1:2,500 diluted in the same buffer) at 4°C overnight. Excess antibody was washed off in PBS/0.1% Tween 20 (three times for 30 min at RT), sections were equilibrated in alkaline phosphatase reaction buffer (100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl 2 , pH 9.5), and then colorized in the same buffer containing 340 μg/ml NBT and 175 μg/ml BCIP overnight at RT. The color reaction was stopped in PBS, sections were dehydrated in ethanol, and then mounted in Eukitt (Kindler). Binding of Fc fusion proteins to transfected CHO and COS cells as well as expression plasmids for chicken CEPU-1 and chicken LAMP have been described previously . Transfection efficiencies and cell surface expression levels were found to be indistinguishable for CEPU-1, LAMP, and NTRA-L, as examined by immunofluorescence analysis using polyclonal antibodies followed by quantification with an image analysis system which has been described previously . To estimate binding constants, transfected CHO cells were exposed to increasing concentrations of purified neurotractin-Fc fusion proteins, followed by an excess of Cy3-conjugated secondary antibody directed to human IgG (Dianova). Fluorescence intensity was quantified within the linear response range of the image analysis system and background fluorescence intensity (measured with the same fusion proteins under the same conditions on untransfected cells) was subtracted. To estimate K D values, binding data were fitted to a linearized form of a titration curve as introduced by Heyn and Weischet and detailed by Bisswanger . To identify novel members of the IgSF in the developing brain we defined sequence patterns characteristic for members of this superfamily which has been described in a previous study . These were used to generate libraries of sequence fragments of neural IgSF members by PCR of reverse-transcribed embryonic chick brain mRNA. Sequencing of one of our initial PCR products indicated that it represents a novel member of the IgSF and it was therefore used to isolate cDNA clones by standard DNA hybridization procedures. Three clones were obtained which encode two isoforms of a novel IgSF protein, termed neurotractin . Both isoforms reveal a hydrophobic COOH-terminal segment which is compatible with anchorage to the plasma membrane via a GPI anchor . The large isoform, referred to as neurotractin-L, is composed of three Ig-like domains and the smaller isoform, neurotractin-S, lacks the third, membrane-proximal, Ig-like domain . The overall domain organization and sequence alignments indicate that neurotractin belongs to a subgroup of neural GPI-linked IgSF proteins, termed IgLON subgroup . Detailed sequence analyses including all known members of this subgroup reveals that neurotractin represents a novel member of this subgroup and can not be considered as the chicken equivalent of one of the mammalian subgroup members . To analyze biochemical features and the histological distribution of neurotractin we used two mAbs, namely NTRA-1 and NTRA-2 . Whereas mAb NTRA-1 reacts with both isoforms, mAb NTRA-2 binds only to the larger form, neurotractin-L, suggesting its epitope is located within the third Ig-like domain. mAb NTRA-1 was used to isolate both isoforms from PI-PLC supernatants of brain plasma membranes by immunoaffinity chromatography. The immunoaffinity isolate was found to comprise two bands, a 50-kD band and a fainter stained 37-kD band that comigrated in SDS-PAGE with neurotractin isoforms expressed in COS cells, suggesting that neurotractin-L is dominating in brain in comparison to the S-form. Members of the IgLON subgroup are found to be highly glycosylated. To determine the degree of N-glycosylation, neurotractin isoforms were subjected to enzymatic deglycosylation which reduced the molecular masses to 38 and 30 kD, respectively . Thus, ∼24% of the molecular mass of glycosylated neurotractin-L represents N-linked carbohydrates. Taken together, neurotractin is a novel GPI-linked member of the neural IgLON subgroup of the IgSF which occurs in two isoforms, a larger 50-kD form and a less abundant small form of 37 kD. To obtain indications of the expression pattern of neurotractin, different brain regions and developmental stages were examined by Western blot analyses. Neurotractin was detectable in embryonic retina, telencephalon, tectum, cerebellum and diencephalon . The highest level of neurotractin expression was found in telencephalon whereas the lowest level was determined in retina. In all brain regions examined, expression of the L-form as well as that of the S-form increases during development. In all regions and at all developmental stages examined, neurotractin-L seems to be more abundant than the S-form which is in line with the relationship observed in the immunoaffinity isolate . To examine whether neurotractin is a brain-specific protein, solubilized samples of embryonic liver, muscle and lung tissue were compared with a total brain sample in SDS-PAGE followed by Western blot analyses. Whereas neurotractin can be identified in embryonic brain it is undetectable in embryonic liver , muscle or lung (not shown) which suggests that neurotractin is a brain-specific protein. To characterize the histological distribution of neurotractin, distinct chicken brain regions of different developmental stages were examined by immunohistochemical analyses using mAb NTRA-1 which is directed to both neurotractin isoforms . At embryonic day 8, neurotractin is expressed on tectofugal axons in the stratum album centrale of the developing tectum mesencephali . By contrast, neurotractin could not be detected in the plexiform layers or the optic fiber layer of the retina (not shown). It was also undetectable on retinal ganglion cell axons on their pathway to the tectum . Therefore, in the retinotectal system at embryonic day 8, neurotractin is restricted to tectal efferents but is lacking on tectal afferents. On their pathway from the retina to the tectum, retinal ganglion cell axons cross the midline at the optic chiasm. In this region, they closely approach another axon tract, the supraoptic decussation which represents a major interhemispheric axon tract in the chicken located at the floor of the diencephalon . Interestingly, neurotractin is found to be strongly expressed on axons of the supraoptic decussation but it is undetectable on the adjacent retinal ganglion cell axons which demonstrates that neurotractin is restricted to subsets of axon tracts . The supraoptic decussation is not the only neurotractin positive axon tract which crosses the midline. Neurotractin was also found on axons of the anterior commissure which is situated in the ventral forebrain and which represents the largest intertelencephalic pathway in chicken . In addition to axons crossing the midline, neurotractin is also expressed in longitudinal axon tracts, for instance on axon bundles in E7 diencephalon or on longitudinal axons in the spinal cord . At embryonic day 9, neurotractin expression is pronounced in a small dorsolateral subpopulation of axons in the spinal cord. Later in development, however, this restriction is lost and neurotractin is found on all longitudinal spinal cord axons (data not shown). As described above, neurotractin occurs in two isoforms which may be differentially expressed in the developing nervous system. Since the L-form–specific mAb NTRA-2 did not recognize the protein in tissue sections we performed in situ hybridizations to investigate a possible differential distribution of the two forms. Two neurotractin probes, one which is L-form–specific and one which detects both forms of neurotractin mRNA were generated. A direct comparison of L-form versus S-form is not possible because the S-form sequence is completely contained within the L-form sequence. Both probes did not reveal detectable differences in the distribution of L-form mRNA versus total neurotractin mRNA in distinct regions of developing chicken brain, for instance in spinal cord , in subpopulations of neurons in ventral telencephalon , in the cerebellum , and in the tectum (data not shown). Thus, the in situ hybridizations suggest that there are no cells which are exclusively expressing the L-form and also support our conclusions drawn from the immunohistochemical analyses that neurotractin expression is restricted to subpopulations of neurons. Taken together, these results demonstrated that neurotractin is expressed by subpopulations of neurons in distinct regions of the developing chicken brain and that it is an axonal glycoprotein that appears to be restricted to subsets of commissural and longitudinal axon tracts. Subpopulations of axons within the anterior commissure, the supraoptic decussation, and longitudinal diencephalic pathways that show prominent neurotractin expression originate and terminate in the telencephalon. This might suggest that neurotractin plays a role in fasciculation and/or elongation of telencephalic axons within these pathways. Thus, to get first insights into the function of neurotractin, we tested if it is able to promote neurite outgrowth of telencephalic neurons. Recombinant forms of neurotractin fused to the Fc domains of human IgG1 were immobilized on tissue culture dishes and were used as substrates for embryonic day 8 telencephalic cells. We examined long-term cell attachment, neurite initiation, and neurite elongation of telencephalic neurons by measuring the number of adhering cells, the number of neurites per 100 cells and average neurite length, respectively, using an automated image analysis procedure (see Materials and Methods). These experiments showed that neurotractin-L fusion protein promotes attachment and neurite extension of telencephalic cells , whereas on Fc control substrate cell attachment was low and neurite outgrowth was undetectable . In contrast, neurotractin-S mediated only weak adhesion which was within the same range as that measured on Fc control substrate and did not induce neurites. Quantification of the neurotractin-L–mediated neurite outgrowth response after 40 h of incubation showed that the number of neurites per 100 cells increased in a dose-dependent manner suggesting that neurotractin modulates neurite initiation. On average only ∼1 of 10 cells was found to elaborate a neurite suggesting that the responsive neurons might represent a subpopulation of telencephalic cells. To investigate if prolonged incubation might recruit a larger neuronal subpopulation to extend neurites, cultures were evaluated after 72 h. However, no additional increase in the number of neurites per 100 cells could be observed for the highest neurotractin concentration that was tested (data not shown). A neurite outgrowth–promoting molecule may regulate the initiation of neurites and/or their elongation. Therefore, we investigated if immobilized neurotractin has an impact on the average length of telencephalic neurites, in addition to its effect on neurite initiation. However, no significant effect of increasing neurotractin-L amounts on the average neurite length could be demonstrated after 40 h or 72 h (data not shown) suggesting that neurotractin primarily influences the initiation of neurites of telencephalic neurons in vitro. Consistently, the average neurite length increased by <10% from 40 to 72 h of incubation (data not shown). As a first step to characterize the cellular receptor on telencephalic neurons responsible for the neurite outgrowth–promoting activity of neurotractin-L, polyclonal antibodies specific for various cell surface proteins were applied in these in vitro assays. We tested antibodies directed to L1 subgroup members (Ng-CAM, neurofascin, and Nr-CAM), F11 subgroup members (F11 and axonin-1), IgLON subgroup members (LAMP and CEPU-1), NCAM, or gicerin. None of these antibodies which have been previously documented to interfere functionally in distinct experimental paradigms using chicken neurons , or to recognize the respective proteins on cell surfaces , blocked neurite outgrowth on neurotractin-L (data not shown). Therefore, the cellular receptor on telencephalic neurons that mediates the neurite outgrowth–promoting activity of neurotractin-L remains unknown at present. To characterize the neurotractin-responsive cells we have analyzed their profile of expression of known adhesion proteins. Consistent with the above-mentioned antibody perturbation experiments, these responding cells are NCAM and F11 positive, but are, however, negative for NgCAM, neurofascin, NrCAM, and axonin-1 (data not shown). Several Ig superfamily members on axons reveal homophilic and/or heterophilic binding to other IgSF members within the same or across plasma membranes to regulate cellular interactions . To further characterize the molecular function of neurotractin isoforms we examined if recombinant neurotractin interacts with itself or with other neural members of the IgSF. To this end, purified Fc fusion proteins of neurotractin-L and -S were incubated with CHO cell transfectants that express putative interaction partners on their surface and, after washing, bound fusion proteins were detected with fluorochrome-conjugated secondary antibodies specific for their Fc portion. Soluble neurotractin-L fusion protein was found to bind strongly to CEPU-1 transfectants and, in comparison, weakly to LAMP transfectants, whereas no homophilic binding to neurotractin-L transfectants could be detected . The S-form of neurotractin also interacts with CEPU-1, however, binding was clearly weaker than that of the L-form while binding to LAMP could not be detected by this method. As a control, interaction of neurotractin-L with other IgSF members was examined under the same conditions but no binding could be observed to F11 , NgCAM, axonin-1, neurofascin (data not shown), or GPI-linked Fc domains alone . As an additional control, CEPU-1–expressing CHO cells were treated with PI-PLC to release CEPU-1 from the cell surface and were then incubated with neurotractin-L fusion protein. A strongly decreased binding to PI-PLC treated transfectants supports the interpretation that neurotractin-L binds to CEPU-1 on the cell surface and not unspecifically to other cell surface components . Furthermore, binding of soluble neurotractin-L to surface-expressed CEPU-1 and LAMP could also be demonstrated with other eucaryotic cells, namely transfected COS cells . To estimate the apparent dissociation constant for the neurotractin-L–CEPU-1 interaction, we quantified binding of neurotractin-L fusion protein to CEPU-1 which was expressed on the surface of transfected CHO cells. In this assay system, one of the interacting proteins is in a native and membrane-bound form and binding of the interacting partner can be monitored by immunofluorescence analysis and quantified by digital image processing. Binding of neurotractin-L fusion protein to CEPU-1–transfected CHO cells was saturable and gave an apparent dissociation constant of 3 × 10 −8 M . The interactions of neurotractin-L with LAMP and of the S-form with CEPU-1 could not be reliably quantified but were estimated to be at least five times weaker than L-form binding to CEPU-1 . Furthermore, no significant fluorescence signal could be observed if soluble Fc control protein was incubated with CEPU-1–transfected cells (data not shown). In conclusion, neurotractin-L interacts with the structurally related molecule CEPU-1 and, though more weakly, with LAMP, whereas neither homophilic binding nor binding to other neural IgSF members could be detected. However, these interactions are not required for neurotractin-L–mediated neurite initiation as revealed by antibody perturbation experiments (see above) which suggests that the neurotractin–CEPU-1 or the neurotractin– LAMP binding might be implicated in other cellular activities. In this study, we identified neurotractin as a novel GPI-linked neural member of the IgSF with three Ig-like domains. It is associated with specific axon tracts and is implicated in neurite initiation as demonstrated by in vitro assays. Comparison of the neurotractin sequence with sequences in GenBank database revealed that it shows 48– 56% sequence identity to members of a neural subfamily of the IgSF which is termed IgLON subgroup . This group comprises limbic system-associated membrane protein (LAMP) , opioid-binding cell adhesion molecule , and neurotrimin in mammalia as well as CEPU-1 , LAMP , GP55-A , and neurotractin (this study) which have been identified in chicken . All IgLON subgroup members share with neurotractin the 3-domain organization, the plasma membrane attachment via GPI, and the restricted expression in partially overlapping regions of the nervous system . Furthermore, neurotractin has 26– 29% sequence identity with distinct Drosophila proteins, including lachesin , amalgam , and klingon . Pairwise comparisons of single domains of IgLON members shows that the corresponding domains are most highly conserved between different molecules (data not shown). This colinear relationship of individual domains suggests an evolutionary origin of these proteins from a common ancestor. LAMP is currently the most thoroughly characterized representative of this subgroup. It reveals a restricted pattern of expression in functionally related areas of the nervous system and might serve as a guidance cue during the development of the septo-hippocampal pathway and the hippocampal mossy fiber projection . Cloning of LAMP from human , rat , and chicken revealed that it is highly conserved in evolution, e.g., human LAMP shows 99 and 91% sequence identity with the rat and chicken proteins, respectively. The structural similarity of vertebrate IgLON molecules and their spatiotemporal expression pattern suggests that they might have related functions in the context of cell–cell interactions in nervous system histogenesis and in the adult brain. Whereas we could demonstrate that neurotractin-L is involved in initiation of telencephalic neurite growth, the axonal receptor protein important for this activity could not be identified at present. Our attempts to characterize proteins that interact with neurotractin resulted in the finding that neurotractin-L Fc fusion protein binds to CEPU-1 and, more weakly, to LAMP . Therefore, it appears to be a characteristic feature of IgLON molecules that some members of this subgroup bind to other members of the same subgroup, however, not to other IgSF proteins. A K D of 3 × 10 −8 M was measured for the binding of neurotractin to the GPI-linked molecule CEPU-1 , an affinity that is lower than that usually reported for soluble polypeptide ligands that bind to cell surface receptors, for instance the neurotrophins . However, cell–cell communication via membrane-bound ligands is likely to depend not only on receptor affinity but also on avidity effects and receptor density. Indeed, other receptors which also interact with membrane-bound ligands have binding constants which are comparable to that measured for neurotractin. For example, Fc fusion proteins of the Eph-like receptor tyrosine kinases that bind to cell surface ligands, termed ephrins, show dissociation constants of 4 × 10 −10 M to 4 × 10 −8 M . Nevertheless, these K D values should not be overinterpreted. On the one hand, potential bivalency of Fc fusion proteins may decrease their dissociation rate constant which may lead to overestimated affinities. On the other hand, analyses using soluble ligands most likely underestimate the true avidity between receptors and membrane bound ligands in their membrane-associated state. Regardless, the interaction of neurotractin with CEPU-1 is of a strength which is of physiological relevance in other receptor/ligand systems. The biological functions of the neurotractin–CEPU-1 or the neurotractin–LAMP interactions are currently unknown. Our antibody perturbations experiments indicate that they are not required for neurite extension of telencephalic neurons on immobilized neurotractin. However, it is conceivable that these interactions are important for other neurons, in contrast with the telencephalic neurons used here. For example, CEPU-1, which is expressed by Purkinje cells, and neurotractin are also colocalized in the molecular layer of the cerebellum suggesting that the neurotractin–CEPU-1 interaction may play a role in development of the Purkinje cell dendritic tree. Cell surface molecules that promote neurite outgrowth can be interpreted as membrane-bound neuronal differentiation factors because neurite initiation and elongation are part of the neuronal differentiation program. For instance, an outgrowth-initiating molecule that is restricted to a particular cortical layer may influence neuronal differentiation of precursor cells invading this layer and switch on dendritic growth. On the other hand, a protein that promotes elongation of neurites may provide a permissive environment for advancing growth cones. Our in vitro analyses suggest that neurotractin-L is more likely to be related to neurite initiation rather than elongation for two reasons. First, within 40 h of incubation we observed a dose-dependent influence on the number of neurites per 100 cells but no significant impact on the average length of the neurites . Second, we did not observe a significant increase of average neurite length between 40 and 72 h of incubation (data not shown). Our observation that neurotractin has a stronger effect on neurite initiation than on neurite elongation appears to be inconsistent with its expression in axon tracts that suggests a role in neurite elongation. However, neurotractin is only one component of a complex network of interacting receptors and ligands that regulate neurite outgrowth in vivo and it is reasonable to assume that other important factors are missing in our in vitro assay system. Thus, a more conclusive interpretation of the role of neurotractin in the context of neurite outgrowth requires additional studies, for instance antibody perturbation experiments in ovo or histological analyses of neurotractin-deficient knockout mice. Other hints to the putative role of neurotractin in neurohistogenesis come from functional analyses of structurally related proteins. Many members of the IgSF that are expressed in the nervous system are implicated in processes like neurite outgrowth , fasciculation , and guidance . In particular, neurotractin is closely related to LAMP , which has been shown to induce neurite outgrowth from specific subpopulations of neurons: transfected CHO cells that express LAMP on their surface have only weak effects on neurons from olfactory bulb or visual cortex but promote neurite outgrowth from perirhinal and hippocampal neurons significantly . This is reminiscent to neurotractin which also promotes outgrowth only of subsets of neurons, in this case subpopulations of telencephalic neurons. Further experiments are needed to characterize the neurotractin-responsive subpopulations of neurons in the developing brain and to identify the receptor(s) involved in the outgrowth response. Neurotractin is expressed in two isoforms, termed L-form and S-form, differing with respect to the presence of the membraneproximal Ig-like domain . Both forms show the same spatiotemporal expression profile, as examined by Western blot analyses and also at the level of in situ hybridization analyses . Alternative splicing of complete Ig-like domains has not been observed previously for IgLON molecules and its functional significance remains unclear at present. One possible reason for expression of different isoforms might be that they differ functionally, for instance with respect to binding of receptors and ligands. In this regard it is of interest that only the L-form has been found to mediate adhesion and neurite initiation of telencephalic neurons and that the L-form binds stronger to CEPU-1 or LAMP than the S-form. This may suggest that these cells express a receptor which binds to the membraneproximal domain of neurotractin which is lacking in the S-form. However, this may be argued against since the membraneproximal domains of IgLON members are least conserved in evolution and ligand or receptor binding sites are frequently located in NH 2 -terminal regions of cell adhesion receptors . Thus, it is also possible that the receptor binding site of neurotractin is located in the aminoproximal domains but may be sterically inaccessible in the S-form Fc fusion protein. In addition to the membrane-bound L- and S-forms, soluble variants of neurotractin may also exist. The immunohistochemical analyses show that there are two aspects of neurotractin expression: First, a prominent labeling of axon tracts and second, for instance in the cerebellum, a weak and diffuse staining (data not shown) which can be confirmed in Western blot analyses and in situ hybridizations . One explanation for the diffuse staining may be that neurotractin is released from the cell membrane by an endogenous phospholipase as it has also been described for axonin-1 , another neurite outgrowth–related IgSF member . In the case of axonin-1, the soluble form may act as a competitive inhibitor of neurite fasciculation . For neurotractin-L, this question will be addressed in future investigations. Western blot analyses of different brain regions showed that neurotractin is expressed in retina, telencephalon, tectum, cerebellum, and diencephalon. Furthermore, analysis of different developmental stages revealed that expression is increasing in all regions during development . Upregulation in development has also been observed for other IgLON molecules, for instance LAMP or CEPU-1 , and suggests that these molecules may also have a function in the mature brain. Consistently, LAMP has also been found in the adult brain of human , rat , and chick . Furthermore, neurotrimin and OBCAM have been detected in postnatal day 20 rat brain . Functions of neurotractin in the adult brain are unknown at present but may include phenomena which are similar to those in the developing brain, for instance functions related to neuronal remodelling and plasticity. | Study | biomedical | en | 0.999997 |
10330413 | RBL-2H3 cells were maintained and harvested as previously described . Mouse monoclonal IgE specific for 2,4-dinitrophenyl (DNP) was purified as previously described ; biotinylated and iodinated or FITC-labeled IgE was used to sensitize cells in some experiments. Mouse monoclonal anti-1,5-dansyl IgE was affinity purified as previously described . Other RBL cell membrane components were labeled with AA4 mAb (a gift from Dr. Reuben Siraganian, National Institutes of Health, Bethesda, MD), specific for the α-galactosyl GD 1b ganglioside derivative; OX-7 ( PharMingen ), specific for the GPI-anchored protein Thy-1; and anti-Lyn (Upstate Biotechnology, Inc., and Santa Cruz Biotechnology ) as previously described . Transferrin receptors (TfRs; CD71) were labeled with a monoclonal antibody from PharMingen , followed by Cy3-goat anti–mouse γ chain (Southern Biotechnology Associates). To remove cholesterol, suspended cells (2–4 × 10 6 cells/ml) were incubated for 1 h at 37°C in the presence or absence of 10 mM MβCD ( Sigma Chemical Co. ) in BSA-containing buffered saline solution (BSA/BSS: 20 mM Hepes, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, and 1 mg/ml BSA), then washed with BSA/BSS before stimulation. For some experiments, cholesterol (Avanti Polar Lipids) was added back to cholesterol-depleted cells (2 × 10 6 cells/ml) in BSA/BSS by incubation for 2 h at 37°C with indicated concentrations of MβCD/cholesterol (8:1, mol/mol) complexes. These complexes were prepared similarly to a previously described procedure . In brief, cholesterol in a chloroform solution was dried under nitrogen in a glass culture tube precleaned with ethanolic KOH. An appropriate volume of sterile-filtered 300 mM MβCD in BSA/BSS was added to the tube, and the resulting suspension was vortexed and bath sonicated until the suspension clarified. The complex was then incubated in a rocking water bath overnight at 37°C to maximize formation of soluble complexes. Suspended RBL-2H3 cells that had been sensitized with anti-DNP IgE and cholesterol depleted/repleted or not were stimulated at a density of 10 6 cell/ml with multivalent DNP-BSA at 37°C for indicated times, lysed by addition of 5× SDS sample buffer (50% glycerol, 0.25 M Tris, pH 6.8, 5% SDS, 0.5% bromphenol blue) and boiled for 5 min and centrifuged for 5 min at 13,000 g . Equal numbers of cell equivalents of lysates (typically, 8 × 10 3 cell equivalents) were electrophoresed on 12% nonreduced SDS polyacrylamide gels, transferred to Immobilon-P membranes ( Millipore Corp. ), and probed with horseradish peroxidase–conjugated antiphosphotyrosine (4G10-HRP; Upstate Biotechnology, Inc.). Enhanced chemiluminescence (Pierce) was used for detection. Phosphorylation as a function of stimulation time was quantified after scanning blots and analyzing with Un-Scan-It (Silk Scientific) and Igor Pro (WaveMetrics). We determined the effect of MβCD on the amount of FcεRI associated with RBL-2H3 cells by two different methods. For some experiments, biotinylated 125 I-IgE was bound to FcεRI under saturating conditions, and these labeled cells were used to monitor the loss of IgE–FcεRI complexes after treatment with or without MβCD as described above. The state of 125 I-IgE released from the cells during MβCD treatment was assessed by collecting the supernatants after cell pelleting (200 g , 5 min) and subjecting these to a high speed centrifugation (250,000 g , 45 min, 4°C). Gamma counting indicated that 54% of the 125 I-IgE was pelleted during the second centrifugation, in contrast to only 6% of the 125 I-IgE that could be pelleted under these conditions for untreated cells. In another set of experiments, FcεRI were saturated with FITC-IgE, and cells were treated with or without MβCD. Receptor-bound FITC-IgE was measured on washed cells with steady-state fluorimetry as previously described . Examination of these cells by fluorescence microscopy in the presence or absence of 15 mM NH 4 Cl to neutralize endosomes showed no evidence for internalization of FITC-IgE after MβCD treatment. To examine the relationship between the density of anti-DNP IgE on the cells and antigen-stimulated tyrosine phosphorylation, FcεRI were saturated with mixtures of anti-DNP IgE and antidansyl IgE in percentage mixtures of 30:70, 50:50, and 100:0. Washed cells were stimulated with 1 μg/ml DNP-BSA at 37°C for various times and tyrosine phosphorylation was analyzed. Competition binding between each of these unlabeled antibodies and FITC-labeled anti-DNP IgE was carried out under identical conditions. With steady-state fluorimetry to quantify the amount of cell-bound FITC-IgE, we confirmed that the percentages of anti-DNP IgE and antidansyl IgE bound to FcεRI in the tyrosine phosphorylation experiments were identical to the percentages added with an uncertainty of ± 8%. Furthermore, fluorimetry experiments with mixtures of FITC anti-DNP IgE and unlabeled antidansyl IgE showed that DNP-BSA binding and cross-linking induced internalization of FITC-IgE–FcεRI proportionally to the amount of FITC-IgE bound in the range of 30–100% occupancy by this IgE . Cholesterol-depleted/repleted or untreated cells were fractionated on sucrose step gradients as previously described , except that the concentration of TX-100 during lysis was 0.04% instead of 0.05%. Aliquots (200 μl) were removed from the top of the gradient, and γ-radiation of biotinylated 125 I-IgE was counted. The gradient fractions were then pooled as indicated, boiled with SDS sample buffer, and blotted as described above, except that the primary antibody was rabbit anti-Lyn antibody (Upstate Biotechnology, Inc.) and the secondary antibody was HRP-conjugated donkey anti–rabbit Ig ( Amersham Pharmacia Biotech ). To determine the location of GD 1b in the gradients, cells labeled with 125 I-AA4 and biotinylated IgE were analyzed as previously described . In some experiments, the pooled gradient fractions were electrophoresed on 12% nonreduced SDS-acrylamide gels and subsequently were silver stained (Daiichi Silver Stain II; Owl Separation Systems). Anti-DNP IgE–sensitized cells were stimulated for the indicated times with DNP-BSA (1 μg/ml) and lysed on ice with TX-100 lysis buffer [10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM Na 3 VO 4 , 30 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.02 U/ml aprotinin, 0.01% NaN 3 , 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.2% TX-100] followed by addition of 10 μM DNP-aminocaproyl- l -tyrosine, as previously described . After centrifugation at 13,000 g for 5 min to remove insoluble material, lysates were incubated with or without 20 μg/ml rabbit anti-IgE . After incubation with protein A–agarose beads (Pierce), samples were centrifuged to pellet the beads and supernatants were removed, boiled in SDS sample buffer, electrophoresed, and blotted with 4G10-HRP as described above. Rabbit anti-IgE/IgE–FcεRI complexes are bound to the protein A–agarose, which result in selective depletion of the FcεRI β and γ bands. The degranulation response that occurs after stimulation with the antigen DNP-BSA or the calcium ionophore A23187 ( Calbiochem-Novabiochem ) for 1 h at 37°C was carried out as described previously , except that cells were treated with or without MβCD immediately before stimulation. Cells sensitized with FITC-IgE were treated with or without MβCD, then washed and incubated with cytochalasin D (1 μg/ml) for 5 min at room temperature to prevent antigen-stimulated IgE–FcεRI internalization and to sustain Lyn co-redistribution with patched IgE–FcεRI at the cell surface. The cells were then stimulated with 1.7 μg/ml DNP-BSA at room temperature for 20 min and subsequently fixed with cold methanol for Lyn labeling or with formaldehyde for GD 1b or TfR labeling as previously described . Confocal fluorescence microscopy was performed as previously described . Cross correlation analysis of the co-redistribution of Lyn or TfR with antigen–cross-linked FITC-IgE–FcεRI were carried out on equatorial images of individual cells using a computational procedure similar to that previously described . Peak values can be calculated from this analysis to a correlation coefficient (ρ) , and these values were averaged for 7–13 cells from each sample for numerical comparison of the degree of co-redistribution. 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*}{\rho}=\frac{\frac{1}{{\mathit{N}}}{ \,\substack{ ^{{\mathit{N}}} \\ {\sum} \\ _{{\mathit{i}}} }\, }({\mathit{x}}_{{\mathit{i}}}-{\langle}{\mathit{x}}{\rangle})({\mathit{y}}_{{\mathit{i}}}-{\langle}{\mathit{y}}{\rangle})}{\sqrt{\frac{1}{{\mathit{N}}}{ \,\substack{ ^{{\mathit{N}}} \\ {\sum} \\ _{{\mathit{i}}} }\, }({\mathit{x}}_{{\mathit{i}}}-{\langle}{\mathit{x}}{\rangle})^{2}}\sqrt{\frac{1}{{\mathit{N}}}{ \,\substack{ ^{{\mathit{N}}} \\ {\sum} \\ _{{\mathit{i}}} }\, }({\mathit{y}}_{{\mathit{i}}}-{\langle}{\mathit{y}}{\rangle})^{2}}}\end{equation*}\end{document} In Eq. 1 , x i and y i are the intensities at each equatorial point of the FITC and Cy3 fluorescence, respectively, and < x > and < y > are the corresponding average values. RBL-2H3 cells were depleted of cholesterol (or not) and repleted (or not) as described above. After treatment, the cells were washed with BSA/ BSS, and then resuspended in methanol. The cell lysate was homogenized with a Duall ground glass tissue grinder, transferred to an ethanolic KOH-cleaned glass vial, and an equal volume of chloroform was added, followed by vigorous vortexing and probe sonication. The samples were rocked overnight at room temperature, then centrifuged at 800 g for 5 min, and the supernatants were transferred to a new glass vial. Chloroform/methanol (1:1, vol/vol) was added to the pellets and the suspension was vortexed vigorously and centrifuged as described above. Supernatants consisting of the total lipid extracts were combined and stored under nitrogen at −20°C. The extent of cholesterol depletion or repletion was measured using a colorimetric cholesterol oxidase assay ( Boehringer Mannheim ) that quantifies total free cholesterol. Total lipid extracts from paired samples of MβCD-treated and untreated cells were assayed simultaneously in these determinations. Thin layer chromatography of the total lipid extracts was carried out on silica gel 60 plates (EM Science) as previously described , with iodine detection. The developing solvent for polar lipids was chloroform/methanol/glacial acetic acid/water (60:50:1:4, vol/vol), and for neutral lipids, hexane/diethyl ether/glacial acetic acid (90: 10:1, vol/vol) . Because cholesterol is a critical component of liquid-ordered DRMs, we investigated whether reduction of the cholesterol content of the RBL-2H3 mast cells affects the tyrosine phosphorylation of FcεRI β and γ subunits. For this purpose, we incubated the cells with 10 mM MβCD for 1 h at 37°C to deplete cellular cholesterol before stimulation with multivalent antigen. Fig. 1 a shows a representative antiphosphotyrosine Western blot of lysates from control and MβCD-treated cells that have been stimulated with an optimal dose of antigen, DNP-BSA, for 0–30 min at 37°C. For the control cells, stimulated tyrosine phosphorylation is maximal after 2 min of stimulation with 1 μg/ml DNP-BSA, and then declines over time as previously reported . For the MβCD-treated cells, there is a substantial reduction in stimulated tyrosine phosphorylation in all bands detected, including the β and γ subunits of FcεRI that are readily observed under these conditions when the 4G10 mAb is used to detect phosphotyrosine. Fig. 1 b quantifies the time course of tyrosine phosphorylation of FcεRI β in this experiment. By this method of analysis, we found that maximal FcεRI β tyrosine phosphorylation is inhibited 95 ± 4% by pretreatment with MβCD in six separate experiments under these optimal conditions. The identity of FcεRI β and γ in the antiphosphotyrosine blots of whole cell lysates was confirmed by specific immunodepletion of these bands with anti-IgE . To further characterize the cells under conditions of MβCD treatment, we determined that untreated cells contain 6.7 ± 0.7 nmol free cholesterol/10 6 cells ( n = 5), and, after incubation with 10 mM MβCD at 37°C for 1 h, the amount of free cholesterol was determined to be 2.7 ± 1.0 nmol/10 6 cells ( n = 5). Thus, the fraction of free cholesterol remaining after treatment is 0.40 ± 0.15 as compared with untreated cells, consistent with previously described levels of cholesterol depletion for a variety of cell types under similar conditions . TLC analysis of cellular lipid extracts showed that there was no detectable difference in phospholipid composition before and after MβCD treatment, and this is also consistent with previous reports from other laboratories for other cell types . In particular, we found that the amounts of phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin present in the total lipid extracts from MβCD-treated cells were not obviously different from untreated cell lipids (data not shown). The amounts of two other unidentified lipid species (one polar lipid species and one neutral lipid species) observed with TLC also were unaffected by MβCD treatment. To further investigate the basis for the dramatic reduction in tyrosine phosphorylation of FcεRI β, we measured the amount of FcεRI before and after MβCD treatment using 125 I-IgE. Under our optimal conditions for inhibition of tyrosine phosphorylation (10 mM MβCD for 1 h at 37°C), we observed a 70 ± 6% ( n = 6) loss of receptor-bound IgE from the cells that could be recovered in the supernatants of the cell washes after MβCD treatment. Similarly, 64 ± 7% ( n = 2) of the ganglioside GD 1b was lost from the cells due to MβCD treatment as detected using 125 I-AA4 mAb. The loss of FcεRI is probably due to vesicle shedding caused by the MβCD treatment because a substantial fraction of 125 I-IgE in the post-wash supernatant after MβCD treatment can be pelleted by high speed centrifugation as membrane vesicles that are detectable by phase contrast microscopy (data not shown). In steady-state fluorescence measurements of FITC-IgE bound to MβCD-treated cells, 65 ± 10% ( n = 3) IgE– FcεRI loss was observed. Qualitatively consistent results were visualized with FITC-IgE and Cy3-AA4 in fluorescence microscopy of labeled cells, and a similar reduction in the cell surface expression of the GPI-anchored protein Thy-1 was also observed (data not shown). Despite these substantial reductions in FcεRI and the outer leaflet markers for DRMs in these cells after MβCD treatment, the plasma membrane expression of Lyn was only modestly reduced as assessed from fluorescence microscopy and Western blot analysis of sucrose gradient fractions (see below). Silver-stained polyacrylamide gels of RBL cell lysates showed no significant alterations in the amounts or composition of proteins detected in this manner (data not shown). These results suggest that the inhibition of antigen-stimulated tyrosine phosphorylation is due in part to a reduction in the amount of FcεRI available for phosphorylation in the MβCD-treated cells. To assess the magnitude of this effect, we determined the relationship between FcεRI tyrosine phosphorylation and the effective concentration of this receptor in the plasma membrane by comparing the amount of FcεRI β tyrosine phosphorylation for cells in which 30% of the receptors were occupied by anti-DNP IgE, and 70% were occupied by anti-1,5-dansyl IgE, which does not bind DNP ligands . Using the same conditions for stimulation and analysis as was used for the MβCD-treated cells, we determined that at the time point for maximal stimulation DNP-BSA caused 66% ( n = 2) less tyrosine phosphorylation of FcεRI β for the cells occupied with 30% anti-DNP IgE compared with those occupied with 100% anti-DNP IgE. Likewise, when FcεRI on cells were occupied by 50% anti-DNP IgE, DNP-BSA caused 41% ( n = 2) less tyrosine phosphorylation of FcεRI β than those occupied with 100% anti-DNP IgE. Assuming that this approximately proportional relationship between receptor number and β phosphorylation is valid for the MβCD-treated cells over this range of anti-DNP IgE densities, then the expected reduction in tyrosine phosphorylation of FcεRI β due to 70% loss of FcεRI should be ∼70%. Thus, the actual reduction in this value (95 ± 4%) represents an 83% inhibition of the stimulated FcεRI β tyrosine phosphorylation expected for the amount of cross-linked receptors present. Because of the nearly complete inhibition of stimulated FcεRI tyrosine phosphorylation caused by MβCD-mediated cholesterol depletion, we examined the effects of this treatment on cellular degranulation as measured by release of β-hexosaminidase. Fig. 2 summarizes the results from three separate experiments and shows that cholesterol depletion does not significantly inhibit degranulation stimulated by an optimal dose of antigen. Furthermore, cholesterol depletion actually enhances the amount of degranulation observed in response to stimulation by the Ca 2+ ionophore, A23187, without altering the amount of β-hexosaminidase released in unstimulated cells. These results indicate that reduction in cellular cholesterol enhances one or more of the downstream events that follow Ca 2+ elevation and lead to degranulation. They also demonstrate that cholesterol depletion under these conditions is not cytotoxic. As seen in Fig. 1 , tyrosine phosphorylation of FcεRI is usually inhibited more strongly than stimulated phosphorylation of some other substrates (e.g., the stimulated bands in the range of 70–100 kD) that are known to be dependent on activation of the tyrosine kinase Syk . These results indicate that relatively small amounts of stimulated tyrosine phosphorylation can result in substantial degranulation responses in the MβCD-treated cells. Consistent with this, stimulation of maximal degranulation requires effective cross-linking of only ∼10% of the IgE receptors on untreated RBL-2H3 cells . To determine the importance of cholesterol in DRM interactions, we examined the distributions of IgE–FcεRI and Lyn across sucrose gradients of cholesterol-depleted or untreated RBL cells. As previously demonstrated for untreated cells , monomeric IgE–FcεRI is found predominantly in the 40% sucrose region of the gradient where cytoplasmic and detergent-solubilized membrane proteins are characteristically observed, and a large percentage of cross-linked IgE–FcεRI is located in the low density region of the gradient where DRM vesicles are found . After MβCD treatment, monomeric IgE–FcεRI is located in the 40% sucrose region, similar to those in the untreated cells. However, cross-linked IgE–FcεRI no longer float to the DRM region, but rather appear in the 50–60% sucrose region where aggregates of IgE– FcεRI characteristically locate in the absence of interactions with DRMs . These results are representative of four experiments, and they show that ∼60% reduction in cholesterol almost completely prevents the association of cross-linked IgE–FcεRI with DRMs. As shown in Fig. 3 b (top), the distribution of Lyn in the gradient fractions of stimulated (+ sAv) and unstimulated (− sAv) cells that were not treated with MβCD is qualitatively similar to that observed by Field et al. , who used higher concentrations of TX-100. In the present experiments, it is notable that the p56 isoform of Lyn is selectively enriched in the low density, DRM region of the gradient (fractions 4–9), whereas the p53 isoform is located predominately in the 40% sucrose region (fractions 10–18). After cholesterol depletion, Lyn no longer localizes in the low density region of the gradient for both stimulated and unstimulated cells, whereas the distribution of p53 Lyn in the 40% sucrose region remains essentially unchanged from the untreated cells . In the cholesterol-depleted cells, it appears that p56 Lyn is relatively enriched in the gradient pellet (bottom fraction), both for the stimulated and the unstimulated cells. Overall, the total amount of Lyn detected in the gradients is only moderately reduced in the MβCD-treated cells compared with untreated control cells. The loss of both aggregated FcεRI and Lyn from the low density region of the gradient raises the question of whether DRMs are disrupted entirely in the cholesterol-depleted cells. To address this question, we investigated the distribution of other DRM markers in sucrose gradients after lysis of cholesterol-depleted cells. As shown in Fig. 3 c, AA4-labeled GD 1b from cholesterol-depleted cells is found almost completely in the low density region of the gradients, indicating that DRMs still exist in some form after ∼60% cholesterol depletion. The shift in GD 1b distribution to a slightly higher density after cholesterol depletion suggests an increase in the protein/lipid ratio, consistent with the substantial loss of cholesterol, a major lipid component of the DRM . These same trends are observed for both stimulated and unstimulated cells. Another DRM marker, the GPI-anchored protein Thy-1 , localizes similarly to GD 1b in the sucrose gradients before and after cholesterol depletion (data not shown). These results indicate that 60% cholesterol depletion can prevent the interactions of some proteins (FcεRI and Lyn) with DRMs without eliminating DRM structure. To evaluate how cholesterol depletion affects FcεRI on intact cells, we used confocal fluorescence microscopy to examine the redistributions of Lyn and GD 1b with cross-linked IgE–FcεRI. Representative images in Fig. 4 , a and b, show that both monomeric IgE–FcεRI (left panels) and Lyn (right panels) are uniformly distributed in the plasma membrane in the absence and presence of MβCD, respectively. When IgE–FcεRI is aggregated by antigen at 22°C for 20 min, small patches of these are formed, and these patches often cluster together on one side of the cell. As see in Fig. 4 c, the concentration of Lyn is enhanced in these regions of patched receptors in the absence of MβCD treatment. For MβCD-treated cells, IgE–FcεRI also redistributes into patches after aggregation by antigen , indicating that lateral mobility is not impeded by cholesterol depletion; however, Lyn does not redistribute with IgE–FcεRI under these conditions . As indicated in the first line of Table I , these differences are statistically significant when quantified by cross correlation analysis of multiple cells. Thus, cross-link– dependent interactions between FcεRI and Lyn on the cell surface are largely prevented by cholesterol depletion, consistent with the loss of interactions of these proteins with DRMs in the sucrose gradient analyses of lysed cells described above. Fig. 4 e shows that, as previously observed , cross-linking of IgE–FcεRI at the cell surface results in co-redistribution of the GD 1b ganglioside that is labeled by Cy3-AA4 mAb. For MβCD-treated cells, we find that co-redistribution of this outer leaflet DRM marker with cross-linked IgE–FcεRI is reduced compared with control cells but not completely disrupted as was the case for Lyn. Fig. 4 f shows an example of this variability, in which one cell exhibits partial co-redistribution of the labeled ganglioside, and the other shows a complete lack of co-redistribution with patched IgE–FcεRI. Under these conditions, ≤20% of the cells exhibited detectable co-redistribution of labeled ganglioside, whereas no detectable co-redistribution of Lyn with IgE–FcεRI patches was observed. From these results, it appears that the interaction between Lyn and FcεRI is more sensitive to cholesterol depletion than is the interaction between the ganglioside and FcεRI in the intact cells, although both are substantially prevented. The difference observed may be related to the greater sensitivity of the Lyn–DRM interactions than GD 1b –DRM interactions, as shown in Fig. 3 (see Discussion). As a further control, we compared the distribution of the transferrin receptor (CD71) to FITC-IgE–FcεRI cross-linked under the same conditions as above. Previous studies showed that this transmembrane protein does not associate with isolated DRMs , nor does it co-redistribute with other DRM-associated proteins when simultaneously but separately cross-linked on BHK and Jurkat T cells . As seen in Fig. 4 , g and h, TfRs do not co-redistribute with cross-linked IgE–FcεRI, and they remain evenly distributed around the periphery of the cell, often in tiny clusters that may reflect interactions with coated pits. Quantitative analysis of the cross correlation of TfR with cross-linked IgE–FcεRI show no appreciable colocalization between these molecules whether or not the cells have been depleted of cholesterol (see Table I ). These results support the significance of the co-redistribution of Lyn with cross-linked IgE–FcεRI described above, as well as its inhibition by cholesterol depletion with MβCD. To investigate the reversibility of cholesterol effects on the functional and structural interactions of FcεRI with Lyn, we restored cholesterol levels in MβCD-treated cells by incubating them with cholesterol–MβCD complexes. The efficiency of repletion is dependent upon the incubation period of the cells with the complex, the molar ratio of cholesterol to MβCD, and the final concentration of MβCD . To optimize repletion, we used several dilutions of cholesterol–MβCD complexes prepared as described in Materials and Methods. In our sequential depletion/repletion experiment, cells were initially left untreated (control samples) or incubated with MβCD to lower cholesterol levels as described above. During the second step, the cells were incubated for 2 h at 37°C at the indicated dilution of cholesterol/MβCD, 3 mM MβCD only, or buffer only. We found that (a) the cholesterol levels of cells depleted by exposure to MβCD in the first step did not change during the subsequent incubation in the absence of MβCD; (b) the presence of 3 mM of MβCD during the second step also did not cause additional cholesterol depletion, nor did it alter the distribution of IgE–FcεRI in the sucrose gradients; and (c) under optimal conditions of cholesterol repletion used (3–6 mM MβCD; 8:1, mol/mol MβCD/cholesterol), the cholesterol content of the repleted cells was 3.0–3.5-fold higher than that in the untreated control cells. Furthermore, TLC analyses of total lipid extracts indicated that cholesterol was the only lipid that changed detectably during the depletion/repletion treatments (data not shown). As shown in Fig. 5 , repletion of cholesterol in MβCD-treated cells results in partial restoration of antigen-stimulated tyrosine phosphorylation. In the experiment shown, maximal recovery of stimulated tyrosine phosphorylation of FcεRI β and other bands was achieved when 1:50 dilution of the preformed 8:1 MβCD/cholesterol complexes was used to give a final concentration of 6 mM MβCD during the repletion step . As seen in Fig. 5 , lane 6, cells that had been treated with MβCD alone during both the depletion and repletion steps have no detectable β phosphorylation, and only a very small amount of stimulated tyrosine phosphorylation is seen in the higher molecular weight bands. Similar results to these were obtained in three separate experiments. Under the conditions of cholesterol depletion/repletion, loss of IgE– FcεRI was determined to be 77 ± 4% ( n = 5), and, based upon the proportional relationship between receptor number and β phosphorylation (above), this leads us to expect that optimal restoration of FcεRI β phosphorylation should be ∼23% of the stimulated control in Fig. 5 , lane 2. The somewhat smaller restoration that is apparent suggests that other factors, such as the loss of other outer-leaflet DRM components during cholesterol depletion noted above, may reduce the maximum restoration achievable (see Discussion). Repletion of cholesterol also results in restoration of cross-link–dependent association of IgE–FcεRI with isolated DRMs. When lysates of cholesterol-repleted cells are analyzed on sucrose gradients, cross-linked IgE–FcεRI migrates to the low density sucrose region, whereas uncross-linked IgE–FcεRI is found in the 40% sucrose region, similar to the gradient distributions from control cells . Cross- linked IgE–FcεRI from cholesterol-repleted cells migrate at slightly lower densities in the sucrose gradients than this complex in the control cells, suggesting that the average density of DRMs in repleted cells is lower than in control cells, possibly due to a decrease in the protein/lipid ratio resulting from an increased cholesterol content. Furthermore, as shown in Fig. 3 b (bottom), p56 Lyn also migrates to the low density region of the gradient (fractions 1–6) after cholesterol repletion in cells with both cross-linked and uncross-linked FcεRI. These results, in parallel with the restoration of stimulated FcεRI tyrosine phosphorylation , provide strong evidence that cholesterol is important for functional coupling of FcεRI with Lyn and for their mutual association with DRMs. Our results demonstrate that cholesterol plays a critical role in the initial step of FcεRI signaling: antigen-stimulated tyrosine phosphorylation of this receptor by the Src family tyrosine kinase Lyn. In parallel with loss of this stimulated phosphorylation , reduction of cellular cholesterol by MβCD causes the loss of association of both Lyn and cross-linked FcεRI with DRMs isolated after cell lysis by TX-100 . Restoration of the cholesterol content of the depleted cells using preformed cholesterol–MβCD complexes restores the association of Lyn and cross-linked IgE–FcεRI with DRMs and also causes partial restoration of antigen-stimulated tyrosine phosphorylation in the cells . These results support the hypothesis that interactions of cross-linked IgE–FcεRI with DRMs are important for the initial coupling of FcεRI and Lyn that results in receptor phosphorylation. On the cell surface, the association of Lyn with aggregated IgE– FcεRI is lost as the result of cholesterol depletion , indicating that the interactions detected in isolated DRMs are relevant to those occurring in intact cells. Furthermore, these microscopy results argue against a direct interaction of cross-linked FcεRI with Lyn as the basis for association of receptors with DRMs, and they support the view that the L o structure of the plasma membrane is important for FcεRI–Lyn interactions. An initially surprising finding in our studies is the apparently selective loss of FcεRI and outer leaflet plasma membrane components of DRMs due to cholesterol depletion by MβCD. As indicated by Western blot analysis and fluorescence microscopy, there is a smaller loss of Lyn due to cholesterol depletion, and there is no detectable loss of other cellular proteins by silver stain analysis of whole cell lysates (data not shown). The mechanism by which cholesterol depletion causes this selective loss is not yet known, but it is interesting to speculate that vesicles containing these components may pinch off from the cells in a mechanism that depends on their local structural environment in the plasma membrane. In a recent study by Ilangumaran and Hoessli , a similar preferential release of DRM components by MβCD treatment was characterized in lymphocytes and endothelial cells, and evidence for release of these components in membrane vesicles was described. Our results suggest that FcεRI may preferentially associate with DRM components on intact cells even in the absence of receptor cross-linking. Consistent with this, Basciano et al. showed that pre-binding of AA4 mAb or its Fab fragment to the α-galactosyl GD 1b antigen on RBL-2H3 cells can effectively inhibit the subsequent binding of IgE to FcεRI . By varying the cell surface density of antigen-specific IgE in the range of 30–100%, we show that the loss of FcεRI due to cholesterol depletion cannot account for the nearly complete inhibition of FcεRI tyrosine phosphorylation that is observed in these cells. Furthermore, the partial restoration of antigen-stimulated tyrosine phosphorylation without an increase in FcεRI expression after cholesterol repletion strengthens the evidence that cellular cholesterol critically regulates the coupling of the remaining FcεRI and Lyn. An important observation by fluorescence microscopy is that cholesterol depletion does not prevent antigen-dependent aggregation of IgE–FcεRI on the cell surface, even though it prevents the co-redistribution of Lyn and inhibits stimulated tyrosine phosphorylation. We previously showed that the cholesterol-binding polyene antibiotic, filipin, prevents anti-IgE–mediated patching of IgE– FcεRI , indicating that it may prevent aggregation of the receptor necessary to initiate signaling. Unlike MβCD, which extracts cholesterol into a water-soluble complex that can be washed away, filipin forms complexes with cholesterol in the membrane that appear to restrict lateral diffusion of at least some membrane proteins, making this reagent less useful for studying the role of cholesterol in signaling by receptors that must aggregate in response to their ligands to be effective. The rapidity with which MβCD can reduce cell cholesterol by substantial amounts without significantly compromising cell integrity, as evidenced by our degranulation results, and the capacity to restore stimulated tyrosine phosphorylation by reintroduction of cholesterol via MβCD complexes, make this an extremely valuable tool for investigating the role of cholesterol in a wide variety of receptor systems. Recent studies used MβCD to investigate the role of cholesterol in signaling by other receptors. Pike and Miller showed that cholesterol depletion by MβCD inhibits EGF- and bradykinin-stimulated phosphatidylinositol turnover, which can be restored by cholesterol repletion with MβCD–cholesterol complexes. These receptors belong to the families of intrinsic tyrosine kinase receptors and G protein–coupled receptors, respectively, suggesting the potentially general importance of cholesterol and the L o structure it confers on the plasma membrane in mediating receptor signaling. Interestingly, cholesterol depletion by MβCD does not inhibit EGF-stimulated tyrosine phosphorylation of its receptor , which probably occurs via a transphosphorylation mechanism . Rather, cholesterol depletion appears to affect the compartmentalization of phosphatidylinositol 4,5-bisphosphate, the primary phospholipase C substrate in the plasma membrane, as revealed by its reduced localization with DRMs in sucrose gradients . In contrast, our results with IgE receptors indicate a role for cholesterol in the initial signaling step in which these receptors are phosphorylated by Lyn, and this finding may have general relevance for other receptors that function by interacting with Src family kinases. Indeed, Xavier et al. and Moran and Miceli showed that pretreatment of T cells with MβCD inhibits T cell receptor for antigen-mediated Ca 2+ mobilization and tyrosine phosphorylation, respectively, providing evidence for an important role for cholesterol in the function of this related multichain immune recognition receptor family member. Our degranulation results suggest that normal levels of cholesterol may negatively regulate downstream signaling or the exocytotic process in the RBL-2H3 cells. The enhancement of degranulation stimulated by Ca 2+ ionophore, as well as the lack of inhibition of antigen-stimulated degranulation, despite the dramatic inhibition of tyrosine phosphorylation by cholesterol depletion, are consistent with this explanation. In addition, preliminary experiments on the effects of cholesterol depletion on Ca 2+ mobilization by antigen indicate that this activity is inhibited less than is stimulated tyrosine phosphorylation of FcεRI, consistent with differential effects of cholesterol depletion on different signaling steps (Holowka, D., and E.D. Sheets, unpublished results). Membrane structural changes involved in exocytosis may also be affected. As described above, the proportionality of FcεRI tyrosine phosphorylation with the number of receptors cross-linked in the range of 30–100% does not hold for more downstream signaling events, as only a small fraction of FcεRI needs to be cross-linked to achieve maximal degranulation. In future experiments, it will be interesting to explore the effects of cholesterol depletion on specific downstream signaling pathways and exocytic membrane events through bypassing receptor-mediated signaling with alternate means of activation. The results described here are consistent with the hypothesis that cross-linked IgE–FcεRI interact with Lyn-containing DRM domains on the cell surface and that these structural interactions are integral to the initiation of signal transduction. Monomeric FcεRI probably interact dynamically with DRM components, and these transient complexes may exist in the plasma membrane of unstimulated cells as small clusters similar to those recently described for certain GPI-anchored proteins . In this hypothesis, cross-linking of IgE–FcεRI on the cell surface causes them to cluster with DRM components, thereby creating larger L o regions containing FcεRI and Lyn that are segregated from more fluid regions of the plasma membrane. It is likely that most transmembrane proteins are more readily accommodated by phospholipids in the more fluid liquid crystalline phase, and these would then segregate from the L o regions containing FcεRI and DRM components. For example, the tyrosine phosphatase CD45 is a transmembrane protein that was shown to be largely excluded from DRMs on T cells and has been recently shown to negatively regulate the Src family kinase Lck . Our microscopy results indicate that segregated DRM domains occupy a large percentage of the cell surface (20–50%) as detectable within the limits of optical resolution, and earlier studies indicated that DRM phospholipids represent a similarly large percentage of plasma membrane phospholipids . Thus, segregation of certain proteins from others may be more important for signaling promoted by DRM interactions of cross-linked FcεRI and Lyn than an increased localized concentration of these DRM-associated proteins within domains. In our model, cholesterol is an essential component for the L o phase, and its 60% reduction, as in the studies presented here, appears to most greatly affect the association of the transmembrane protein FcεRI and the inner leaflet component Lyn with DRMs . Outer leaflet DRM components are retained in the low-density, TX-100–insoluble membrane vesicles , and they still maintain a small but detectable association with cross-linked IgE–FcεRI on intact cells , which may reflect the continued presence of an L o environment in the outer leaflet under conditions of diminished cholesterol. It is possible that sphingomyelin and other sphingolipids enriched in the outer leaflet of the plasma membrane cause a preferential retention of cholesterol in this leaflet of the bilayer under conditions of limiting cholesterol, since this particular class of phospholipids may interact preferentially with cholesterol . Thus, cholesterol-dependent associations at the inner leaflet of the plasma membrane may be more sensitive to cholesterol depletion than are such interactions in the outer leaflet. As an alternative explanation for our results, it is possible that cholesterol serves as a critical boundary lipid for FcεRI that facilitates a direct interaction with Lyn. However, this explanation would not account for the association of these components with DRMs and the correlation between loss of this structural association and loss of functional coupling. The involvement of DRMs in functional coupling between FcεRI and Lyn as a means of promoting the proximity of these proteins while excluding transmembrane tyrosine phosphatases such as CD45 is an attractive hypothesis that warrants further examination. | Study | biomedical | en | 0.999997 |
10330414 | Human peripheral blood monocyte-derived macrophages were prepared as previously described ( 2 ). The human erythroleukemic cell line K562 transfected with cDNA encoding α v β 3 (Kα v β 3 ), α v β 3 in which the tyrosine residue at position 747 or 759 was mutated to phenylalanine (Kα v β 3 Y747F and Kα v β 3 Y759F, respectively) and Tacβ 3 (chimera of the extracellular and transmembrane domain of the IL2 receptor α-chain [Tac subunit] and the cytoplasmic tail of β 3 , KTacβ 3 ) were derived and maintained as described ( 2 , 3 ). In addition K562 cells were similarly transfected with cDNA encoding Tacβ 3 in which the serine residue at position 752 was replaced with proline (KTacβ 3 S-P), cysteine (KTacβ 3 S-C), alanine (KTacβ 3 S-A), or glutamic acid (KTacβ 3 S-E). Expression of all Tacβ 3 S-X chimeras was equivalent to Tacβ 3 ( 3 ) as determined by flow cytometry as described (2; see Table I ). For construction of Tacβ 3 S-X, the HindIII and XhoI fragment of pTacβ 3 encoding the CT of β 3 ( 3 ) was ligated into HindIII-XhoI–digested pBluescript (Stratagene), creating pBSKSPB3TAIL. This contruct was subjected to PCR using a 5′ T7 oligonucleotide (Stratagene) with the 3′ oligonucleotide (5′-CCCCCCTCGAGTTAAGTGCCCCGGTACGTGATATTGGTGAAGGT-XXX-CGTGGC-3′) where XXX is AGG for S 752 P, ACA for S 752 C, GCT for S 752 A, and TTC for S 752 E. The resulting products were digested with HindIII-XhoI and ligated into pTacβ 3 digested with HindIII-XhoI, creating pTacβ 3 S-P, pTacβ 3 S-C, pTacβ 3 S-A, and pTacβ 3 S-E, respectively. K562 cells also were transfected with full-length α v β 3 in which the serine at position 752 of β 3 was mutated to alanine as described for the mutation of β 3 tyrosine residues ( 4 ). In brief, nested PCR was performed on pBLY100 using the overlapping oligonucleotides 5′-GAGGCCACGCCTACCTTCACCAATATCACG-3′ and 5′-CTCCGGTGCGGATGGAAGTGGTTATAGTGC-3′ encoding the S-A mutation with oligonucleotides in the mutation cassette ( 4 ). After the nested PCR reaction, the wild-type β 3 CT was replaced with the S-A mutant CT by NdeI-NheI restriction. Transfection, selection, and fluorescent cell sorting for expression levels of α v β 3 S752A equivalent to wild-type α v β 3 was as described previously, resulting in Kα v β 3 S752A (Table I ). Modified cDNAs were verified by dideoxy nucleotide sequencing. Phagocytosis assays were performed as described ( 2 ) by flow cytometry using either FITC-FN– or FITC-mAb16 (anti-α 5 )–coated 3.0-μm beads. Data are presented as a Phagocytic Index, the number of beads internalized per 100 cells. Chemotaxis assays were performed in modified Boyden chambers (Neuroprobe) using 14.0-μM polycarbonate filters as described ( 19 ). Vitronectin (VN), fibronectin (FN), and BSA were added to basal chambers at 5 μg/ml and mAb at 10 μg/ml were added to apical chambers coincident with cells. Cells in Iscove's Modified Eagle's Medium (IMDM) adjusted to 1 mM Ca 2+ and 1 mM Mg 2+ with 0.5% human serum albumin and 2.0 mM Mn 2+ Cl were incubated for 4 h at 37°C in a humidified 5% CO 2 atmosphere for migration. Migration was quantitated by counting the number of cells per high power field on the underside of the filter after Giemsa staining. Adhesion assays were performed as described in FN-coated (10 μg/ml) microtiter wells ( 2 ). Data are presented as the percent of added cells adherent after 1 h at 37°C. Transfected K562 cells or monocyte-derived macrophages were stimulated as described in the text, washed once by centrifugation in ice-cold IMDM and suspended in ice-cold homgenization buffer containing Hepes (50 mM), EDTA (4 mM), EGTA (2 mM), sucrose (0.25 M), dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (0.2 mM), Na 3 VO 4 (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), and leupeptin (10 μg/ml), pH 7.5. Suspended cells were sonicated on ice and assayed for CamKII activity against the synthetic substrate autocamtide II (KKALRRQETVDAL) ( 21 ). An aliquot of cell extracts was used for protein determination by BCA. Parallel aliquots were assayed for CamKII activity in a 25-μl reaction mixture containing Hepes (50 mM), magnesium acetate (10 mM), Na 3 VO 4 (2.0 mM), NaF (5.0 mM), phenyl-arsine-oxide (10.0 mM), CaCl 2 (1 mM), calmodulin (0.1 μM; Sigma Chemical Co. ), autocamtide II (20 μM), and γ-[ 32 P]ATP (0.1 mM, 3,000 cpm/pmol). The reaction was initiated by ATP addition and terminated by addition of trichloroacetic acid to a final concentration of 10%. The reaction mixture was centrifuged through phosphocellulose separation units (Pierce) and washed as described ( 26 ). CamKII activation results from phosphorylation events that result in kinase activity which is no longer dependent upon exogenous calcium or calmodulin. CamKII activity in cellular extracts was measured by quantitating the incorporation of radioactive phosphate into a synthetic CamKII substrate (autocamtide-2) in the presence (calcium/calmodulin-independent + calcium/calmodulin-dependent activity) or absence (calcium/calmodulin-independent activity) of calcium and calmodulin. The activation of CamKII (autonomous activity) is expressed as a direct percentage of the total cellular CamKII activity ( 1 ) in which: \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*}\%\;CamKII\;activity=\frac{Autonomous\;activity\;}{Total\;activity}{\times}100\end{equation*}\end{document} where autonomous activity equals the CamKII activity without calcium or calmodulin and total activity equals CamKII activity with calcium or calmodulin. CamKII expression levels in transfected cell lines was assessed by immunoprecipitation as previously described, followed by Western blot analysis ( 1 ). Kα v β 3 , KTacβ 3 , and KTacβ 5 were infected with a replication defective adenovirus in which the E1 region was replaced with the CMV early promoter and the cDNA for a constitutively active CamKII (AdCMV.CKIID3) or β-galactosidase (AdCMV.gal) and viral stocks propagated and titered as described ( 1 ). Transfected K562 cells at 5 × 10 6 /ml in IMDM were infected with recombinant adenovirus at a multiplicity of infection of 100 for 1 h followed by the addition of normal growth medium to dilute cells to a concentration of 0.5 × 10 6 /ml. After 4–6 h, cells were harvested for analysis of CamKII activity or functional assay as described in Results. Viability of all infected cell types exceeded 85% at the initiation, and 70% at the conclusion of experimental time courses. FN was purified by gelatin affinity and VN by heparin affinity as previously described ( 2 , 3 ). Monoclonal antibodies 7G2 (anti–human β 3 ), W6/ 32 (anti–human HLA), IC12 (anti–human α v ), AP3 (anti–human β 3 ), P1F6 (anti–human β 5 ), 4E3 (anti-IL2Rα, gp55, TAC), and mAb16 (anti– human α 5 ) have been previously described and were used in excess at 5.0 μg/ml unless otherwise indicated ( 2 , 3 ). The kinase inhibitors H7 (50 nM), KN62 (2.5 μM), KN04 (5.0 μM), KT5926 (20 nM), and ML-9 (2 μM) were included in some assays where indicated and all were from LC Laboratories (Woburn, MA). All other reagents were from Sigma Chemical Co. unless otherwise indicated. Data are presented as the mean ± SEM from at least three replicates for all studies. Significance was determined by analysis of variance followed by Duncan's comparison testing. A minimum confidence interval of 95% was employed for all studies. We have previously described a phenomenon, termed integrin crosstalk, in which ligation of α v β 3 prevents α 5 β 1 -mediated phagocytosis in macrophages and in K562 cells expressing transfected α v β 3 . To determine if integrin crosstalk regulated α 5 β 1 functions other than phagocytosis, we evaluated the effects of α v β 3 ligation on the migration of K562 cells on the α 5 β 1 ligand FN. K562 cells did not migrate specifically to FN in IMDM containing 1 mM Ca 2+ and 1 mM Mg 2+ . However, addition of 2 mM Mn 2+ or the α 5 β 1 conformation-stabilizing mAbs 8A2 or A1A5 at 5.0 μg/ml greatly enhanced the FN-specific migration of these cells, consistent with a requirement for high affinity α 5 β 1 in migration (data not shown). As shown in Fig. 1 A, the migration of untransfected K562 to FN in the presence of 2 mM Mn 2+ was enhanced sixfold over migration to the nonspecific protein casein; this migration was completely inhibited by mAb to α 5 β 1 (data not shown). We also examined α v β 3 -mediated migration in K562 expressing this transfected integrin in addition to the endogenous α 5 β 1 . Kα v β 3 migrated in response to VN ; migration response to VN was inhibited by mAb to α v or β 3 (data not shown). However, migration of Kα v β 3 to FN was severely impaired compared with untransfected or mock transfected K562 . Migration of Kα v β 3 to FN was restored by the addition of the ser/thr kinase inhibitor H7 (50 nM), while addition of H7 had no effect on Kα v β 3 migration to VN (data not shown). Restored migration of Kα v β 3 to FN in the presence of H7 was completely inhibited by mAb to β 1 (data not shown). These results completely parallel the previously described α v β 3 -mediated crosstalk which inhibits α 5 β 1 -mediated phagocytosis ( 3 ) and support the hypothesis that the coligation of α v β 3 by FN regulates α 5 β 1 -mediated K562 cell migration to FN because this function, like phagocytosis, requires a high affinity form of α 5 β 1 . To demonstrate definitively that α v β 3 regulation of α 5 β 1 -mediated migration was another example of integrin crosstalk, we examined migration to FN in KTacβ 3 and KTacβ 5 , K562 cells expressing chimeric molecules comprised of the extracellular domain of the IL2 receptor and the cytoplasmic tail domain of the β 3 or β 5 integrin, respectively. Expression of Tacβ 3 , but not Tacβ 5 , leads to constitutive inhibition of α 5 β 1 -mediated phagocytosis in K562 cells . Expression of Tacβ 3 , but not Tacβ 5 or Tac lacking a cytoplasmic tail , completely inhibited α 5 β 1 -mediated migration to FN. The constitutive inhibition of migration to FN in KTacβ 3 was reversed by the addition of 50 nM H7 . These studies demonstrate that α 5 β 1 -mediated migration and α 5 β 1 -mediated phagocytosis are similarly regulated by α v β 3 or the isolated β 3 CT and that this regulation is dependent upon a ser/thr kinase regulated by H7. These data suggest that both α 5 β 1 -mediated migration and α 5 β 1 -mediated phagocytosis are regulated by α v β 3 -initiated crosstalk. We have previously demonstrated that expression of the isolated β 3 cytoplasmic tail is sufficient for initiation of α v β 3 crosstalk . To further delineate the required sequence elements of this unique regulatory pathway, we introduced point mutations in the β 3 cytoplasmic tail and analyzed their effects upon α v β 3 -initiated crosstalk to α 5 β 1 -mediated migration. In a spontaneously occurring Glanzmann's Thrombasthenia mutation, the serine residue at position 752 of the β 3 CT is mutated to proline ( 6 ). This mutation results in loss of platelet β 3 function and a severe bleeding disorder. In vitro study has shown that Ser752 of the β 3 CT is required for the conformational change associated with elevated affinity of β 3 for ligand ( 8 ). To test whether Ser752 also is required for integrin crosstalk, we expressed an α v β 3 receptor in K562 cells in which Ser752 of β 3 was mutated to Ala . While the ligation of wild-type α v β 3 blocked α 5 β 1 -mediated migration on FN , the S752A mutant migrated as well as the untransfected cells. In addition, the S752A mutant migrated as well as wild-type α v β 3 on VN , consistent with reports that this mutation does not affect ligand binding by β 3 integrins ( 8 ). This demonstrates that failure of the S752A mutant to initiate crosstalk did not result from an inability to recognize ligand. Recently, a tyrosine in the β 3 cytoplasmic tail, Tyr747, has been implicated in activation-dependent α v β 3 adhesion to VN ( 4 ). In contrast to the S752A mutation, Y747F had no effect on α v β 3 -initiated integrin crosstalk . Consistent with the previous report of a requirement for this tyrosine in firm adhesion, the Y747F mutation did abolish migration of Kα v β 3 Y747F to VN . Mutation of Tyr759 to Phe (Y759F) did not affect either crosstalk or the migration function of α v β 3 . These data demonstrate that the crosstalk signaling and adhesive functions of α v β 3 have distinct and independent sequence requirements in the β 3 cytoplasmic tail. To evaluate further the requirement for β 3 S752 in integrin crosstalk, additional mutations at that position were made in the consititutively inhibitory Tacβ 3 construct. Mutation of Ser752 to Glu, Pro, or Cys as well as Ala abolished the inhibitory activity of Tacβ 3 on α 5 β 1 -dependent migration and α 5 β 1 -dependent phagocytosis . Like the wild-type β 3 cytoplasmic tail, none of the mutants affected K562 binding to FN-coated surfaces, a function that does not require the high affinity state of α 5 β 1 . The addition of H7 reversed the Tacβ 3 inhibition of α 5 β 1 -mediated migration and phagocytosis . α v β 3 ligation inhibits the α 5 β 1 high affinity functions of phagocytosis and migration, without effect upon α 5 β 1 -mediated adhesion. Alterations in α 5 β 1 affinity can be regulated by calcineurin, a calcium/calmodulin-dependent phosphatase and CamKII (calcium/calmodulin-dependent protein kinase II; reference 1 ). Recently, inhibition of CamKII activity by α v β 3 ligation in smooth muscle cells was reported ( 1 ). Therefore, we evaluated α v β 3 regulation of CamKII as a potential mediator of α v β 3 -initiated crosstalk. CamKII activity was measured in human monocyte-derived macrophages in the presence and absence of an α 5 β 1 -specific phagocytosis target (mAb-16–coated latex beads) ( 3 ). Ligation of macrophage α 5 β 1 with mAb-16 beads enhanced CamKII activity twofold, while ligation with a control target (W6/32 beads) had no effect . Both basal and stimulated CamKII activities were decreased by the CamKII inhibitor KN62, but not the structurally related, but non-inhibitory KN04. Ligation of α v β 3 with soluble mAb 7G2 prevented the rise in CamKII activity induced by mAb-16 beads . No additional decrease in CamKII activity was detected when KN62 and 7G2 were combined. Thus, β 3 ligation prevented the α 5 β 1 -induced rise in CamKII activity. To explore further the hypothesis that CamKII mediates α v β 3 regulation of α 5 β 1 , we evaluated the regulation of CamKII in Kα v β 3 . Binding of mAb-16 beads to Kα v β 3 , and to vector-transfected K562 (data not shown), resulted in an increase in CamKII activity that was not seen when Kα v β 3 were incubated with W6/32 beads that bound to the cells equivalently. As in macrophages, the α 5 β 1 -mediated rise in CamKII activity was prevented by ligation of α v β 3 with soluble mAb 7G2 and by 7G2 Fab fragments or Arg-Gly-Asp peptide . As seen in macrophages, inhibition of the α 5 β 1 -induced increase in CamKII activity by α v β 3 ligation was blocked by KN62, but not KN04. Previously we have demonstrated that the cytoplasmic tail of β 3 is both necessary and sufficient for α v β 3 inhibitory crosstalk to α 5 β 1 . In the presence of mAb-16 beads, expression of Tacβ 3 , but not Tacβ 5 , prevented the α 5 β 1 -mediated rise in CamKII activity . These results indicate that α 5 β 1 and α v β 3 differentially regulate CamKII activity in macrophages and K562 cells. To determine if the failure of the Kα v β 3 S752A to initiate crosstalk was related to an inability to regulate CamKII, we evaluated CamKII activity after mAb-16 bead binding in K562 cells transfected with wild-type α v β 3 and the S752A and Y747F mutants. While ligation of α v β 3 with the β 3 -specific mAb 7G2 suppressed mAb-16 bead-induced activation of CamKII, mutation of Ser752 of β 3 prevented the suppression of CamKII activity seen upon α v β 3 ligation . However, mutation of Tyr747 or Tyr759 (data not shown) did not affect α v β 3 regulation of CamKII. Thus, Ser752 is required for both α v β 3 inhibitory crosstalk to α 5 β 1 and α v β 3 regulation of CamKII . To determine the effect of α v β 3 and mutant β 3 on expression of CamKII, immunoprecipitates of CamKII were analyzed by Western blot with CamKII-specific Ab. As shown in Fig. 4 B, cellular expression of CamKII (see arrow) was unchanged by the expression of α v β 3 and mutants in transfected K562 cells. Suppression of the α 5 β 1 -dependent increase in CamKII activity by α v β 3 ligation or by Tacβ 3 expression suggested that CamKII regulation could have a role in α v β 3 crosstalk to α 5 β 1 . To determine the role of CamKII in α v β 3 crosstalk to α 5 β 1 , Kα v β 3 cells were incubated with the α 5 β 1 phagocytosis target, mAb-16 beads, or control target, P1F6 (anti-α v β 5 ) beads. Phagocytosis was measured in the presence and absence of 7G2 to ligate α v β 3 , the CamKII inhibitor KN62, or control KN04. As reported previously, phagocytosis via α 5 β 1 was inhibited upon α v β 3 ligation with mAb 7G2 ( 2 , 3 ). α 5 β 1 phagocytosis also was inhibited by KN62 , but not KN04. Combining 7G2 and KN62 resulted in no further decrease in α 5 β 1 phagocytosis. Under all conditions, there was no significant internalization of P1F6 beads. To determine the dependence of α 5 β 1 phagocytosis on CamKII activation, we evaluated phagocytosis in untransfected K562 cells which express α 5 β 1 , but not α v β 3 . The absence of α v β 3 in these cells permitted the use of FN-coated beads as a phagocytosis target for α 5 β 1 rather than the more selective mAb-16 beads used when α v β 3 is present. K562 phagocytosis of FN-coated beads via α 5 β 1 was inhibited by the CamKII inhibitor KN62 , but not the control KN04. Thus, enhanced CamKII activity, initiated by α 5 β 1 binding of mAb-16 beads, appears to be required for α 5 β 1 phagocytosis. These data support the hypothesis that ligation of α 5 β 1 stimulates CamKII activity and that α v β 3 -mediated suppression of this activity is at least in part responsible for its inhibition of α 5 β 1 -mediated phagocytosis. To demonstrate that a similar mechanism was responsible for the inhibitory β 3 crosstalk to α 5 β 1 during migration, we evaluated the effects of the CamKII inhibitor KN62 on K562 cell migration in response to FN. KN62, but not the inactive analogue KN04, inhibited the FN-induced migration of mock transfected K562 cells and KTacβ 5 . The presence of KN62 did not further attenuate the minimal migration of Kα v β 3 or KTacβ 3 cells. To test the hypothesis that α v β 3 crosstalk to α 5 β 1 was a result of CamKII downregulation by β 3 , K562 cells were infected with an adenovirus-directing expression of a constitutively active form of CamKII ( 1 ). Expression of this construct in untransfected K562 cells resulted in an eightfold increase in CamKII activity over a control viral construct encoding β-galactosidase . Next, KTacβ 3 and KTacβ 5 infected with virus encoding either β-galactosidase or constitutively active CamKII were assayed for their ability to migrate in response to FN. Expression of the active kinase specifically overcame the constitutive inhibition of α 5 β 1 -mediated migration in KTacβ 3 , without any effect on migration in KTacβ 5 . Thus, expression of active CamKII overcame α v β 3 -mediated suppression of α 5 β 1 high affinity functions. Unfortunately, safety concerns precluded testing the effect of the constitutively active CamKII in the phagocytosis assay. Integrin crosstalk is an important mechanism for coordinating signals from multiple simultaneously ligated integrins on a single cell for a functional response to extracellular matrix. Although sometimes called “transdominant inhibition,” crosstalk may induce, as well as suppress functions of the target integrin, so we believe the more general term, preferable ( 9 ). Although the number of examples of integrin crosstalk has rapidly expanded in the past few years, little is known concerning the molecular mechanisms by which one integrin affects the function of another. K562 cells have proved a valuable model for examination of integrin crosstalk because these cells express a single integrin, α 5 β 1 , permitting a wide variety of genetic experiments exploring the basis of integrin crosstalk. In this system, we have previously shown that ligation of transfected α v β 3 inhibits the high affinity phagocytic function of α 5 β 1 without effect upon low affinity α 5 β 1 -mediated adhesion and that the β 3 cytoplasmic tail is both necessary and sufficient for this effect. We now have used this model to explore the biochemical mechanisms involved in crosstalk. Based on a previous report, we examined a potential role for CamKII in α v β 3 -mediated suppression of the high affinity functions of α 5 β 1 , and performed structure-function analysis of the β 3 cytoplasmic tail to further delineate the required structures for this unique signaling event. In this study, we show that either α v β 3 ligation or expression of the isolated β 3 cytoplasmic tail exerts an inhibitory effect upon α 5 β 1 -mediated migration as well as phagocytosis. Since both α 5 β 1 migration and phagocytosis are events that require the high affinity state of the integrin, and since the α v β 3 -mediated inhibition of α 5 β 1 is reversed by KN62 in both cases, these data suggest that a common signaling mechanism is responsible for these crosstalk events. Based on the data in this report, we propose the hypothesis that CamKII, a ser/thr kinase with multiple intracellular substrates, is an important regulator of α 5 β 1 function and a target of integrin crosstalk. First, ligation of α 5 β 1 by specific antibody- or ligand-coated beads enhances the activity of CamKII in both macrophages and K562 cells. Second, activation of CamKII by ligation of α 5 β 1 is required for both phagocytosis and migration. In contrast CamKII inhibitors do not affect adhesion which can be effected by low affinity α 5 β 1 . Thus, the requirement for CamKII activation appears to be specific for the high affinity functions of α 5 β 1 . Coligation of α v β 3 , or exposure of the isolated β 3 cytoplasmic tail, prevents α 5 β 1 -induced rise in CamKII activity. Since the β 3 integrin and the CamKII inhibitor have the same effect on α 5 β 1 function, the data suggest that suppression of the ability of α 5 β 1 to activate CamKII may be an important mechanism of integrin crosstalk. A role for CamKII suppression in integrin crosstalk is supported by the reversal of crosstalk inhibition of migration with constitutively active CamKII. Thus, our data support the hypothesis that α 5 β 1 -mediated CamKII activation is required for the high affinity functions of migration and phagocytosis and that α v β 3 -activated crosstalk suppresses these functions through inhibition of CamKII activation. Thus, α 5 β 1 and α v β 3 have opposing effects on CamKII activity. Neither the upstream events regulating CamKII nor its downstream effector are yet known. Tyrosine kinase inhibitors have no effect either on the high-affinity functions of α 5 β 1 or on suppression by α v β 3 , suggesting the possibility that the entire pathway is independent of the well-known effects of integrin ligation on several tyrosine kinases ( 2 , 3 ). Indeed, the independence of integrin crosstalk from the phosphorylation of Tyr747 further suggests that the signaling involved in the regulation of CamKII may be completely independent of these pathways. A recent report by Wu et al. ( 25 ) demonstrates that ligation of α 5 β 1 and α v β 3 have opposite effects on plasma membrane calcium channel activity. Since calcium is an important regulator of CamKII, this voltage gated calcium channel may be important in the differential regulation of CamKII by these two integrins. Based on our preliminary pharmacologic data, one likely effector for CamKII in α 5 β 1 high affinity function is myosin light chain kinase (MLCK). MLCK inhibitors KT5926 and ML9 both reverse α v β 3 inhibition of α 5 β 1 -mediated phagocytosis and migration without affecting inhibition of CamKII activation by α v β 3 ligation (Blystone, S.D., and E.J. Brown, unpublished data). MLCK phosphorylation by CamKII is known to inhibit MLCK activity, leading presumably to decreased myosin-induced cell traction ( 21 ). This integrin-mediated modulation of myosin function is consistent with the known role for myosin in phagocytosis and migration. Analysis of structural requirements in the β 3 cytoplasmic tail in α v β 3 -mediated crosstalk reveals that Ser752 of the β 3 cytoplasmic tail is required for inhibition of CamKII and for initiation of integrin crosstalk, while crosstalk is independent of either of the β 3 cytoplasmic tail tyrosines. The requirement for β 3 Ser752 in crosstalk is unexpected. The importance of Ser752 was suggested by a mutation to proline in a patient with Glanzmann's Thrombasthenia which abolished high affinity binding of fibrinogen by platelet α IIb β 3 ( 6 , 8 ). However, detailed analysis has shown that mutation of Ser752 to Ala does not affect ligand binding by α IIb β 3 ( 8 ). The failure of the Ser752 to Ala β 3 mutation to affect ligand binding is supported by our studies in K562 which demonstrate normal adhesion, normal migration , and normal generation of the ligand-induced binding site (LIBS) recognized by the antibody LIBS-1 in response to RGD peptide in this mutant (data not shown). In contrast, integrin crosstalk is entirely abolished by the S752A mutation, as it is by mutation to Pro (the original Glanzmann's mutation), to Glu (to mimic a potential phosphorylation), and to Cys (as a conservative mutation). Thus, it appears that Ser is absolutely required at this position. While this suggests the possibility of Ser phosphorylation in integrin crosstalk, we have been unable to demonstrate such phosphorylation so far. In contrast, Tyr747, which is absolutely required for stimulated adhesion and for α v β 3 -mediated migration in K562 cells, is not involved in integrin crosstalk. Thus, these two amino acids, closely spaced in the relatively short cytoplasmic domain of one chain of an integrin, mediate two entirely distinct signaling cascades. In a recent report, Bouvard et al. ( 5 ) showed that increased CamKII levels resulted in a decrease in the affinity of α 5 β 1 for FN. In this in vitro system, CamKII and the phosphatase calcineurin regulate α 5 β 1 affinity. Because the β 3 suppression of α 5 β 1 phagocytosis occurs subsequent to α 5 β 1 binding of ligand ( 3 ), it is possible that repeated cycling of α 5 β 1 affinity is required for phagocytosis and migration. Binding of ligand-coated beads to high affinity α 5 β 1 would activate CamKII, which would then decrease integrin affinity. This hypothesis predicts that integrin crosstalk from α v β 3 which blocks CamKII activation would prevent α 5 β 1 movement to the low affinity state. This is entirely consistent with reports of receptor activation rather than inactivation by integrin crosstalk ( 14 , 24 ) which measured ligand binding rather than functions that require affinity modulation. Finally, these data demonstrate that, while increased CamKII activity is required for α 5 β 1 -mediated phagocytosis and migration, α v β 3 can perform these same functions independent of any increase in CamKII. This is a startling example of the diversity of signaling and function among the integrins. It suggests that there may be fundamental differences within this family of closely related receptors in how they mediate even their most basic functions. While many studies have emphasized common features of integrin α- and β-chains in association with cytoskeleton, calreticulin, and signaling molecules, the differences between α v β 3 and α 5 β 1 in requirements for phagocytosis and migration suggest that there will be profound differences among integrins even as they perform similar functions. | Study | biomedical | en | 0.999995 |
10330415 | HECs were purified from human tonsils as described previously . Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics. Total RNA was isolated from HECs, HUVECs, and human tonsillar lymphocytes by lysis and extraction with RNAZol (Tel-Test, Inc.). Approximately 45 μg RNA was obtained from 7 × 10 6 cells. First strand cDNA was made from 2 μg total RNA primed with random hexamers using AMV reverse transcriptase (RT; Life Technologies, Inc.). PCR reactions were carried out in a total volume of 10 μl of 1× KlenTaq buffer ( Clontech ) containing 400 nM primers, 200 μM dNTPs, 0.2 μl KlenTaq Advantage DNA polymerase mix (KlenTaq polymerase; Clontech ), and 1.0 μl of twofold serially diluted cDNA as template. Cycling conditions were as follows: 1 min at 94°C; 30 cycles of 30 s at 92°C followed by 1 min 15 s at 68°C; one cycle of 10 min at 68°C. The following primers were used: for HEC-GlcNAc6ST, 5′-AAACTCAAGAAGGAGGACCAACCCTACTATGTGATGC-3′ and 5′-GTGGATTTGCTCAGGGACAGTCCAGCTAGACAGAAGAT-3′, which amplify a 456-bp fragment corresponding to nucleotides (nt) 884–1339 in Fig 3 a; for hypoxanthine phosphoribosyltransferase (HPRT), 5'-CCTGCTGGATTACATCAAAGCACTG-3′ and 5′-TCCAACACTTCGTGGGGTCCT-3′. The resulting amplified DNA was electrophoresed and visualized by ethidium bromide. A cDNA expression library was prepared from human HECs using the SMART cDNA technology ( Clontech ), which incorporates a long-distance PCR amplification step of first strand cDNA. 1 μg of total HEC RNA prepared as above was mixed with a modified oligo (dT) primer (1 μM) containing a NotI site and a universal site for 3′ priming of the PCR reaction and the SMART oligonucleotide (1 μM), which provides a universal site for 5′ priming of the PCR reaction. This mixture was heated at 72°C for 2 min to disrupt RNA secondary structure, and first strand cDNA was synthesized by Moloney murine leukemia virus (MMLV) RT in a total volume of 10 μl. 2 μl of this reaction mixture was subjected to 18 cycles of long-distance PCR primed by the universal primers using KlenTaq polymerase ( Clontech ). The PCR reaction mixture was incubated in the presence of proteinase K for 1 h at 45°C to destroy the KlenTaq polymerase activity, followed by heat inactivation at 90°C for 10 min. The double-stranded cDNA was polished by treatment with T4 DNA polymerase at 16°C for 30 min, followed by ligation to EcoRI/BstXI adaptors (Invitrogen) overnight at 16°C in the presence of T4 DNA ligase. Adaptor-ligated cDNAs were digested with NotI and then phosphorylated. The double-stranded cDNA was purified by size fractionation, ligated into EcoRI/ NotI-digested pCDNA1.1 (Invitrogen), and introduced into Escherichia coli MC1061/p3 (Invitrogen) by electroporation. The library contained 500,000 independent clones with an average insert size of 1.1 kb. HEC-GlcNAc6ST (human) was cloned from the HEC cDNA library by modification of a pool selection procedure . In brief, an aliquot (comprising 400,000 colony forming units) of the amplified bacterial stock of the HEC cDNA library was plated onto 200 LB plates and grown for ∼18 h at 37°C. Each pool of 2,000 colonies was harvested and grown for an additional 2 h at 37°C, and glycerol stocks were made. PCR analysis was performed, using the HEC-GlcNAc6ST–specific primers described above, to identify positive pools. 1 of the 9 positive pools was titered and plated onto 40 plates to yield 100 colonies per plate. These pools were expanded and analyzed as in the first round. A single positive subpool was titered and plated onto 20 plates of 10 colonies each. Analysis of individual colonies by PCR resulted in a single positive clone, which was sequenced . To clone the murine HEC-GlcNAc6ST, a 241-bp probe (nt 26–267) was amplified from the EST clone and used as probe for screening a bacterial artificial chromosome (BAC) library from the C57BL/6 mouse (Genome Systems, Inc.). From the single positive clone, DNA was purified and sequenced directly, using primers derived from EST AA522184 (forward: 5′-TGGGTCAGCATGCCTTCCATACTAAC-3′; reverse: 5′-TTCTAAGATTCCGGTTGCTTCTCCGTGGAC-3′) and then obtaining sequence upstream and downstream (582 nt). The resulting 1926 nt sequence was confirmed by resequencing in both directions. A human fetal brain library (λ ZAP; Stratagene) was the kind gift of Dr. Marc Tessier-Lavigne at the University of California, San Francisco. Approximately 10 6 plaques were screened with a probe consisting of a 730-bp HindIII/BamHI restriction fragment from IMAGE Consortium clone 40604 using standard techniques . 18 independent positive plaques were identified after the second round of hybridization. Cloned fragments were excised and sequenced as above. The probe for HEC-GlcNAc6ST consisted of a 496-bp fragment from LifeSeq clone no. 2620445, corresponding to nt 1021–1516 of the cloned cDNA . The probe was labeled with [α- 32 P]dATP ( Amersham Pharmacia Biotech ) by the random decamer priming method (Strip-EZ DNA kit; Ambion, Inc.). Multiple Tissue Northern blots ( Clontech ) containing poly(A) + RNA from various human tissues were hybridized at 60°C overnight in Rapid-Hyb ( Amersham Pharmacia Biotech ) and then washed twice at room temperature for 15 min in 2× SSC/0.1% SDS followed by two 15-min washes at 60°C in 0.1× SSC/0.1% SDS and autoradiography. For the Northern blot to establish expression in HECs, poly(A) + RNA was prepared from 1.5 × 10 7 HECs and 2.0 × 10 7 HUVECs, respectively. Isolation of the poly(A) + RNA with oligo(dT) latex beads was performed according to the manufacturer's protocol (Oligotex Direct kit; QIAGEN, Inc.). Approximately 2 μg poly(A) + RNA was loaded per lane. The RNA was separated by electrophoresis in a 1% denaturing agarose-formaldehyde gel and transferred to positively charged nylon filters (Hybond N+). The filters were hybridized and washed as for the Multiple Tissue blots. Blots were stripped using the Strip-EZ DNA kit (Ambion, Inc.), according to the manufacturer's protocol. Paraffin sections (5 μM) from C57BL/6 mice were deparaffinized, fixed in 4% paraformaldehyde, and treated with proteinase K. After washing in 0.5× SSC, the sections were covered with hybridization solution (50% formamide, 300 mM NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, 1× Denhardt's, 10% dextran sulfate, 10 mM DTT), prehybridized for 1–3 h at 55°C, and hybridized overnight with sense or antisense 35 S-labeled riboprobe transcribed from the IMAGE consortium clone 851801 which had been modified by digestion with SacI followed by religation. After hybridization, sections were washed at high stringency, dehydrated, dipped in photographic emulsion NTB2 ( Eastman Kodak Co. ), stored at 4°C for 2–8 wk, developed, and counterstained with hematoxylin and eosin. For generation of recombinant GlyCAM-1/IgG fusion protein, COS-7 cells were grown to 80% confluency in a T162 culture flask (Corning-Costar) and transfected with 8 μg of a plasmid encoding GlyCAM-1/IgG and 8 μg of plasmid encoding either HEC-GlcNAc6ST (pCDNA1.1), KSGal6ST (pCDNA3.1), or the empty vector (pCDNA1.1) using Lipofectamine (Life Technologies) in Opti-MEM (Life Technologies). The cDNA encoding the GlyCAM-1/IgG chimera was constructed by amplifying the entire coding sequence of murine GlyCAM-1 by PCR and cloning the resulting fragment into the pIG1 vector . Cells were grown for 12 h after transfection in DME containing 10% FBS, then cell layers were washed once with PBS and cultured for 72 h in serum-free medium (Endothelial SFM, 16 ml per flask; GIBCO BRL ) supplemented with Na 2 35 SO 4 (0.25 mCi/ml, 1,400 Ci/ mmol; ICN). Recombinant GlyCAM-1/IgG fusion protein was isolated from the conditioned medium (CM) by affinity chromatography on protein A–agarose . The protein was then transferred into 50 mM ammonium bicarbonate on a Centricon 30 concentrator (Amicon, Inc.). 1% of each sample was analyzed by SDS-PAGE, and the remaining samples were lyophilized for subsequent acid hydrolysis and positional analysis. The lyophilized recombinant GlyCAM-1/IgG samples were hydrolyzed in 0.2 M H 2 SO 4 , and the hydrolysates were prepared for analysis by high pH anion exchange chromatography (HPAEC) essentially as described previously , with the following modifications: (a) after 30 min hydrolysis in 0.2 M H 2 SO 4 at 96°C, and before the initial gel filtration step, excess sulfate was precipitated by addition of an equivalent amount of Ba(AcO) 2 ; (b) the second ion exchange column (DEAE-Sepharose) was equilibrated in 2 mM pyridine-acetate, pH 5.0, and eluted with a gradient of 2–500 mM pyridine-acetate. The monosulfated oligosaccharides eluted from this column between 50 and 250 mM pyridine acetate. The eluate was lyophilized, redissolved in 100 μl of water, and 30-μl samples of the resulting solution were subjected directly to HPAEC. HPAEC was performed using a Dionex DX 500 chromatographic analysis system and a Carbopac 1 column (Dionex Corp.) essentially as described , with column elution performed isocractically with 150 mM NaOH in 400 mM NaOAc at 1 ml/min. The authentic carbohydrate standards used in this analysis were obtained as described . CHO/FTVII/C2GnT cells were grown to 80% confluency in T75 flasks (6 × 10 6 cells per flask; Nalge Nunc) and then transfected with plasmids encoding C2GnT (1 μg, pCDNA1.1), fucosyltransferase VII (FTVII; 1 μg, pCDNA3.1), human CD34 (2 μg, pRK5), sulfotransferases (at the concentrations indicated for each experiment), and vector plasmid (pCDNA3.1) to achieve 8 μg total DNA per flask, using Lipofectamine. 48 h after transfection, the cells were harvested in 0.6 mM EDTA in PBS without calcium and magnesium, washed once in assay buffer (0.1% BSA in PBS), and resuspended at 4 × 10 6 /ml in assay buffer. 25 μl of this cell suspension was added to wells of 96-well plates containing 50 μl of L-selectin/IgM chimera, or assay buffer. Cells were incubated on ice for 30 min, washed twice in assay buffer, and resuspended in 50 μl assay buffer containing secondary staining reagents. Cells were incubated for another 30 min, washed twice in assay buffer, and then resuspended in 50 μl assay buffer containing tertiary staining reagents and/or directly conjugated primary mAbs. For the sialyl 6-sulfo Le x or overall sLe x determinations, cells were incubated with G72 mAb or HECA 452 ( PharMingen ), respectively, diluted in assay buffer, and then reacted with rabbit anti– mouse IgG-FITC ( Zymed Laboratories, Inc. ) (for G72) or mouse anti–rat IgM-FITC ( PharMingen ) (for HECA 452). The cells were incubated for 30 min, washed twice, and fixed in 1.5% paraformaldehyde in PBS, pH 7, for 20 min before transfer into 300 μl of assay buffer. Cells were analyzed on a FACSort™ ( Becton Dickinson ) flow cytometer using CELLQuest software ( Becton Dickinson ). To produce the L-selectin/IgM chimera , COS cells (10-cm plates) were transfected with L-selectin/IgM cDNA in pCDM8 (8 μg cDNA, 50 μl Lipofectamine per plate) and incubated in Opti-MEM for 10 d, at which time the CM was harvested and clarified by centrifugation. The CM was concentrated to half its original volume, titered, and used neat. Biotinylated goat anti– human IgM (μ) and streptavidin-FITC were purchased from Caltag Laboratories. The L-selectin mAb Mel-14 was purified by ammonium sulfate precipitation from hybridoma cell culture supernatant, followed by immunoaffinity purification with mouse L-selectin/IgG. The control for Mel-14 was a rat IgG2a myeloma ( Zymed Laboratories, Inc. ). For the two-color analysis, we used mouse anti-CD34 phycoerythrin (PE) (QBend10-PE; Coulter Corp.) and the isotype-matched control, mouse IgG1-PE (Caltag Laboratories). Our previous structural analysis of the carbohydrate chains of GlyCAM-1 indicated that the Gal-6-sulfate and GlcNAc-6-sulfate modifications accounted for essentially all of the sulfation of GlyCAM-1 . The previously cloned chicken chondroitin/keratan sulfate sulfotransferase (C6/KSST) has been shown by Habuchi et al. to catalyze sulfation at C-6 of galactose in Siaα2→ 3Galβ1→ 4GlcNAc, which is a core structure within the capping groups shown in Fig. 1 . We used the cDNA sequence encoding C6/KSST to probe the National Center for Biotechnology Information (NCBI) dbEST and LifeSeq (Incyte Pharmaceuticals, Inc.) human EST databases for related sequences. When we examined expression of transcripts corresponding to the ESTs by Northern analysis, one was of particular interest, because its tissue distribution was highly restricted . To determine whether this gene was expressed in HECs, a cell type in which L-selectin ligands are elaborated, we carried out a semiquantitative RT-PCR analysis on HEC cDNA with primers derived from this EST. HECs were purified, as described previously, from human tonsils using immunomagnetic separation with MECA-79 as the probe . The resulting cells had a purity of >99% and represented <1 per 1,000 of the stromal cells in tonsils. In parallel, we prepared cDNA from HUVECs and tonsillar lymphocytes. As shown in Fig. 2 A, the primers specific for the novel gene amplified a fragment of 456 bp from HEC cDNA, but failed to amplify this product from the HUVEC or lymphocyte cDNA. Since we had a relatively small number of HECs available, we used a PCR-based technique (SMART technology; Clontech, Inc. ) to produce cDNA from total RNA, from which we prepared a plasmid expression library. Using PCR amplification of the 456-bp fragment to identify positive pools, we isolated a full-length cDNA clone from the library by a pool selection procedure, as described in Materials and Methods. Initially, 200 pools of 2,000 colony-forming units/pool were screened. After two additional rounds of screening, a single positive clone was obtained. The cDNA corresponding to this clone contains a single open reading frame of 1,158 nucleotides. The cDNA is apparently full-length as indicated by the presence of an upstream stop codon and a Kozak sequence surrounding the first ATG, and a poly(A) tail at the 3′ end. This open reading frame predicts a type II transmembrane protein of 386 amino acids with 3 potential sites for N-linked glycosylation. The new cDNA sequence was used to probe the human databases for additional matching ESTs. One EST was identified in the LifeSeq database that mapped to the new gene at the 5′ end of its protein coding region. When the clone corresponding to this EST was fully sequenced, its sequence completely matched the original cDNA sequence within the coding region. There were two base changes in the 3′ untranslated region and divergence in the 5′ untranslated region. We present the sequence corresponding to LifeSeq clone no. 2617407 , since the library from which it was cloned was created without a PCR amplification step. The predicted amino acid sequence of this novel gene is 31% identical to C6/KSST , the chicken enzyme used in the original EST search. We have termed this novel gene HEC-specific GlcNAc-6-sulfotransferase (HEC-GlcNAc6ST) on the basis of the characterization described below. Using the human HEC-GlcNAc6ST cDNA sequence as a probe, we identified a highly related mouse sequence in the NCBI dbEST database . A 241-bp probe based on this EST was used for screening of a BAC library from mouse (C57BL/6). The probe was found to hybridize to a single BAC. The genomic clone within this BAC contained an open reading frame of 1167 bp , which is 77% identical at the nt level to the coding region of the human HEC-GlcNAc6ST. No introns were detected. The sequence predicts a protein of 388 amino acids , which is 73% identical to that of human HEC-GlcNAc6ST. We term this apparent mouse homologue mHEC-GlcNAc6ST. The expression of HEC-GlcNAc6ST was confirmed to be highly restricted by further analysis. On a conventional Northern blot (human), transcripts corresponding to this gene were absent from most tissues . Low levels of a 2.4-kb transcript were apparent in lymph node, liver (adult and fetal), and pancreas. A prominent band of the same size was detected at relatively high levels in mRNA from HECs, but was undetectable in HUVEC mRNA . An additional transcript at ∼6 kb was expressed in liver and pancreas, and trace levels appeared to be present in lymph node, HECs, and HUVECs . Using an antisense riboprobe based on the original mouse EST, we carried out in situ hybridization on mouse tissues. Strikingly, mHEC-GlcNAc6ST transcripts were detected only in the HEVs of lymph node . No hybridizing signal was found in other cell types of the lymph node, or in several other organs, including spleen, thymus, liver, skeletal muscle, pancreas, stomach, and kidney (data not shown). A weak signal was detected in gut intestinal epithelium (data not shown). The sense control did not yield signal in any tissue. Our screening of the human EST databases with the chicken C6/KSST led to the identification of multiple ESTs that mapped to another gene. We cloned a cDNA for this gene by conventional techniques from a fetal brain library as described in Materials and Methods. While this work was in progress, the same cDNA (with only two base changes in untranslated regions) was published independently by Fukuta et al. . The expressed protein was characterized as a keratan sulfate sulfotransferase with Gal-6-sulfotransferase activity (KSGal6ST) . The predicted protein is a type II transmembrane protein of 411 amino acids with 32% identity to HEC-GlcNAc6ST. Fukuta et al. reported the expression of KSGal6ST in many tissues, including lymph node. We confirmed these results; in addition, we detected its expression in HECs, albeit at apparently low levels, by performing RT-PCR with HEC cDNA and the HEC-cDNA library (data not shown). To test whether KSGal6ST and HEC-GlcNAc6ST (human) were capable of sulfating an L-selectin ligand, we transfected COS cells with a cDNA encoding a GlyCAM-1/IgG chimera and a cDNA encoding one or the other sulfotransferase. The transfected cells were cultured in the presence of [ 35 S]sulfate, and radiolabeled GlyCAM-1/IgG was purified from the CM on protein A–agarose. As shown in Fig. 5 A, there was substantial incorporation of radioactivity into GlyCAM-1/IgG when either KSGal6ST or HEC-GlcNAc6ST cDNA (but not empty vector) was included in the cotransfection. To establish the regiochemistry of sulfation on radiolabeled GlyCAM-1/IgG, samples resulting from the two sulfotransferase transfections were subjected to hydrolysis and Dionex HPLC analysis according to our previously established procedures . As shown in Fig. 5 B, transfection with KSGal6ST resulted in sulfated mono- and disaccharides that comigrated with [SO 3 → 6]Gal and [SO 3 → 6]Galβ1→ 4GlcNAc, confirming this enzyme as a Gal-6-sulfotransferase . In contrast, transfection with HEC-GlcNAc6ST resulted in products that corresponded to [SO 3 → 6]GlcNAc and Galβ1→ 4[SO 3 → 6]GlcNAc. Thus, this enzyme was established to be a novel GlcNAc-6-sulfotransferase. To determine whether the HEC-GlcNAc6ST could contribute to the generation of sialyl 6-sulfo Le x (Table I ), we took advantage of the G72 mAb, which recognizes this structure in a sulfate-dependent manner . We carried out transfection experiments with CHO cells, termed CHO/FTVII/C2GnT, which had been stably transfected with (a) fucosyltransferase VII (FTVII) to provide α1→ 3 Fuc, and (b) core 2 β1→ 6 N -acetylglucosaminyltransferase (C2GnT) to provide a core structure for O-linked glycans upon which extended chains with sLe x capping groups can be elaborated . The modified CHO cells were transiently transfected with HEC-GlcNAc6ST or KSGal6ST cDNA as a control. Transfection of the CHO cells with HEC-GlcNAc6ST cDNA resulted in significant staining by G72 as measured by flow cytometry , whereas transfection with KSGal6ST did not yield staining (not shown). Similar results were obtained with G152 , a related mAb (data not shown). Inclusion of a CD34 cDNA in the transfection did not significantly enhance staining by G72, indicating that an exogenous mucin scaffold was not required for the expression of this epitope. Since HEC-GlcNAc6ST and KSGal6ST are both expressed in HECs and are capable of sulfating GlyCAM-1/IgG in transfected cells, they are candidates to participate in the biosynthesis of L-selectin ligands. Our previous structural analysis indicated that Gal-6-sulfate and GlcNAc-6-sulfate are present equally in native GlyCAM-1 oligosaccharides. However, as reviewed above, the relative contribution of the two sulfate esters to L-selectin binding affinity has been a matter of considerable uncertainty. To test whether these enzymes can contribute to the generation of ligand activity, we carried out further flow cytometry experiments with an L-selectin/IgM chimera as a probe. The CHO/FTVII/C2GnT cells were transiently transfected with cDNAs for the two sulfotransferases (singly or in combination) plus a CD34 cDNA. As shown in Fig. 7 A, no binding of the chimera was detected in the absence of the sulfotransferases. Transfection with either sulfotransferase yielded positive staining, both in terms of the proportion of positive cells and their mean fluorescence intensity (MFI). Strikingly, the combination of KSGal6ST and HEC-GlcNAc6ST cDNAs produced strongly enhanced binding of L-selectin/IgM, which greatly exceeded the sum of the signals from the individual sulfotransferases . This apparent synergistic effect was evident over a considerable range of cDNA concentrations in the transfection mixtures (Tables II and III ). The binding of the L-selectin/IgM chimera induced by the sulfotransferases was confirmed to be specific as indicated by its calcium dependence (data not shown) and complete inhibition binding by a function-blocking anti–L-selectin mAb . The effects of the sulfotransferases on the L-selectin/ IgM chimera were not due to differences in transfection efficiency, as the CD34 expression levels were essentially constant, irrespective of the combinations of sulfotransferase cDNAs in the transfection mixtures (Table II ). Furthermore, to control for the possibility that the sulfotransferases might cause changes in essential glycosylation parameters that could affect L-selectin binding, we stained the transfected cells with the HECA 452 mAb. This antibody recognizes sLe x -related structures and is widely used as a reporter for glycosylation modifications (sialylation and fucosylation) pertinent to selectin ligands . Because this antibody reacts equally well with sulfated (at Gal-6, GlcNAc-6, or both) and nonsulfated sLe x structures , it was of particular utility for detecting the overall presence of sLe x on the cell surface of the transfectants. As shown in Table III , the expression of the HECA 452 epitope was not significantly altered (<25% variation) by transfection with the sulfotransferase cDNAs in any combination. The final issue we investigated was the contribution of CD34 to the expression of L-selectin ligand activity. In striking contrast to the results with G72, the binding of L-selectin/IgM was dependent on the presence of the CD34 protein scaffold, as there was a nearly complete loss of staining when the CD34 cDNA was omitted from the transfection . Interestingly, among the CD34-positive population, only those cells expressing the highest levels stained with the L-selectin/IgM chimera . Sulfation plays a central role in the interactions of L-selectin and P-selectin with their physiological ligands . PSGL-1, a leukocyte ligand for both P-selectin and L-selectin , possesses a cluster of sulfated tyrosine residues, which facilitate binding of both selectins . As reviewed above, L-selectin recognition of its cognate HEV ligands requires sulfation, as well as fucosylation and sialylation, for optimal binding. For the molecularly defined HEV ligands, sulfation is on sugar moieties rather than tyrosine residues . Our objective has been the molecular identification of the sulfotransferases that participate in the biosynthesis of these endothelial ligands. The specificities of the desired sulfotransferases were dictated by the analysis of the oligosaccharides of L-selectin–reactive GlyCAM-1, which revealed the presence of Gal-6-sulfate and GlcNAc-6-sulfate in the context of sLe x (sialyl 6′-sulfo Le x and sialyl 6-sulfo Le x , respectively). Similarly, the functional glycoforms of CD34 also contain Gal-6-sulfate and GlcNAc-6-sulfate in approximately equal representations (Hemmerich and Rosen, unpublished observations). Histochemical evidence with carbohydrate-directed mAbs shows that sialyl 6-sulfo Le x is prominently displayed on the HEVs of human peripheral lymph node , further implicating a GlcNAc-6-sulfotransferase activity in the biosynthesis of L-selectin ligands. Bowman et al. recently characterized such an activity from porcine lymph node, which was highly enriched in HECs. To identify the relevant sulfotransferases at the molecular level, we probed human EST databases for homologues of the chicken C6/KSST, a bona fide Gal-6-sulfotransferase. Here we report the cloning of two carbohydrate sulfotransferases. One is indeed a Gal-6-sulfotransferase, which was independently discovered by Fukuta et al. and named KSGal6ST. The second enzyme, which we have termed HEC-GlcNAc6ST, is a novel GlcNAc-6-sulfotransferase. These two sulfotransferases, together with the chicken C6/KSST , and the recently reported human chondroitin-6-sulfotransferase (C6ST, specificity for C-6 of GalNAc) and GlcNAc-6-sulfotransferase (GlcNAc6ST) constitute a family of highly conserved enzymes. Overall, amino acid identities within the family range from 27 to 42%. These enzymes are type II transmembrane proteins with short cytoplasmic tails, features that are typical of glycosyltransferases and carbohydrate sulfotransferases. Within this new family of carbohydrate sulfotransferases, there are three regions of amino acid sequence in which amino acid identity ranges from 45 to 54% and similarity from 80 to 90% . Regions one and two contain elements that conform to the recently described consensus binding motifs for the high energy sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate. These elements are found in all sulfotransferases characterized to date . In addition, regions one and three contain two stretches of sequence of 11 amino acids each (corresponding to amino acids 124–134 and 328–339, respectively, in the HEC-GlcNAc6ST sequence) that are highly conserved (>90% similarity). It is possible that these two elements contribute to a binding pocket that interacts with the 6-hydroxyl group of an appropriate oligosaccharide acceptor (Gal, GalNAc, or GlcNAc) to bring it into apposition with the donor phosphosulfate group. Our characterization of the KSGal6ST and HEC-GlcNAc6ST supports their involvement in the synthesis of L-selectin ligands. KSGal6ST has a wide tissue distribution which includes HECs. HEC-GlcNAc6ST shows a highly restricted, although not absolute, localization to HECs. Second, the sulfotransferases catalyze the two specific sulfation modifications on a recombinant L-selectin ligand that have been established to occur on native ligands. In the case of HEC-GlcNAc6ST, we have used the G72 mAb to show that transfection with the cDNA leads to 6-sulfation of sLe x on the cell surface of CHO/FTVII/ C2GnT cells. This finding establishes that recombinant HEC-GlcNAc6ST can be used to generate a highly specific sulfated structure that is present on lymph node HEVs . Using a flow cytometry assay based on the binding of an L-selectin/IgM chimera to transfected CHO/FTVII/C2GnT cells, we showed that each enzyme imparts L-selectin binding. Control experiments established that the effects of the sulfotransferases were not due to indirect effects on transfection efficiencies or to global changes in glycosylation parameters of the cells. In contrast to the generation of the G72 epitope, L-selectin/IgM binding required cotransfection of the cells with a CD34 cDNA. These results indicate that a specific protein scaffold, not present endogenously in CHO cells, is needed for optimal ligand activity, although at least one of the relevant sulfated carbohydrate epitopes (sialyl 6-sulfo Le x ) can be formed without the provision of such a scaffold. This finding is analogous to the situation with P-selectin, in which sLe x determinants can be formed by transfection of COS cells with an appropriate fucosyltransferase cDNA, but ligand activity requires the inclusion of PSGL-1 cDNA . As noted above, a shared feature of the molecularly defined HEV-associated ligands for L-selectin is the presence of a mucin region . This feature provides the potential for multivalent presentation of carbohydrate recognition determinants, which is thought to be important for enhancing the avidity of L-selectin interactions . Thus, the CD34 contribution to ligand activity seen in the present study may be primarily due to its mucin character. The ability of other mucins to perform this postulated scaffold function and their possible distinct usage of the different sulfotransferases are questions for future investigation. While each sulfotransferase was capable of conferring L-selectin binding onto CD34 in the CHO/FTVII/C2GnT cells, the greatest effect was clearly produced by the combined action of the two sulfotransferases. The level of binding achieved in the cells transfected with both sulfotransferases could not be attained with either sulfotransferase alone, over a wide range of cDNA concentrations. These findings argue that optimal binding to L-selectin requires both the Gal-6-sulfate and GlcNAc-6-sulfate moieties. It is possible that this synergy arises through dual recognition of separate monosulfated chains, for example one chain capped by sialyl 6-sulfo Le x and the other by sialyl 6′-sulfo Le x . This mechanism would fit a model of selectin binding proposed by Varki , in which a specific cluster of adjacent O-linked chains comprises the full recognition determinant. Alternatively, individual chains containing both modifications may underlie the synergistic effect. In this regard, it should be noted that most of the sulfated chains within GlyCAM-1 contain two or more sulfates, although the structure of these chains has not been solved . It is not yet clear whether sialyl 6′,6-disulfo Le x (Table I ) exists as a capping group on these multisulfated chains or whether there are extended chains containing sulfates on internal Gal or GlcNAc residues. Additional analysis of natural ligands will be required to resolve these important structural questions. L-selectin normally functions in tethering and rolling of lymphocytes on HEVs under conditions of blood flow . Future experiments must determine the impact of the two sulfation modifications on the kinetic parameters of L-selectin binding to ligands, as these parameters are the key determinants of the dynamic interactions of lymphocytes with HEVs . We are currently addressing these issues using a parallel plate flow chamber to recapitulate physiological flow dynamics within blood vessels . On the basis of its activities and highly restricted expression pattern, HEC-GlcNAc6ST is likely to be specifically involved in the biosynthesis of the sialyl 6-sulfo Le x epitope and related structures within L-selectin ligands. Ultimately, it may be necessary to inactivate the gene in mice by gene knockout to define the biological roles of this enzyme. Experiments of this type were required to demonstrate the critical role for FTVII in the generation of selectin ligands, including the HEV ligands for L-selectin . We cannot rule out the possible involvement of another recently cloned GlcNAc-6-sulfotransferase identified in mice and humans in ligand biosynthesis. As revealed by in situ hybridization, transcripts encoding the latter enzyme were present in HECs of mouse lymph nodes, although the overall tissue expression pattern was extremely broad in both species, suggesting other functions as well. In this regard, it should be noted that 6-sulfated GlcNAc residues are present in the GAG chains of keratan sulfate and a variety of glycoproteins, including the N-linked oligosaccharides of thyroglobulin , gp120 of HIV , and porcine zona pellucida . Furthermore, the GlcNAc-6-sulfate motif in the context of the core-2 structure has been identified in respiratory mucins from cystic fibrosis patients , and a GlcNAc-6-sulfotransferase activity, which can act on mucins, has been detected in human respiratory mucosa . With respect to the sialyl 6′-sulfo Le x epitope, our data on the acceptor specificity and expression pattern of KSGal6ST are consistent with a possible role for this enzyme in the generation of the epitope within HECs. However, this enzyme , like the GlcNAc-6-sulfotransferase described by Uchimura and colleagues , is broadly distributed, being expressed in all of the major organs. In analogy with HEC-GlcNAc6ST described in the present study, one might predict the existence of an HEC-specific, or more highly restricted, Gal-6-sulfotransferase that sulfates L-selectin ligands in vivo. L-selectin–dependent leukocyte trafficking into lymphoid organs and inflammatory sites is likely to be subject to complex regulation, which may be affected at the level of L-selectin ligand biosynthesis through the availability of protein scaffolds or by posttranslational modification. Given the dramatic sulfate dependency of the interaction between L-selectin and its ligands, and the high [ 35 S]sulfate incorporation seen in HEVs and HEV-like vessels , it is tempting to speculate that regulation of the expression or activity of one or both of the relevant sulfotransferases represents critical control points for this process. It is further plausible that the differential expression of the sulfotransferases may contribute to the apparent variation in the posttranslational modifications of L-selectin ligands in different lymphoid organs . The expression of KSGal6ST and HEC-GlcNAc6ST, as well as other candidate sulfotransferases, should also be examined in HEV-like vessels that are induced at sites of chronic inflammation . Endothelial ligands for L-selectin are also inducible on flat-walled vessels in vivo, although the biochemical nature of these ligands is poorly understood . In vitro models of endothelial activation suggest that sulfation is required for the activity of these ligands . Endothelial sulfotransferases that are implicated in inflammatory leukocyte trafficking would clearly assume importance as potential targets for antiinflammatory therapeutics. | Study | biomedical | en | 0.999996 |
10330416 | Fasciculin 2 was obtained from Sigma Chemical Co. and α-bungarotoxin (BTX) was purchased from Biotoxins, Inc. Tetramethylrhodamine-conjugated fasciculin 2 and Oregon green 488-conjugated BTX were prepared using the corresponding FluorReporter Protein Labeling Kit (Molecular Probes) following the manufacturer's recommended procedures and the unreacted dyes removed using BioGel P-2 spin columns (Bio Rad Laboratories). Heparin was obtained from Sigma Chemical Co. The globular and collagenic-tailed AChE forms were isolated from tissue-cultured quail myotubes by detergent/high salt extraction followed by preparative sucrose gradient sedimentation as previously described ( 55 ). The pooled fractions from several gradients containing the G4 tetramers and the G2 dimers (globular forms) or A12 collagenic-tailed AChE forms were purified on an immunoaffinity column containing mAb 1A2 anti-avian AChE antibody ( 54 ) covalently attached to Sepharose CL-4B at a concentration of 1 mg/ml. The bound AChE was eluted with 0.1 M triethylamine, pH 11, in 1 M NaCl and 0.5% Triton X-100, and neutralized with Tris-HCl to pH 7. The AChE concentration was estimated using radiometric assay ( 33 ). The myotomal region of Xenopus laevis embryos was excised and dissociated to make muscle cultures according to a previously published method ( 47 ). To induce the formation of clusters of AChR or AChE, muscle cells were cocultured with spinal cord neurons to establish the NMJ, or treated with 10-μm polystyrene latex beads coated with recombinant heparin-binding growth-associated molecule (HB-GAM) which also induces the formation of postsynaptic specializations ( 49 ). To visualize endogenous AChE, muscle cultures were labeled with R-fasciculin 2 at a concentration of 150 nM for 0.5–1 h and then examined by fluorescence microscopy either in the living state or after fixation with 95% ethanol at −20°C. For most experiments, the cultures were double-labeled with OG-BTX (at 150 nM) to visualize AChRs. To study the binding of exogenous AChE to the surface of Xenopus muscle cells, cultures were incubated with either purified quail A12 collagenic-tailed AChE (at 0.1–0.2 ng/ml) or G2/G4 globular forms of AChE (at 0.5 ng/ml) for 1 h. The transplanted quail AChE was then examined by labeling Xenopus cultures with mAb 1A2 followed by fluorescently conjugated secondary antibody. HSPG at the cell surface was detected with mAb HepSS-1, an anti-heparan sulfate monoclonal antibody (Seikagaku Corp.). A polyclonal anti-perlecan antibody ( 27 ), a generous gift of Dr. J. Hassell (Shriners Hospitals for Children, Tampa, FL), was used to label this HSPG in Xenopus cells. The localization of DG was studied with a monoclonal anti–β-DG antibody (Novacastra Laboratories). The HSPG and perlecan labeling was done on live cultures, but the DG labeling was carried out after cell fixation and permeabilization since the antibody recognizes an intracellular epitope of the transmembrane protein. To label Xenopus myotomal muscle fibers in vivo, the tail of the larva was skinned, fixed, and incubated with the antibody. Alternatively, the fibers within the tail musculature were first dissociated with collagenase and then immunolabeled. Purified anti-avian perlecan antibody mAb 33 ( 6 ) was prepared from ascites fluid obtained by using the original hybridoma cell line (a generous gift from Dr. Douglas M. Fambrough, Johns Hopkins University, Baltimore, MD). The purified antibody was absorbed onto protein A–Sepharose CL-4B beads ( Sigma Chemical Co. ) to saturation, and the beads washed extensively to remove unbound antibody. The beads were then used to capture perlecan secreted by quail myotube cultures. Myotube-conditioned medium from 5-d-old cultures was centrifuged 30 min at 12,000 g and 500-μl aliquots of supernatant were incubated with 10 μl of the mAb 33 beads overnight. After extensive washing with PBS containing 5 mM EDTA and 0.5% BSA, the beads were treated with 1 μM diisopropylfluorophosphate to irreversibly inhibit any trace amounts of endogenous AChE already bound to perlecan. After washing with PBS, EDTA, and BSA, aliquots of immobilized perlecan beads were diluted in microfuge tubes with 500 μl PBS, 0.5 M NaCl, 1 mM EDTA, 0.5% BSA, and 0.5% Triton X-100 containing either 0.1–0.2 ng purified A12- or G4/G2-AChE or buffer alone. After 1-h incubation at 5°C, the salt concentration was decreased to 0.3 M NaCl and incubation continued overnight. The next day the beads were washed three times with PBS, 5 mM EDTA, and 0.5% BSA and assayed for AChE activity using a radiometric assay as previously described ( 54 ). AChE-perlecan binding was also assayed by the surface plasmon resonance biosensor technology ( 42 ). A BIAcore X instrument (BIAcore. Inc.) was used in this study. Perlecan purified from Englebreth-Holm-Swarm tumor ( 37 ), kindly provided by Dr. J. Hassell (Shriners Hospitals for Children, Tampa, FL), was conjugated to BIAcore sensor chip CM5 with carboxylated dextran surface. The chip surface was first treated with a mixture of N -hydroxysuccinimide (NHS, 50 mM) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC, 200 mM) in Hepes-buffered saline (HBS, 10 mM Hepes, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4). After washing with HBS, the perlecan sample, diluted to a concentration of 30 ng/ml with 100 mM Na-acetate buffer at pH 5, was injected into the flow cell to effect coupling and this process was terminated with 1 M ethanolamine (pH 8.5). Samples were then injected into the flow cell to study their binding to perlecan. Globular G2/G4 AChE was diluted to a concentration of 10 ng/ml and asymmetric A12 AChE was diluted to 2.3 ng/ml with HBS immediately before injection and 20 μl of each sample was injected into the flow cell. The change in resonance units (RU), indicative of the binding and dissociation, was continuously recorded with a computer. The data were analyzed with BIAevaluation software supplied by the manufacturer and plotted with SigmaPlot software (SPSS, Inc.). Fasciculin 2, a 61–amino acid snake α-neurotoxin isolated from the venom of African mambas, binds specifically and tightly to the catalytic subunit of AChE ( 9 , 34 ). The crystal structure of the AChE-fasciculin 2 complex has been determined and the molecular interactions between the toxin and the enzyme are well characterized ( 9 , 19 , 26 ). To study the distribution and fate of AChE, we synthesized a tetramethylrhodamine conjugate of fasciculin 2 to label the enzyme on muscle cells. When the cutaneous pectoris muscle of the frog ( Rana pipiens ) was double-labeled with R-fasciculin 2 and OG-BTX, a precise colocalization pattern of AChE and AChR was observed. Fig. 1 , A and B show a single muscle fiber imaged in whole mount. Like AChRs, AChE labeling also exhibits a banding pattern typical of the frog NMJ. Detailed analyses of the R-fasciculin 2 labeling pattern by conventional and confocal microscopy have shown a more precise registration of AChE and AChR at the NMJ than the pattern hitherto observed with histochemical or immunocytochemical methods (Rotundo, R.L., and H.B. Peng, manuscript in preparation). Since Xenopus myotomal muscle cells were used to study AChE clustering in this study, we also examined AChE distribution in the tail musculature of the larva with R-fasciculin 2. As shown in Fig. 1 (C and D), R-fasciculin 2 labeling was colocalized with sites of AChR clustering at NMJs revealed by OG-BTX labeling. In addition to the NMJ, R-fasciculin 2 also labeled myotendinous junctions (MTJs) which are located adjacent to the NMJs at the intersomitic area . The MTJ labeling showed up as a series of streaks oriented longitudinally at the ends of the myotome. These streaky structures correspond to membrane invaginations where myofibrils insert into the sarcolemma ( 13 , 46 ). This MTJ localization of AChE revealed by R-fasciculin 2 is consistent with previous histochemical results ( 16 , 41 ). To study the clustering of AChE, cultured Xenopus myotomal muscle cells were labeled with R-fasciculin 2. As shown in Fig. 2 (A and B), R-fasciculin 2 labeling was observed at spontaneously formed AChR clusters on these muscle cells, and the pattern of AChE labeling closely resembled that of the AChR. Virtually all AChR hot spots observed were associated with AChE. Previous studies have shown that AChR clusters at the NMJ are derived, at least in part, from the preexisting pool of cell surface receptors by lateral migration ( 2 ). To determine whether preexisting AChE molecules could also contribute to synaptic clusters, muscle cells were prelabeled with R-fasciculin 2 and then cocultured with spinal cord neurons or treated with beads coated with HB-GAM, which mimic the nerve in inducing postsynaptic specializations ( 49 ). As shown in Fig. 2 , preexisting AChE labeled with R-fasciculin 2 became concentrated at bead-induced AChR clusters (C and D) and at the developing NMJ (F–H) marked by OG-BTX. These results were based on observations made on a total of six separate nerve–muscle and bead–muscle cocultures encompassing >100 cell pairs each. Consistently, AChE clustering as evidenced by R-fasciculin 2 labeling was detected at a much smaller percentage of nerve- or bead-induced AChR clusters in 1-d cocultures (∼20%) than in 2-d cocultures (>70%). This suggests that AChE clustering lags behind AChR clustering by ∼1 d. The fluorescence intensity of R-fasciculin 2 labeling at the cluster was generally several-fold less than that of the corresponding OG-BTX labeling when each was normalized with respect to the background. This suggests that the site density of AChE at the cluster is significantly less than that of the receptor. Since AChE is a secreted molecule, it is possible that most of the molecules are secreted into the medium and not captured by the cell surface acceptors (to be described below) in tissue culture. Alternatively, it is possible that not all AChE molecules are available for fasciculin 2 labeling, and that the toxin's affinity for AChE decreases with time. More importantly, however, these data show that, like AChRs, preexisting cell surface AChE molecules can be recruited to form clusters and suggest that they are capable of undergoing lateral migration at the cell surface to become aggregated at sites of synaptic stimulation. To identify the molecules on the cell surface that can serve as acceptors for AChE during the process of synaptic localization, we transplanted exogenous AChE to cultured Xenopus muscle cells by adapting a method previously used to study the localization of this enzyme at the NMJ in vivo ( 56 ). The collagenic-tailed A12 AChE form or the globular AChE forms consisting of dimers (G2) and tetramers (G4) of catalytic subunits were purified from cultured quail myotubes and applied to cultured Xenopus muscle cells at a concentration of 0.1–0.4 ng/ml. Their binding to the cell surface was then detected with mAb 1A2 which specifically labels the catalytic subunit of quail AChE but not the Xenopus enzyme ( 54 , 56 ). The transplanted collagenic-tailed A12 form of AChE bound to the surface of Xenopus muscle cells in a clustered manner , where they often colocalized with AChR hot spots , or sometimes more diffusely . Although the pattern of the AChE clusters bore similarity to that of AChRs, they were not congruent with each other. The AChE cluster generally occupied a larger area than the AChR cluster. As it is known that the A12 AChE binds to heparin via its collagen-like tail to be sequestered on the muscle cell surface and at the NMJ ( 30 , 53 , 56 ), we examined whether transplanted A12 AChE was associated with HSPG on the cell surface. Pretreatment of muscle cells with heparin at a concentration of 20 μg/ml abolished the binding of A12 AChE to the cell surface (data not shown). Perlecan is an abundant HSPG on the surface of skeletal muscle and appears to play an important role in muscle differentiation ( 31 , 45 , 50 , 51 ). We thus examined its relationship to AChE. Although this molecule is generally considered as an ECM-bound HSPG, our recent study has shown that a pool of perlecan is actually associated with the cell membrane by interacting with α-DG in skeletal muscle cells ( 50 ). In fact, the bulk of perlecan on cultured Xenopus muscle cells is cell membrane-associated since these cells secret relatively small amount of matrix molecules and do not form organized basal lamina under culture conditions of this study. To determine whether AChE codistributed with perlecan, anti-avian AChE mAb 1A2 and a polyclonal anti-perlecan antibody ( 27 ) were used to double-label A12 AChE-treated muscle cells . Both clustered and diffusely distributed quail A12 AChE molecules were precisely colocalized with perlecan. In the clustered state , the patterns of AChE and perlecan labeling coincided nearly perfectly while even in the diffuse state , the puncta of labeling by these two antibodies also showed precise registration. These data, based on six transplantation experiments, thus strongly suggest that A12 AChE binds to perlecan. In contrast to A12 AChE, globular G2/G4 AChE forms, which do not have the collagen-like tail and do not interact with HSPGs, showed little binding to the cell surface when applied at similar concentrations . Moreover, the binding of these globular AChE forms bore no relationship to the pattern of perlecan labeling on the cell surface . To determine whether AChE could bind directly to perlecan, purified A12 or globular G2/G4 AChE forms were incubated with perlecan immobilized on mAb 33-conjugated Sepharose beads. Beads incubated with bovine serum albumin, rather than perlecan-containing conditioned medium, were used as an additional control for nonspecific binding in these experiments. As shown in Fig. 6 A, the purified A12 AChE was bound to isolated perlecan. That the binding occurred in 0.3 M NaCl, which was necessary to prevent aggregation of the A12 AChE, suggests that it is with high affinity. In contrast, the globular G2/G4 oligomeric forms showed binding levels similar to the albumin control. It could be argued that A12 AChE binds indirectly to the antibody-perlecan beads via other molecules in the conditioned medium. Thus, a second set of binding studies using surface-plasmon resonance (BIAcore) technology was conducted. In these experiments, purified perlecan was covalently linked to the sensor chip. Samples of G2/ G4 and A12 AChE were then injected into the experimental chamber (flow cell) sequentially to study their interactions with the bound perlecan. In this assay, the binding is measured optically and expressed as the net increase in resonance units (RU) at the termination of sample injection. As shown in Fig. 6 B, buffer injection did not result in any increase in RU. Using this as a baseline, G2/G4 (at 0.2 ng in 20-μl sample volume) did not show any significant binding to perlecan. When A12 was injected (at 0.05 ng in 20-μl sample volume), a 270-fold change in RU over the G2/G4 value was seen, indicative of its binding to perlecan. This binding was not reversed by the running buffer that had 150 mM NaCl. Significant dissociation of A12 from the perlecan surface was only observed after a buffer with 1–2 M NaCl was injected into the flow cell as shown in Fig. 6 B. These biochemical measurements thus show that A12 AChE binds directly to perlecan through its collagen-like tail and provide further support to our conclusion that cell surface perlecan serves as an acceptor for the synaptic A12 form of AChE. They are consistent with the cellular binding studies described above . The fact that these two molecules are only dissociated under high salt condition is consistent with the notion that the binding is mediated by the heparan-sulfate glycosaminoglycan chains on perlecan. To determine whether the transplanted AChE can be recruited to form clusters at the NMJ, we innervated muscle cells pretreated with quail A12 AChE. As shown in Fig. 4 (C and D), this exogenous AChE was also clustered at developing NMJs revealed by R-BTX labeling. Again, the organization of the AChE cluster was not precisely aligned with that of the AChR clusters with the AChE cluster generally occupying a larger area than the AChR cluster. In contrast to the endogenous AChE, the NMJ localization of the transplanted AChE was clearly detectable in 1-d nerve–muscle cocultures (see Discussion). Thus, similar to endogenous AChE, the transplanted AChE can also be clustered by lateral migration at the cell surface. Accompanying the clustering of AChE, HSPGs also became concentrated at the NMJ ( 3 , 6 , 50 ). To determine whether preexisting perlecan molecules can undergo lateral migration and become clustered, cultured muscle cells were labeled with anti-perlecan antibody and then treated with HB-GAM–coated beads followed by fluorescently conjugated secondary antibodies. As shown in Fig. 7 (A and B), preexistent perlecan was indeed clustered in response to the bead stimulation. Together with AChE binding to perlecan described above, these results suggest that the AChE-perlecan complex at the cell surface can be recruited to form synaptic clusters. As neither AChE nor perlecan is a transmembrane protein, an integral membrane linker for this complex would be necessary to effect its lateral migration at the cell surface. The core protein of perlecan contains three globular domains at its COOH terminus that are also shared by laminin A-chain and agrin ( 32 ). Recently, we have shown that, like laminin and agrin, perlecan can bind directly to α-DG which is the extracellular component of the transmembrane DG glycoprotein complex and that these two proteins cocluster in response to synaptic stimuli such as spinal neurons and HB-GAM–coated beads ( 50 ). An example of this coclustering induced by beads is shown in Fig. 7 (C and D). Here both perlecan and DG become clustered at the bead–muscle contact and there is a high degree of registration between clusters of these two molecules. To determine whether AChE was also colocalized with DG, we double-labeled bead-treated muscle cells and nerve–muscle cocultures with R-fasciculin 2 and anti–β-DG antibody. As shown in Fig. 8 , AChE and DG also appeared to be precisely coclustered at sites of nerve–muscle contacts (A and B) and at bead-induced clusters (C and D). To correlate these data obtained from cultured muscle cells with AChE clustering in vivo, we examined the relationship between AChE, perlecan, and DG in whole mounts of myotomal muscle. As shown in Fig. 9 (A and B), AChE and perlecan are colocalized at both the NMJ and the MTJ. Double-labeling myotomal muscle with anti–β-DG antibody and fluorescent BTX revealed that DG is also clustered at the MTJ in addition to its being present at the postsynaptic membrane . Finally, double-labeling myotomal muscle with β-DG antibody and R-fasciculin 2 showed colocalization of DG and AChE at intersomitic junctions . At higher magnification, the colocalization of these two molecules at ends of the muscle fiber, where NMJs are located, and along MTJ invaginations became more evident . These data thus suggest that the perlecan-DG complex can serve as an acceptor for the collagenic-tailed form of AChE and allow it to assume an association with the muscle membrane. This membrane association may provide the structural basis for the observed clustering of preexistent membrane-bound AChE to the synaptic site. In this study, we used fluorescently conjugated fasciculin 2 to follow the clustering of AChE during NMJ formation. This probe has many of the same advantages as fluorescent α-bungarotoxin which has offered an extremely powerful tool for visualizing AChRs ( 1 ). Its compact size allows it to penetrate more deeply into tissues than antibody reagents. Its specificity and 1:1 stoichiometry in binding to the catalytic site of AChE ( 9 ) enables high-resolution imaging of AChE distribution on muscle cells. Iodinated fasciculin 2 has recently been used to quantify AChE site density in mammalian muscle by EM-autoradiography ( 4 ). To our knowledge, the current work is the first to utilize fluorescent fasciculin for optical imaging of AChE. We have shown that a preexistent, membrane-bound collagenic-tailed form of AChE, either endogenously deposited or experimentally transplanted can be recruited to form clusters at the postsynaptic membrane. This suggests that AChE is capable of lateral migration on the cell surface and becomes immobilized at sites of synaptic differentiation. Thus, AChE clustering seems to bear similarity to the much studied AChR clustering process which can be explained by a diffusion-mediated trapping mechanism of AChRs preexistent on the cell surface ( 20 , 35 ). Since AChE in muscle is not a membrane-bound protein, this necessitates one or more acceptor molecules to link it to the cell surface. The immunocytochemical colocalization and binding studies presented here show that perlecan is one such acceptor for A12 AChE. Perlecan is one of at least two modular HSPGs on the surface of skeletal muscle cells, the other being muscle agrin ( 32 ). Although the bulk of perlecan is associated with ECM, our recent studies have shown that a pool of this molecule is associated with the cell surface in association with α-DG ( 50 ). This work suggests that this cell surface pool is, at least in part, also involved in AChE anchorage. As AChE is secreted, this membrane-bound perlecan could readily capture and sequester it on the cell surface. This scheme is consistent with previous findings that the heparin-binding property of A12 AChE is essential for its localization on the cell surface ( 53 ). The heparin-binding motifs within the collagen-like tail of this AChE form have recently been elucidated ( 18 , 36 ). The interaction between this motif and the heparan-sulfate side chain on the perlecan molecule seems to be the basis for the localization of this enzyme to the cell surface ( 53 , 56 ). Despite our focus on perlecan, muscle agrin, which also becomes concentrated at sites of synaptic differentiation ( 38 ), may also be an acceptor for A12 AChE. Our preliminary studies based on immunological labeling have shown that agrin can coexist with perlecan at the same loci on the cell membrane, albeit at a lower concentration. Previous studies have shown that α-DG can interact with ECM-bound molecules that have G-domain motifs, such as laminin, agrin, and perlecan ( 10 – 12 , 22 , 28 , 50 , 58 ). On the other hand, β-DG, the transmembrane component of the DG complex, interacts with dystrophin or utrophin via its cytoplasmic tail ( 28 ). Thus, the DG complex is capable of mediating the transmembrane linkage between ECM and the cytoskeleton. This study suggests a new role for DG as a component of the machinery for cell surface sequestration and clustering of AChE and other ECM components in skeletal muscle during synaptogenesis as depicted in a model in Fig. 10 . The transmembrane nature of the DG complex may allow it to undergo lateral movement within the plane of the membrane in the nonclustered state and thus to move its bound HSPG-AChE complex in a manner similar to the AChRs . The lateral mobility of DG is also supported by the recent demonstration that exogenously applied laminin induces clustering of DG ( 15 ). The mechanism for DG clustering at the synaptic site is unknown, although it may also be a cytoskeleton-mediated process as is the case of AChR clustering ( 8 ). For AChR clustering, there is compelling evidence to suggest that synaptogenic stimuli induce the formation of a postsynaptic cytoskeletal scaffold which serves to immobilize freely diffusing receptors. Lateral diffusion of AChRs, with a diffusion coefficient estimated to be on the order of 10 −10 –10 −9 cm 2 /s, can account for the rate of AChR clustering induced by synaptogenic signal ( 35 , 48 ). The cytoskeletal specialization underlying the postsynaptic membrane, including F-actin, utrophin/dystrophin, and the transmembrane sarcoglycan complex ( 25 , 39 ) may be involved in the clustering and/or stabilization of the DG-HSPG-AChE complex . The coclustering of AChE and AChR suggests that their clustering processes may share common determinants. Subtle differences, however, must also exist as shown by the lack of congruency between these two types of clusters with the AChE cluster being larger than the AChR cluster. In the same manner, it has been shown that clusters of dystroglycan and HB-GAM, which binds to HSPG, are also more extensive in area than AChR clusters despite their colocalization ( 14 , 49 ). Recent studies have shown that postsynaptic specializations, including both AChR and AChE clusters, still form in utrophin and dystrophin double-knockout mice despite their severe muscular dystrophy ( 17 , 23 ). However, AChE appears to be “more scattered” according to one study ( 17 ). This could be due to two reasons. First, the postjunctional folds are greatly reduced in these animals. Because synaptic AChE is associated with the basal lamina both on the top of and along the folds in normal muscle ( 44 ), their reduction should result in a significant deficit of AChE. Furthermore, the level of DG at the NMJ also seems to be reduced, although the exact level seems to vary between the two studies ( 17 , 23 ). This could also reduce the total amount of AChE at the NMJ according our model. Nevertheless, the DG remaining at the NMJ may offer the structural basis of AChE localization even in these animals. Our results have shown that the clustering of endogenous AChE lags behind that of AChRs by about a day in tissue culture. However, transplanted AChE becomes detectable at newly established NMJs on the same day as the AChRs. This suggests that the machinery for AChE clustering is activated at the time of synaptic stimulation, but other factors may limit the rate of AChE accumulation in cultured muscle cells. A previous study has shown that DG clustering is detectable on cultured Xenopus muscle cells within the first 1–2 h after nerve contact ( 14 ). Thus, the delay in AChE accumulation seems to be quantitative in nature due to the site density of this molecule on the muscle surface. As described above, the fluorescence intensity of R-fasciculin 2 labeling of AChE at clusters is generally much lower than that of AChR labeling. In fact, the site density of AChE at frog NMJ is estimated to be ∼600 sites/μm 2 as compared with 10,000 sites/μm 2 for AChRs ( 5 , 21 ). A factor that could limit the amount of membrane-bound AChE is the number of sites available for its binding on the heparan-sulfate chains of HSPGs. In addition to AChE, these chains also offer a substrate for other heparin-binding molecules such as several ECM components ( 59 ) and growth factors ( 49 ) for their localization at the cell surface. The low site density may thus account for the length of time necessary for its accumulation to detectable level at synaptic sites. Transplantation of exogenous AChE greatly increases its site density on the cell surface as shown by the visualization of diffusely distributed molecules . The clustering of AChE is an example of the specialization of synaptic basement membrane during NMJ formation. A recent study using DG-null embryoid bodies has shown that this protein plays a central role in the assembly of the basement membrane ( 29 ). In addition to AChE, the scheme presented in this work based on molecular interaction with HSPG-DG complex may also find application in the formation of other specializations involving heparin-binding synaptic molecules such as neuregulin and peptide growth factors ( 40 , 49 ). | Other | biomedical | en | 0.999998 |
10330431 | Soluble, biotinylated class I monomers, comprising the murine D b molecule, human β2-microglobulin, and lymphocytic choriomeningitis virus (LCMV) 1 peptide p33, were generated as described previously ( 24 , 25 ). Tetrameric complexes were subsequently generated by stepwise addition of PE-labeled streptavidin ( Sigma ) to the biotinylated monomers at a 1:4 molar ratio. Single-cell suspensions were prepared from spleens and incubated with PE-conjugated tetramers at 37°C for 15 min. Allophycocyanin-conjugated anti-CD8 antibodies were added subsequently on ice for 30 min. Cells were washed and analyzed on a FACScan™ ( Becton Dickinson ) or sorted using a MoFlo cell sorter (Cytomation). Naive T cells were harvested from unprimed transgenic mice expressing a TCR specific for peptide p33 in association with H-2D b ( 26 ). Experiments with two founder lines revealed similar results. To generate effector and memory T cells, spleen cells (10 6 ) were adoptively transferred into normal C57BL/6 mice, which were immunized with 200 PFU of LCMV WE sprain 2 h later. Effector cells were harvested on days 7–9 after infection. Memory cells were harvested on days 30–90 after infection ( 27 ). Spleen cells were stained for the expression of CD8 (FITC; PharMingen ), Vβ8 (PE; PharMingen ), and Vα2 (biotin, followed by streptavidin coupled to Tricolor; PharMingen ). To analyze expression of Lck, cells were stained for surface expression of CD8 (PE) and Vα2 (biotin, followed by streptavidin coupled to Tricolor). Cells were fixed and subsequently permeabilized, then stained with a polyclonal rabbit anti-Lck serum ( PharMingen ) followed by FITC-conjugated anti–rabbit antibodies. Alternatively, normal C57BL/6 mice were infected with LCMV (200 PFU), and spleen cells were isolated 8 or 45 d later and analyzed by tetramer staining. EL-4 target cells were pulsed with peptide p33 or KAVVNIATM at a concentration of 10 −7 or 10 −5 M, respectively, for 90 min at 37°C in the presence of [ 51 Cr]-sodium chromate in IMDM medium supplemented with 10% FCS. Cells were washed three times, and 10 4 cells were transferred to a well of a round-bottomed 96-well plate. Ex vivo–isolated spleen cell suspensions were adjusted to the same number of TCR transgenic cells, serially diluted, and mixed with peptide-pulsed target cells. Plates were centrifuged and incubated for various time-spans at 37°C. At the end of the assays, 70 μl of supernatant was counted in a γ-counter. Spontaneous release was determined by adding medium instead of effector cells. Total release was determined by adding 2 M HCl instead of effector cells. Percent specific release was calculated as follows: 100 × (experimental release − spontaneous release)/(total release − spontaneous release). Naive, effector, and memory T cells were generated as described above. For proliferation, numbers of specific T cells were determined by flow cytometry after staining for Vα2 (FITC; PharMingen ) and Vβ8 (PE; PharMingen ) and adjusted to 10 6 specific T cells/ml. Cells were stimulated with peptide p33 (KAVYNFATM)– or A4Y (KAVANFATM)–pulsed macrophages. Proliferation was assessed 30 h later by pulsing cultures with [ 3 H]thymidine for 6 h. Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4)–Ig fusion molecules and anti-CD8 antibodies 53.6.72 ( 28 ) were added at a final concentration of 10 or 3 μg/ml, respectively. Intracellular Ca 2+ ([Ca 2+ ] i ) was measured as described ( 29 ) using p33-pulsed thioglycollate-stimulated macrophages as APCs. Induction of TCR and CD8 downregulation was assessed as described ( 29 ). T cells were lysed for 30 min at 4°C in 1% Brij96 buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA) in the presence of protease and phosphatase inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM Pefabloc-SC, 50 mM NaF, 10 mM Na 4 P 2 O 7 , and 1 mM NaVO 4 ). CD8 immunoprecipitations and Lck immunoblottings were performed as described ( 39 ) using the following antibodies: 53.6.72 (rat IgG, anti-CD8), and 3A5 (IgG2b, anti-Lck; Santa Cruz Biotechnology ). For total Lck immunoblotting, the cell populations were stained for Vα2 and Vβ8 and sorted by FACS Vantage™ ( Becton Dickinson ). CD8 + T cells were purified by depleting CD4 + and class II + cells using magnetic beads ( Dynal ) according to the manufacturer's instructions. T cells were stained for 30 min at 4°C with FITC-conjugated anti-CD8α antibody ( PharMingen ), washed, and then incubated at 37°C for 1 h. Cells were then settled onto poly- l -lysine–coated slides and analyzed by confocal microscopy. For Lck staining, T cells were settled onto poly- l -lysine– coated slides and stained for Vα2 (biotin, followed by streptavidin coupled to Texas red; PharMingen ). The cells were then fixed in 3.7% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After saturation with 1% BSA in PBS, the cells were incubated with the anti-Lck antibody (3A5; Santa Cruz Biotechnology ), washed, and incubated with an FITC-conjugated anti– mouse IgG2b (Southern Biotechnology Associates). The cells were analyzed by confocal microscopy. Double stainings were performed with FITC-conjugated anti-CD8 antibodies and anti-Lck antibody (3A5; Santa Cruz Biotechnology ) followed by Texas red–conjugated anti–mouse IgG2b (Southern Biotechnology Associates). Nuclei were stained using propidium iodide (PI). A proper comparison of memory T cells with naive T cells at the single cell level has proven difficult for technical reasons. To be able to compare naive T cells with effector and memory T cells expressing a single defined TCR, we used an adoptive transfer system ( 19 , 27 ). Spleen cells (10 6 cells) derived from transgenic mice expressing a class I–restricted TCR (Vα2Vβ8) specific for the LCMV-derived peptide p33 were adoptively transferred into normal C57BL/6 mice, which were infected 2 h later with live virus. The adoptively transferred naive T cells expanded dramatically within 1 wk, differentiated to effector cells, and exhibited high ex vivo cytolytic activity . The number of specific CD8 + T cells subsequently declined, but memory T cells remained at a high frequency in the host for at least 90 d . Compared with the effector cells isolated on day 8 after infection, the memory T cells exhibited at least 100-fold reduced ex vivo cytolytic activity . Moreover, although 95% of the effector cells expressed low levels of CD62L, 70% of the memory T cells had reverted back to a CD62L high status . Similar results were obtained when CD44 expression was analyzed (not shown). Thus, the transferred naive T cells were rapidly activated in vivo after infection, then proliferated and differentiated to lytic effector cells. After elimination of the virus at day 6 to day 8 after immunization (not shown), a proportion of the cells survived as memory cells which exhibited a more quiescent status than the effector cells. The requirements for the activation of ex vivo–isolated naive, effector, and memory cells were analyzed in vitro. We found that the three populations required the same density of the virus-derived peptide p33 to proliferate, when B7 + thioglycollate-elicited macrophages were used as APCs, confirming earlier results (19; not shown). However, when B7-mediated costimulation was inhibited by a CTLA-4–Ig fusion molecule, proliferation of naive cells was strongly reduced, whereas almost no effect could be observed for effector and memory cells . Similar results were obtained with different peptide concentrations (not shown). As shown previously for naive T cells ( 30 ), proliferation of effector and memory T cells was affected more severely by blocking CD28 during stimulation with the weak agonist A4Y . These results indicate that, when stimulated by professional APCs, naive T cells do not require higher doses of antigen or higher levels of TCR engagement than effector and memory cells. However, in contrast to effector and memory T cells, naive T cells require the triggering of CD28 in order to be maximally activated. CD28 reduces the threshold for T cell activation by amplifying the signals delivered by the TCR, thus inducing a stronger phosphorylation of many proteins involved in T cell activation ( 29 , 31 , 32 ). To investigate whether effector T cells exhibit enhanced TCR-mediated signaling, induction of Ca 2+ flux was assessed in naive and effector cells after stimulation with p33-pulsed APCs. Elevation of free intracellular Ca 2+ was significantly enhanced in effector cells compared with naive T cells, indicating that the TCR in effector T cells is more efficiently coupled to the signaling machinery than in naive T cells . Similar results were obtained using various peptide concentrations (10 −6 to 10 −9 M; not shown), whereas intracellular free Ca 2+ was low in both naive and effector cells in the absence of specific peptide . Together with the observation that effector and memory T cells have a reduced requirement for the signal amplification by CD28, these results suggest that another mechanism might be able to substitute for CD28 in effector and memory cells. Such a mechanism may be of physiological relevance, since it would allow for the activation of memory T cells in peripheral tissues, where B7 expression is scarce. Interestingly, we observed that, in contrast to effector and memory cells, proliferation of naive T cells could be inhibited by anti-CD8 antibodies . This effect cannot be accounted for by a decreased stability of the TCR–peptide/MHC interaction, since (a) the affinity of the complex was the same in all the cells; (b) proliferation of naive, effector, and memory cells was inhibited by anti-CD8 antibodies when the APCs were pulsed with a weak agonist ; and (c) anti-CD8 antibodies did not inhibit T cell–APC conjugate formation (not shown). The contribution of coreceptors to T cell activation is not only due to their capacity to bind to the same MHC molecule engaged by the TCR and thus stabilize the TCR–MHC/peptide interaction ( 33 – 36 ), but also to their association with the tyrosine kinase Lck ( 37 ), which is involved in many early signaling events ( 38 ). It has been shown that in specific human T cell–APC conjugates, the coreceptors are recruited to triggered TCRs and are downregulated and degraded with identical kinetics and with fixed stoichiometry ( 39 ). It was possible that the distinct susceptibilities of naive versus effector and memory T cells to anti-CD8 blocking might be due to the distinct association of CD8 with the triggered TCR. Therefore, the association and the comodulation of TCR and CD8 in naive, effector, and memory cells were assessed in the next experiment. Fig. 4 A shows the expression levels of TCR and CD8 on naive, effector, and memory T cells before stimulation. Similar levels of TCR were expressed on the surface of all cell types, whereas CD8 expression was reduced by ∼50% on ex vivo–isolated effector cells. Specifically triggered TCRs have been shown to be internalized rapidly ( 40 ). In addition, CD8 has been shown to be associated and co-downmodulated with the TCR in T cell clones ( 39 ). To test whether the distinct susceptibility to anti-CD8 antibodies of naive versus effector and memory T cells might reflect a distinct association and co-downmodulation of CD8 with the TCR, the various cell types were stimulated with specific peptide, and downregulation of the TCR and CD8 was monitored at different time points. The TCR was specifically internalized in all three different cell types. In contrast, CD8 expression was not affected in naive T cells, whereas a proportion of CD8 was internalized in effector and memory T cells . The downmodulation of coreceptor induced by TCR triggering is the consequence of binding of the coreceptor-associated Lck to the CD3 complex ( 39 ). Indeed, it has been shown that truncated CD4 molecules that fail to associate with Lck are not downregulated with triggered TCRs ( 39 ). This prompted us to analyze whether the lack of CD8 downregulation in naive T cells after triggering is due to a less extensive association of their coreceptors with Lck. Immunoprecipitation of CD8 and probing for the presence of Lck by immunoblotting showed that their association was much higher in both effector and memory T cells compared with naive T cells . Flow cytometry analysis showed that surface CD8 expression was comparable in naive and memory cells and was only twofold reduced in effector cells . Further, total cellular amounts of Lck were not different in the various cell types . These data rule out the possibility that CD8–Lck association is driven by changes in expression levels. Interestingly, after triggering of effector T cells with peptide-pulsed APCs, the amount of CD8-associated Lck decreases in a time-dependent way correlating with TCR internalization, suggesting that the CD8-associated Lck is preferentially used by the triggered TCRs . Taken together, these results demonstrate that in naive cells only a few CD8 molecules are associated with Lck, and that specific priming results in redistribution of the kinase, which becomes preferentially associated with the coreceptors. These results suggest that the different localization of Lck might be responsible for the functional difference between naive and effector/memory T cells. In other words, the less stringent requirements in terms of costimulation for activation of memory and effector T cells may be explained by an Lck-mediated amplification of TCR signaling in these cells. It has been shown that the association of Lck to the coreceptor prevents its targeting to the endocytic pathway and its recycling ( 41 , 42 ). Therefore, we analyzed the CD8 endocytosis in naive, effector, and memory cells. The cells were stained at 4°C with an FITC-conjugated anti-CD8 antibody and than warmed to 37°C for 1 h. In effector and memory cells, the CD8 staining was distributed on the membrane and was completely absent in the cytoplasm of the cells. In contrast, in naive cells a clear process of endocytosis occurred, as demonstrated by the intracellular distribution of the fluorescence . Thus, while in effector and memory cells the coreceptor is stably expressed on the membrane, in naive cells CD8 is constitutively internalized , correlating with the lack of CD8–Lck association . The differential association of Lck with CD8 in naive versus effector and memory T cells might reflect a different distribution of the kinase inside the cell. To investigate this question, we compared the cellular distribution of Lck in naive, effector, and memory T cells. We found that in effector and memory cells, Lck was localized at the plasma membrane, whereas in naive cells Lck had a more homogeneous cytosolic distribution . In contrast, CD8 was present only in the cell membrane . Thus, the different association of Lck with the CD8 coreceptor in naive versus effector and memory cells is due to a distinctive targeting of the kinase inside the cells. To reveal whether the distinct distribution of Lck was also observed in memory T cells isolated from normal, non-TCR transgenic animals, the recently developed tetramer technology was used for the isolation of virus-specific effector and memory T cells. Mice were infected with LCMV, and peptide p33–specific effector and memory T cells were stained using H-2D b tetramers pulsed with peptide p33 . As a control, TCR transgenic memory T cells generated as described above were also stained. Specific cells were isolated by cell sorting, and the distribution of Lck was analyzed by confocal microscopy. To facilitate analysis of the intracellular distribution of Lck, the nuclei were separately stained using PI . As a source of naive T cells, CD8 + , tetramer-negative T cells were isolated from uninfected animals. The intracellular localization of Lck was clearly visible in naive T cells, whereas it was predominantly in the cell membrane in effector and memory T cells. Using an adoptive transfer system, we compared early and late events after TCR triggering in naive, effector, and memory cells. In naive T cells, TCR triggering induced a weak calcium flux, and optimal proliferation required costimulatory signals via CD28. In contrast, CD28 costimulation did not affect effector and memory T cell responses. These results strongly support the concept that T cell priming requires the presentation of antigenic peptides by professional APCs, which express high levels of costimulatory molecules, whereas activation of effector and memory cells can also be achieved by nonprofessional APCs. Since naive, effector, and memory T cells expressed the same TCR, our results indicate that the reduced requirement for costimulation and the enhanced TCR signaling are not due to increased TCR affinities but rather reflect an improved signaling cascade. Moreover, many recent findings indicate that CD28 engagement, in addition to delivering accessory signals, facilitates T cell activation by enhancing the signals delivered by the triggered TCRs ( 29 , 32 , 39 ). We therefore suggest that effector and memory T cells do not require the CD28-mediated costimulation because they acquire an improved signal transduction machinery amplifying TCR signaling by a different mechanism. In effector and memory cells, Lck, a kinase critically involved in TCR signaling ( 38 ), is targeted to the membrane and associated with the coreceptor. Surprisingly, however, in naive cells only a few CD8 molecules are associated with Lck and the kinase is homogeneously distributed inside the cell. Thus, in effector and memory T cells, Lck is located in a strategically important position at the cell surface and therefore is able to rapidly react to TCR triggering. In support of this notion, it has been recently shown that Lck must be localized to the plasma membrane to function properly in TCR signaling ( 43 ). The mechanism of this developmentally regulated targeting of Lck to the plasma membrane in effector and memory T cells is presently not known. It is possible that membranes of effector and memory T cells may be enriched in glycolipid microdomains, which preferentially bind src kinases ( 44 , 45 ). However, preliminary evidence suggests that effector but not memory T cells are enriched in such glycolipid microdomains (not shown), indicating that a different mechanism is responsible for the targeting of Lck to the cell membrane in memory T cells. Interestingly, it has recently been shown that targeting of Lck to the plasma membrane requires the attachment of S-acyl groups to Cys3 and Cys5 of the molecule ( 43 ). Therefore, it is possible that in T cells the posttranslational S-acylation of Lck is the mechanism that regulates the intracellular localization of Lck. This notion is supported by the observation that palmitylation of other src family members can occur reversibly and therefore may represent an important mechanism for the regulation of their activity ( 46 , 47 ). Moreover, it has recently been shown that various palmitylated signaling molecules are recruited to glycolipid microdomains in the plasma membrane upon activation of T cells ( 48 , 49 ), further indicating that T cell activation may alter the intracellular distribution of signaling molecules. During the interaction of T cell clones with APCs, the coreceptors are recruited to triggered TCRs and are downregulated with identical kinetics. This process is the consequence of binding of coreceptor-associated Lck to ZAP-70/ζ and takes place whenever the TCR is triggered ( 39 ). We found that in naive mouse T cells, the CD8 coreceptor molecules are not downregulated with the triggered TCRs. Nevertheless, the results do not exclude that few CD8 molecules are associated with Lck and are recruited to the TCR also in naive cells. However, the limited sensitivity of the method does not allow the measurement of their downregulation or degradation. This is also suggested by the fact that proliferation of naive T cells induced by the wild-type agonist can be selectively inhibited by anti-CD8 antibodies. The fact that the responses of effector and memory cells to the strong agonist are not affected by anti-CD8 antibodies is in line with previous findings using T cell clones ( 39 ). Indeed, the intracellular interaction between Lck and the CD3 components does not require the extracellular binding of CD8 to the cognate MHC molecule, probably because in mouse ( 50 ) as well as in human ( 39 ) T cells, a fraction of TCRs are constitutively associated with the coreceptor via Lck. The fact that proliferation of naive T cells can be inhibited by anti-CD8 antibodies might therefore be explained by the lack of constitutive association between CD8 and TCR/CD3 in these cells. Therefore, few Lck-associated CD8 molecules are not easily available and likely need to bind the MHC molecules to be recruited to the T cell–APC contact region. It has been shown that effector T cells ( 21 ) and also memory T cells ( 19 ) are more rapidly committed for proliferation than naive T cells. According to our findings, effector and memory T cells might reach thresholds for proliferation more rapidly than naive T cells, due to the Lck-mediated amplification of the signals delivered by the TCRs. In addition, in contrast to naive T cells, memory T cells have been reported to receive a survival signal from noncognate MHC class I molecules ( 14 ), a finding which is possibly also related to the enhanced TCR signaling machinery present in memory T cells. In B cells, the high rate of somatic mutation in the Ig variable regions may lead to the generation of memory cells with high-affinity receptors. This study indicates that in T cells, the “memory bonus” is not offered by the receptor itself but by its enhanced capacity to transduce activation signals. To our knowledge, this finding represents the first evidence of a biochemically controlled process that functionally differentiates memory from naive T cells. | Study | biomedical | en | 0.999997 |
10330432 | Lines of transgenic mice were generated that express a membrane form of hen egg-white lysozyme (mHEL) containing Hb(64–76) or one of five different APLs of Hb(64– 76) as an epitope tag. The self-peptide Hb(64–76) is derived from the minor d allele of the β chain of mouse Hb. When presented by I-E k , this epitope is recognized by the 3.L2 T cell hybridoma and clone ( 49 ). Many of the APLs recognized by 3.L2 T cells involve substitutions at the 72 position of the Hb(64–76) peptide, the TCR contact residue in the middle of the MHC/peptide complex (P5 position) as determined by crystallography ( 50 ). The specific ligands and APLs studied in this report are Hb(64–76), Hb(64–76) N72T, Hb(64–76) N72I, Hb(64–76) N72A, Hb(64– 76) N72Q, and Hb(64–76) N72E. Henceforth these ligands are called N72(wt), T72, I72, A72, Q72, and E72. The epitope tags were placed between amino acids 43 and 44 of mHEL, and the transgenic mice were generated as described previously ( 51 ). Expression of each transgenic construct was controlled by the MHC Eα promoter, limiting expression to all class II–positive cells ( 43 , 52 ). Founders were obtained, characterized, and bred to the 3.L2tg mouse, which is specific for Hb(64–76)/I-E k ( 53 ), and the 3A9 TCR transgenic mouse, which is specific for HEL(46–61)/I-A k ( 54 ). Progeny were screened by PCR analysis of purified tail digest DNA, and all mice used in experiments were heterozygotes for TCR and mHEL/APL transgenes. The peptides used in this study were synthesized, purified, and analyzed as described previously ( 48 ). The peptide sequences, in single letter amino acid code, are: GKKVITAFNEGLK [N72(wt)]; GKKVITAFQEGLK (Q72); and NTDGSTDYGILQINSR [HEL(46–61)]. T cell hybridomas were used to detect the presence of stimulatory ligands on splenocytes from transgenic mice as described ( 51 ). In brief, increasing numbers of splenocytes were incubated with 10 5 hybridoma cells for 24 h. Stimulation of each hybridoma was then determined by measuring the level of IL-2 produced using the IL-2–dependent cell line CTLL-2 as described ( 55 ). The hybridomas used in these assays were 3.L2.12 ( 56 ), 3A9 ( 57 ), QC6.2, and QC85.5. The latter two hybridomas were generated by immunizing CE/J mice with the Q72 peptide, and each hybridoma was subcloned twice before use in this study. The QC6.2 hybridoma is specific for Q72, whereas the QC85.5 hybridoma is stimulated by Q72, A72, T72, and N72(wt). Primary T cells were stimulated with Hb(64–76) peptide as described ( 58 ). In brief, 5 × 10 5 splenocytes/well were incubated with increasing amounts of peptide for 48 h, pulsed with 0.4 μCi of [ 3 H]TdR for 18 h, and harvested. Proliferation was measured as cpm incorporated (mean of triplicate wells). Results from several experiments were then averaged to obtain the data shown in Fig. 4 . The antibodies used were PE-conjugated anti– mouse CD4 ( PharMingen ); FITC-conjugated anti–mouse CD8a ( PharMingen ); biotinylated 3.L2 clonotypic antibody ( 53 ); biotinylated 3A9 clonotypic antibody ( 51 ); biotinylated F10.6.6 ( 59 ); and biotinylated 14-4-4S ( 60 ). Cells stained with biotinylated antibodies were subsequently incubated with Tricolor-streptavidin (Caltag) or PE-streptavidin (Caltag). Single-cell suspensions of thymocytes or splenocytes were stained in FACS buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide) using the following protocol. Aliquots of cells (10 6 /sample in 100 μl FACS buffer) were placed in polypropylene culture tubes (12 × 75 mm; VWR) and incubated on ice for 1 h with the biotinylated or directly labeled antibodies. Cells were then washed once with 3 ml of FACS buffer and incubated for 30 min on ice with the streptavidin-fluorochrome conjugate where appropriate. Cells were washed again, fixed for 18–24 h in FACS buffer plus 1% paraformaldehyde, and analyzed on a FACScan ® ( Becton Dickinson ) flow cytofluorometer using CELLQuest ® ( Becton Dickinson ) software. Samples were gated on live cells, and 10 5 live cell events per sample were collected. Thymi from 8–16-wk-old transgenic mice and nontransgenic littermates were excised, embedded in OCT compound (Tissue-Tek), and frozen in liquid nitrogen. Thymic tissue was then cut into sagittal sections (4 μm thick), mounted on glass slides, and fixed in acetone for 10 min at room temperature (RT). Sections were stored at −70°C. Before immunolabeling, fixed tissue sections were allowed to reach RT and were washed three times in PBS. To block binding of antibody reagents to endogenous Fc receptors, either supernatant from the 2.4G2 hybridoma was applied (neat) for 15 min at RT or purified anti–mouse CD16/CD32 ( PharMingen ) diluted 1:200 (2.5 μg/ml) in PBS blocking buffer (PBS, pH 7.4, with 0.1% BSA, 5% normal mouse serum) was applied for 15 min at RT. Tissues were then washed three times in PBS. To block nonspecific binding of secondary streptavidin reagents to endogenous biotin, sections were incubated in avidin D and biotin blocking solutions (Vector Labs), respectively, for 15 min. Tissues were washed three times in PBS before and after each blocking application. Biotin-conjugated primary antibodies were diluted in PBS blocking buffer and applied to tissues for overnight incubation in a humidified chamber at 4°C. To detect mHEL/APL transgene expression, biotinylated F10.6.6 was diluted 1:25 (33 μg/ml) and applied as above. For detection of MHC class II expression, biotinylated 14-4-4S was diluted 1:50 (10 μg/ml) and applied as above. Tissues were then washed three times in PBS. Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) was diluted in PBS blocking buffer 1:1,000 (1 μg/ml) and applied for 30 min at RT. After tissues were washed three times in PBS, coverslips were mounted to slides in PBS/glycerol (1:1). To control for irrelevant binding of F10.6.6, thymi from nontransgenic littermates were stained according to the above protocol. To control for nonspecific binding of Cy3-conjugated streptavidin, anti-TNP IgG 1 ( PharMingen ) and anti-TNP IgG 2 ( PharMingen ) were diluted 1:25 in PBS blocking buffer and applied as primary antibodies. Immunofluorescence was visualized on a Zeiss Axiophot microscope. Electronic images were captured by a Spot camera (Diagnostic Images, Inc.) and processed using Northern Eclipse version 2.0 software. These experiments are designed to study the functional consequences for T cells when endogenous APLs were expressed in the thymus and in the periphery. The system is based on the self-antigen Hb, and uses the 3.L2 TCR transgenic mouse (3.L2tg), which is specific for Hb(64–76)/I-E k ( 53 ). The 3.L2tg mouse is ideally suited for these studies, as positive selection of the transgenic receptor is moderate in H2 k mice, making it possible to observe enhanced positive as well as negative selection. To the 3.L2tg mouse, we have genetically introduced APLs of Hb(64–76). These APLs differ by as much as 150,000-fold in their relative activity towards mature 3.L2 T cells ( 48 ), and the relative activity correlates with the half-life of the TCR–ligand complex (47; Table I ). Ligand levels in the APLtg mice are clearly physiologic, and peptide presentation is achieved through normal antigen processing pathways. Thus our approach should be distinguished from experiments using mice which express a single peptide covalently attached to the class II β chain ( 32 , 43 ). In this latter approach, ligand levels are much higher than those achieved under normal circumstances, and the covalent linker may potentially influence binding of the peptide to the class II molecule. Our strategy for introducing these ligands into the 3.L2 TCR transgenic mouse was recently published for mice expressing the antagonist A72 ( 51 ). In brief, the APLs are introduced as an epitope tag contained within a carrier protein in transgenic mice. This carrier protein is a membrane form of HEL whose expression is controlled by an MHC Eα promoter. The Eα promoter was derived from the established expression vector pDOI-5, which has been shown to efficiently target expression of transgenes to all class II– positive cells in the thymus and the spleen ( 43 , 52 ). As integral membrane proteins, all transgenes are also targeted to the class II antigen processing and presentation pathway ( 61 , 62 ). The APLtg mice are then bred to the 3.L2tg mice, and the progeny are analyzed. Thus, the net effect of this approach is to add one specific ligand for 3.L2 T cells to the pool of endogenous ligands already present in the B6.AKR background. The specific Hb(64–76)–related ligands described in this report are N72(wt), the natural epitope with an Asn at position 72; T72, a weak agonist; I72, an antagonist; Q72, a “null” ligand with no effect on mature T cells; and E72, also a null ligand. Note that we also include new data from the A72tg mouse, as it is instructive to consider these data in the context of the spectrum of APLs described in this report. Thymic expression of the mHEL/APL proteins and I-E k from each transgenic line was assessed by immunofluorescence of frozen sections using mAbs. Results shown in Fig. 1 , A–F, demonstrate that each line expresses the mHEL/APL protein in both the thymic cortex and medulla. Furthermore, the pattern of expression observed is identical to that seen for MHC class II . Peripheral expression of the transgenes was determined by two-color FACS ® analysis of splenocytes. The results shown in Fig. 2 demonstrate that each line expresses the mHEL/APL protein in all MHC class II–positive cells. This population represents ∼60% of splenocytes and is largely B220 + (not shown). Importantly, none of the transgenes appears to influence MHC class II expression in the thymus or in the periphery, as the data demonstrate that class II expression in these six transgenic lines is identical . Within a given line, identical levels of mHEL/ APL expression are seen in splenocytes from 3–4-wk-old mice and in the 6–12-wk-old mice used in this study (data not shown). This implies that any potential differences in the timing of embryonic transgene expression could not influence T cell development in mature mice. However, when comparing one line to another, slight variability in the level of transgene expression was noted. Using both biotinylated and directly labeled bivalent F10.6.6, we estimate that at most there is a three- to fivefold difference in cell surface expression of the various chimeric proteins, with N72 the highest, Q72 the lowest, and ranked as follows: N72(wt) > T72 > E72 > I72 > A72 > Q72 . These data demonstrate expression of the mHEL/APL proteins at similar levels and in all MHC class II–positive cells in both the thymus and the periphery. We tested the effect of each endogenous APL on 3.L2 T cell development by crossing 3.L2tg mice with APLtg mice, and then performing three-color FACS ® analysis on thymocytes. All mice examined were heterozygotes for the TCR and/or mHEL/APL transgenes, approximately equally distributed between male and female, and ranged from 6 to 12 wk of age. Where possible, 3.L2tg-only littermates were used as positive controls; otherwise, age-matched 3.L2tg heterozygotes were selected. For each cross, we examined at least 8 mice, and in the case of Q72tg × 3.L2tg we examined 16 mice (Table II ). CD4 single-positive (SP) cells were analyzed for their expression of the 3.L2 clonotypic receptor. For each experiment, the location of the quadrants, the CD4-SP region, and the marker delineating CD4-SP, 3.L2 clonotype hi antibody (CAB) hi population of cells was set using the 3.L2tg control. These same markers or gates were used to analyze the experimental population. The data are summarized in Table II . Note that the 3.L2tg phenotype is based on analysis of 36 thymi and 26 spleens. For each cross, examples of CD4 versus CD8 dot plots and histograms showing CAB expression in the CD4-SP population are shown in Fig. 3 . In the histograms, the 3.L2 control is shown as a dotted line, and the experimental condition as a heavy solid black line. The ligands are listed in order of their effect on mature T cells ( 48 ): N72(wt), the strongest agonist; T72, a weaker agonist; I72, an antagonist; A72, a weaker antagonist; Q72, a ligand with no effect on mature 3.L2 T cells; and E72, a ligand with no effect on mature T cells and with a negatively charged residue replacing the normal asparagine. As expected, we find that expressing the 3.L2 TCR agonist N72(wt) in the thymus of 3.L2tg mice results in complete negative selection of all thymocytes with the transgenic receptor . Overall, the thymus size is about half that seen in 3.L2 controls, there are fewer CD4 + CD8 + double-positive (DP) and CD4-SP thymocytes, and increased numbers of double-negative (DN) cells (Table II ). There are no CD4-SP cells with the clonotypic receptor. The weaker agonist T72, a ligand that is 50-fold less active ( 48 ), has an identical effect when expressed in the thymus of 3.L2tg mice. Most surprisingly, thymic expression of the antagonist I72 also results in complete negative selection of specific T cells. The weaker antagonist A72 produces only modest negative selection, as described previously ( 51 ). We conclude that, for the 3.L2 TCR, the range of negatively selecting ligands in vivo is broad, and includes ligands that function as agonists and as antagonists of mature T cells. Of significant interest, adding the ligand Q72 to the pool of endogenous ligands already present on the B6.AKR background results in an increase in the percentage of CD4-SP cells with high levels of the 3.L2 TCR over that seen in 3.L2tg control mice (Table II ). This difference is 12% and is statistically significant, with P < 0.01. Also note that the percentage of thymocytes in the CD4-SP quadrants of these two groups is the same, and that the overall size of thymi in the two groups is also approximately the same. Thus, this increase in the proportion of CD4-SP, CAB hi cells is likely to represent an increase in the absolute number of cells with the 3.L2 TCR. When Q72 is replaced by E72, this effect is not observed and 3.L2 × E72 mice are identical to 3.L2 mice, both in terms of the number of CD4-SP thymocytes and the percentage of CD4-SP thymocytes with high levels of the clonotypic receptor. It is important to note that the Q for N substitution at position 72 conserves charge while lengthening the amino acid side chain by one methyl group. This is in contrast to the E for N substitution, which both lengthens the side chain and replaces the polar amide with a negatively charged carboxyl group. Taken together, these data suggest that ligands capable of positively selecting the 3.L2 TCR are weak ligands like Q72, with no activity on mature T cells but capable of forming specific interactions. To further control for the effect of expressing a transgenic membrane protein in all APCs, we also crossed 3.L2tg mice to a line of mice which express mHEL only, that is, they lack the APL epitope tag but use the same Eα promoter. We find that mice expressing the 3.L2 TCR and mHEL are identical to 3.L2 mice (Table II ). For example, in these mice there are 9.7% CD4-SP thymocytes. Of these CD4-SP cells, 59.3% are CAB hi . This experiment provides evidence that the effects we have observed are specific for the epitope tags, and not a result of the HEL components of the transgenes interfering with or altering processing and presentation of endogenous epitopes. Splenocytes from the mice analyzed above were also examined by three-color FACS ® . The data demonstrate that when considering naive T cells, the peripheral phenotype is largely determined by selection events occurring in the thymus. For example, the percentage of CD4 + splenocytes in N72tg × 3.L2tg mice is roughly one half that seen in 3.L2 controls (Table II ). This decrease represents the loss of all cells with high levels of 3.L2 TCR . This same phenotype is also seen in T72tg × 3.L2tg and I72 × 3.L2tg mice. In A72tg × 3.L2tg mice, a reduced number of CD4 + CAB + cells is seen. In the case of Q72tg × 3.L2tg mice, a small increase in the percentage of CD4 + CAB + splenocytes is seen compared with 3.L2 controls. This increase is not statistically significant. In E72tg × 3.L2tg mice, a slight reduction in the percentage of CD4 + CAB + splenocytes is seen. Finally, there is no statistically significant difference between mHEL-only × 3.L2tg mice and 3.L2tg controls (Table II ). Again, this implies that epitopes from the mHEL portion of the constructs have not influenced the results. Interpretation of the data in negatively selecting mice is relatively straightforward. Nearly all cells with the 3.L2 clonotypic receptor are eliminated in the thymus, and no cells with high levels of this receptor are found in the spleen. For the weaker ligands, interpretation of the data is more complicated. There are many determinants, other than genetic factors, that influence the size of the spleen and the relative size of the CD4 + and CD8 + compartments. In previous studies with the A72tg × 3.L2tg mice, we found that CD4 + CAB + cells in the periphery were naive. Yet antigen-induced expansion of any particular population of splenocytes could clearly influence the relative numbers of those cells. In this set of experiments, we did not routinely examine the CD4 + CAB + or the CD4 + CAB − populations of cells for activation markers, making the potential contribution of such an event difficult to determine. Also, we have not examined what influence, if any, these ligands have on the survival of 3.L2tg T cells. These caveats notwithstanding, the peripheral phenotypes are generally consistent with those observed in the thymus, in terms of the size of the CD4 + compartment and the percentage of CD4 + CAB + cells. We wished to determine the functional state of naive T cells selected on the various APL backgrounds. For these experiments, we added increasing amounts of Hb(64–76) peptide to splenocytes as described ( 51 ). Each curve represents the average cpm incorporated at each antigen dose for all experiments performed. As shown in Fig. 5 (top), splenocytes from the negatively selecting backgrounds are tolerant. They proliferate only weakly to high concentrations of Hb(64–76) peptide. For N72 × 3.L2 mice, a response is first measurable at 1.0 μM, compared with 0.003 μM for 3.L2 controls. Overall, these data demonstrate a 500-fold shift in the dose–response curve. T72tg × 3.L2tg and I72tg × 3.L2tg mice are also tolerant, with 500- and 100-fold shifts in their response curves, respectively. The response of A72tg × 3.L2tg splenocytes to Hb(64–76) peptide was recently published ( 51 ). In that report, we found that the presence of the endogenous antagonist A72 inhibited the proliferative response of 3.L2tg T cells. Using an adoptive transfer approach, we have also observed peripheral antagonism with A72 in vivo ( 63 ). The reduced response shown in Fig. 5 (bottom left) reflects both this peripheral antagonism and the reduced numbers of CD4 + CAB + cells present when all experiments are averaged. The response of Q72tg × 3.L2tg T cells is identical to 3.L2 controls at low and intermediate antigen doses. At doses above 1.0 μM, the Q72tg × 3.L2tg response is greater than that seen in 3.L2tg controls, reflecting greater numbers of specific T cells proliferating. The response observed in E72tg × 3.L2tg mice is identical to 3.L2 controls, within the error of the experiments. We conclude from the above that 3.L2tg mice selected on the endogenous ligands N72, T72, and I72 are tolerant to Hb(64–76). A72 produces a degree of tolerance by peripheral antagonism as described ( 51 ). Expression of Q72 in the thymus and spleen of 3.L2tg mice results in functional T cells and a proliferative response slightly greater than control mice. Finally, E72 has no effect on the response of naive 3.L2 T cells to Hb(64–76) peptide. It was possible that differential processing and presentation of the Hb-related APL epitopes in each founder line could influence our interpretation by allowing us to either underestimate or overestimate the effect of any particular ligand. Many of the ligands examined are not agonists for 3.L2tg T cells, and there is no single assay available to detect presentation of these APL epitopes. Instead, we have used a combination of T cell hybridomas and TCR transgenic mice to provide convincing evidence that all chimeric proteins are equivalently processed and presented in both the thymus and the spleen of APLtg mice. Processing and presentation of the relevant epitopes in each chimeric protein were demonstrated by hybridoma stimulation assays using splenocytes as APCs. The 3.L2.12 hybridoma is specific for Hb(64–76), and is weakly stimulated (50-fold reduction) by T72 ( 48 ). As expected, as few as 3 × 10 3 N72tg splenocytes stimulate the 3.L2.12 hybridoma . At higher numbers of splenocytes per well (10 5 –10 6 ), T72tg splenocytes will also stimulate the 3.L2.12 hybridoma, although this stimulation is reduced 35–50-fold compared with that achieved with N72tg splenocytes and is not visible in the scale of the graph. In a similar fashion, the hybridoma QC85.5 is stimulated most strongly by the Q72 peptide, but will also produce IL-2 when stimulated by N72, T72, and A72. When splenocytes from each transgenic mouse line were used as APCs, the response observed was consistent with this observation, confirming the presence of these ligands . Again, the response to N72tg and A72tg splenocytes, while fivefold above background, is not visible in the scale of the Q72tg response. The presence of the Q72 ligand was also demonstrated by using Q72tg splenocytes to stimulate the QC6.2 hybridoma , which is specific for Q72 and no other APL of Hb (64–76). Finally, APCs from all transgenic lines, regardless of which specific APL of Hb(64–76) they express, should also process and present epitopes from the common HEL portion of the chimeric protein. This expectation was tested with the 3A9 T cell hybridoma, which is specific for HEL(46–61)/I-A k . We find that splenocytes from all transgenic lines stimulate the 3A9 hybridoma, and that the induced responses are equivalent . Thymic expression, processing, and presentation of mHEL/ APL transgenic proteins were demonstrated by breeding each line of APLtg mice to 3A9 TCR transgenic mice. T cells in 3A9 TCR transgenic mice are specific for HEL(46–61)/ I-A k , and therefore should be negatively selected in the presence of this strong agonist ligand. As shown in Table III and Fig. 7 , this is indeed the case. When thymocytes from mHEL/APL +/− , 3A9 +/− mice were examined by FACS ® analysis for CD4, CD8, and the 3A9 clonotypic receptor, there were virtually no DP cells. The number of CD4-SP (CD4 + CD8 − ) cells was reduced by ∼75%, and in this latter population no cells expressed the 3A9 clonotypic TCR . Consistent with this observation, the size of thymi in 3A9 × mHEL/APL mice was reduced by ∼80% relative to 3A9 littermate controls. In these mice, no splenocytes were found with the 3A9 clonotypic receptor, and naive splenocytes failed to proliferate to the HEL(46–61) peptide (not shown). We conclude 3A9 × mHEL/APL mice are tolerant to HEL(46–61), and the mechanism for this tolerance is negative selection, demonstrating the presence of properly processed and presented HEL(46–61) epitopes in the thymi of all mHEL/APL mice. In summary, these data clearly show that the APL portion of each chimeric transgenic protein is processed and presented by APCs in vivo. For those hybridomas stimulated by more than one APL, the relative magnitude of the response observed with splenocytes as APCs compares favorably with the response observed with purified peptide in a standard hybridoma proliferation assay (48, and data not shown). When considered together, these data strongly suggest that each transgenic line expresses similar numbers of I-E k /APL complexes on APCs in both the thymus and the periphery. In this study, we have determined the effect of a broad spectrum of APLs on the selection of an MHC class II– restricted, antigen-specific transgenic T cell in vivo. We find a strong correlation between the effect of the ligand on thymocyte selection and the relative biological activity of the ligand. Specifically, all ligands with measurable relative activities in vitro produce some level of negative selection in vivo. When combined with our previous observation that the relative biological activity is kinetically related to half-life of the TCR–ligand complex ( 47 ), the data demonstrate that all ligands examined with a half-life >2 s in vitro produce complete negative selection in vivo. Thus, these results establish a kinetic threshold for the negative selection of an MHC class II–restricted TCR. Negatively selecting ligands include both weak agonists and antagonists of mature T cells. Our results also identify one ligand (Q72) that appears to enhance the positive selection of specific thymocytes. Q72 has no effect on mature 3.L2 T cells, and no binding can be detected in BIAcore™ experiments. However, when expressed in the thymus of 3.L2tg mice, this ligand results in increased numbers of CD4-SP thymocytes with high levels of the 3.L2 clonotypic receptor. E72, another ligand with no effect on mature 3.L2 T cells, has no effect on 3.L2tg T cell development. Taken together, these results clearly illustrate an ordered progression from negative selection to positive selection to no effect, as the potency of the ligand is decreased. When combined with the affinity measurements of ligand binding, they suggest that there are kinetic thresholds for negative and positive selection which are related to the half-life of the TCR–ligand complex. Specific interactions between TCR and ligand of sufficient longevity will result in negative selection, whereas weaker (shorter) interactions may be sufficient for positive selection. The results also suggest that positive selection involves specific interactions between TCR and ligand, as only one of the two weak or null ligands examined increased the percentage of specific T cells. For the ligands used in this study, the correlation between the biological activity on mature T cells and the half-life of the TCR–ligand complexes in vitro is compelling. Accordingly, we have chosen to interpret the in vivo selection data using a kinetic model of thymocyte selection, as this model best fits all of the available data ( 47 ). Other interpretations are possible, particularly when using negative selection as the sole parameter to be correlated with the kinetic measurements. Specifically, on the basis of negative selection alone, one could use an occupancy model to interpret the data. In this model, negative selection of 3.L2tg T cells would result for all ligands that bind the TCR with an equilibrium binding affinity of 170 μM or greater, the limit of detectable binding in our experiments. Recent data from a single peptide/MHC class II complex system suggest that a substantial increase in the concentration of a positively selecting ligand can result in negative selection ( 39 ). We have no direct information on how the small differences in the level of expression of endogenous membrane proteins in this study might influence the number of available peptide/MHC complexes derived from these proteins. Data using an mAb to detect specific MHC/ peptide complexes on the cell surface have suggested that only a fraction of the class II molecules on an APC may be available for loading with a single epitope. These experiments also suggested that there may be an upper limit to the fraction of class II that can be loaded from endogenously expressed membrane proteins ( 64 ). The 3A9 T cell hybridoma assays described above demonstrate that, for at least this particular functional assessment, the consequences of slight differences in expression are minimal. One interpretation is that we have achieved maximal or near-maximal levels of the appropriate complexes using our approach of continuously delivering the transgenic membrane protein to the class II antigen processing pathway in all APCs. Furthermore, in a kinetic model, outcome is determined primarily by the TCR dissociation rate, which does not depend on ligand concentration. This important distinction makes it unlikely that the three- to fivefold difference in expression of the APL transgenes would have any effect on negative selection, where the association between APL and TCR–ligand half-life is strongest. Additionally, data from our laboratory using an inducible system to control expression of N72(wt) support this conclusion (not shown). Therefore, for example, the significant differences between the effects of I72 and A72 on negative selection cannot be explained by small differences in ligand concentration. The relationship between TCR–ligand kinetics and positive selection remains speculative, as the half-lives of the relevant complexes cannot be accurately measured with current technology. Furthermore, practical considerations in this study have limited our investigations to one level of expression for each ligand; thus, the influence of ligand density on positive selection remains to be determined. It is possible, for example, that the A72 ligand expressed at lower levels could give a result similar to that seen with Q72, as predicted by an occupancy model of thymic selection. One way to integrate occupancy and kinetic models is to postulate that signaling in both positive and negative selection requires a certain number of complexes with an appropriate half-life. Once the occupancy threshold is exceeded, the relevant kinetic parameter becomes the stability of the TCR–ligand complex. Short-lived complexes could result in contact caps with a different composition of signaling molecules than complexes with longer half-lives ( 65 , 66 ), thereby altering subsequent events in the signaling cascade. Differential, ordered phosphorylation of the TCR ζ chain has been observed in mature T cell signaling ( 24 ), and it is not unreasonable to postulate a similar process in developing thymocytes. Thus, positive and negative selection may involve distinct signaling pathways ( 67 ). It is important to note that adding one specific ligand to the pool of endogenous ligands is not a definitive test of positive selection per se. On any particular background, positive selection may already be maximized, so that the only additive effect that could be observed would be negative selection. We feel that, as indicated by the data presented here and elsewhere ( 53 ), this is not the case for 3.L2tg T cells. Therefore, the observed transition from negative selection to positive selection to no effect matches the predicted outcome as the potency of the ligand is decreased. In summary, we have presented data examining the role of APLs in thymic selection of 3.L2 T cells in vivo. Our system individually adds one specific ligand at one fixed concentration to MHC class II–positive cells in the thymus and spleen. Under these conditions, we find that all ligands with measurable effects on mature T cells produce at least some level of negative selection. We also find that one particular null ligand, Q72, enhances the development of 3.L2tg T cells. These observations lead us to conclude that there is a direct correlation between the biological activity of the APLs of 3.L2tg T cells and their effects on thymic selection. We have previously demonstrated that these biological effects reflect the stability of the interaction between 3.L2tg T cells and each MHC/peptide complex ( 47 ). The logical conclusion is that there are kinetic thresholds for positive and negative selection related to the half-life of the TCR–ligand complex. | Study | biomedical | en | 0.999996 |
10330433 | After appropriate informed consent, subjects underwent an excisional lymph node biopsy under local anesthesia in an outpatient operating room. The tissue was obtained from levels 3 and 4 of the posterior cervical chain, with the second biopsy obtained from the contralateral site. In each case, the largest palpable lymph node was selected for excision. The freshly excised lymph node tissue was immediately cut into pieces, and a measured portion was snap frozen under OCT compound as a block of tissue; the remaining portion was separated into a single-cell suspension by mechanical disaggregation. The single-cell suspension was counted and used for several different analyses, including the preparation of microtiter wells used for a limiting dilution analysis (LDA) of vRNA-expressing cells. Probes for ISH of HIV-1 RNA were produced from three separate plasmids that were linearized, and single-stranded RNA molecules were produced by in vitro transcription. During the transcription, digoxigenin-UTP ([uridine 5′-triphosphate]; Boehringer Mannheim ) or 35 S-UTP was directly incorporated into the sequence as described ( 12 ). For the digoxigenin-labeled probes, three separate antisense probes were made, quantified by trace labeling with 3 H during transcription, and hydrolyzed for 10 min in 20 mM NaHCO 3 , pH 10.2, to yield labeled fragments of ∼300 bp. The three probe preparations were then mixed at equal molar amounts and used at 0.1 fm/μl in hybridization buffer for ISH. The procedure was performed as previously reported ( 11 – 13 ). The color reaction was stopped by rinsing the slides in Tris-EDTA buffer, pH 8.0, and the sections mounted in aquamount. The frequency of HIV RNA + cells was counted by direct microscopic observation, using a calibrated ocular grid, by an observer blinded to the other analyses performed on the same specimen. The frequency of individual vRNA + cells was counted in multiple adjacent sections, and the total area occupied by lymphoid tissue and density of cells was determined in each section. In each case, the number of HIV RNA + cells and the total lymphoid tissue area was determined for each section. The number of adjacent sections analyzed depended on the frequency of positive cells and ranged from 5 to 100 sections per lymph node. The density of cells was determined by counting nuclei in an adjacent hematoxylin and eosin– stained section for each case. Among these specimens, the mean number of cells (nuclei)/mm 2 was 12,500. By comparing the number of positive cells with the total amount of lymphoid tissue examined, the frequency of positive cells per 10 6 total cells was calculated. ISH analysis of lymph node tissue from HIV − individuals, using riboprobes with the same HIV-1 sequences in sense orientation or probes specific for murine sequences, failed to identify any positively stained cells (data not shown). HIV RNA was quantitated by a modification of the procedure described previously ( 14 ). The synthetic competitor for HIV analysis was constructed by ligation of the 5′ and 3′ respective oligonucleotide templates into the general cloning vector pQPCR1 as described ( 14 ). This pQPCR.HRV2 competitor contained several primer sets, one of which was a site in the HIV-1 pol gene designed to amplify a 415-bp fragment extending from position 2713 to 3127 in the HIV genome . All competitors were used to generate single-stranded competitor RNA molecules, which were purified based on a specific hybridization tag 3′ to the reverse primer cassette and quantitated by trace labeling with a known specific activity of 3 H-UTP. The single tube quantitative PCR technique, a modification of the multi-tube quantitative PCR technique ( 14 ), was performed by adding GITC extract containing an unknown amount of HIV-1 RNA to a competitor cocktail containing five different synthetic HIV competitors at a series of concentrations differing by 0.4 log 10 multiples. For example, competitor cocktail 3 consists of competitor A at log 10 3 (1,000 copies), competitor B at log 10 3.4 (2,510 copies), etc. Each competitor differed from the others by an internal 25-bp segment of DNA that is the basis for differential hybridization and detection. Extraction, cDNA generation, and quantitative PCR were performed as described ( 14 ). The enzyme immunoassay detection procedure was also carried out essentially as described ( 14 ), except that each PCR product was plated into 12 microtiter wells instead of 4. Two wells each were probed with detection oligonucleotides for the HIV-1 gene and stuffer detection oligos specific for competitors A, B, C, D, and E. Calculation of endpoints was performed as described ( 14 ). The pol gene primers used were: upstream oligo, 5′-CATACAATACTCCAGTATTTGCCA; and pol downstream oligo, 5′-AAGTCAGATCCTACATACAAATCA. PCR conditions for pol amplification were: 0.8 μM primers, 2.6 mM Mg 2+ , and 50°C annealing for 35 cycles. The detection oligonucleotide for hybridization of the internal HIV sequence was 5′-TGGATGTGGGTGATGCATATTTTTCAGTTC; each different competitor species was detected with a distinct 25-mer sequence using standard hybridization conditions ( 14 ). For determination of the bulk amount of vRNA in tissue, 20–40 4-μm sections were cut from the frozen tissue block adjacent to other sections used to perform ISH and immunohistochemical analysis. This material was immediately solubilized by adding 10 μl GITC/section to a tube containing the frozen sections and was used for the QC-RT-PCR analysis as described above (termed tissue homogenate). A measured portion of each lymph node biopsy specimen was separated in the operating suite and transported immediately to the containment lab in ice cold balanced salt solution. The tissue was separated into a single-cell suspension by standard mechanical disaggregation techniques, washed, and counted. Limiting diluted wells were prepared by dispensing 500, 1,000, 5,000, or 10,000 cells into 36 replicate microtiter wells in 10 μl HBSS with 10% FCS, and then 100 μl GITC was added to each well. Individual wells were analyzed by QC-RT-PCR as described using a competitor cocktail with the lowest individual competitor concentration at 1,000 copies/reaction. The logarithm of the fraction of negative wells (<1,000 vRNA) was plotted against the number of cells per well. Regression lines passing through the origin were calculated and used to estimate the precursor frequency of positive cells. When the frequency of negative wells is >40%, most of the positive wells contain only a single positive cell and, therefore, the copy number directly determined by the QC-RT-PCR procedure in those positive wells is also a direct measure of the copy number per replication-active cell in vivo. The analysis contained herein was applied to cervical lymph node biopsy specimens obtained from nine patients with a wide spectrum of plasma HIV RNA levels, six of whom had serial biopsies performed before and after the introduction of highly active antiretroviral therapy (combinations of HIV-1 protease inhibitors and two reverse transcriptase inhibitors). All of the subjects were in relatively advanced stages of HIV disease, with <350 CD4 T cells/μl. The plasma vRNA levels and the absolute blood CD4 cell counts concurrent with the biopsies, the timing of the biopsies relative to therapy, the specific antiretroviral regimens involved, and the quantitative results obtained in this analysis are shown in Table I . Fundamental to these studies was the development of a procedure to accurately quantify the HIV vRNA from tissue specimens. Using primers specific to various regions of the HIV genome detecting only full length, unspliced message (see Materials and Methods), a modification of the previously described QC-RT-PCR assay was employed ( 14 ). To verify the accuracy of our QC-RT-PCR assay, we compared this assay to two other independent methods for HIV RNA quantitation, using a series of plasma specimens obtained from patients in various clinical stages of HIV infection as well as a standard HIV RNA preparation obtained from the National Institutes of Health AIDS reagent repository. The results of this analysis demonstrated excellent concordance between the UAB QC-RT-PCR and the Roche Amplicor HIV Monitor ® assay ( 15 ), whereas comparison with the branched chain DNA (bDNA; Chiron) assay ( 16 ) using the same specimens revealed the previously described slight systematic bias toward lower viral load results in the bDNA assay (data not shown). Thus, the independently constructed QC-RT-PCR assay provides results equivalent to the Roche assay over a wide range of RNA concentrations. By using dilutions of specimens containing varying concentrations of HIV vRNA, this QC-RT-PCR ( 14 ) assay has a sensitivity of ∼100 copies/reaction (data not shown). The goal of these studies is to accurately quantitate the amount of virus contained in HIV-infected cells and to cross-validate the results using independent procedures. The sensitivity of RT-PCR allows single-cell analysis; therefore, the current study began by assessing the vRNA content of individual HIV-infected cells. To directly determine the frequency and vRNA content of lymph node cells actively producing virus, five individual biopsy specimens obtained before combination antiretroviral therapy were subjected to LDA. Both the frequency of individual cells expressing detectable vRNA and the copy number per cell were directly determined. The mean copy number of vRNA per cell for each patient is remarkably consistent (3,900 or 3.6 log 10 copies/cell [range: 3.5–3.7 log 10 copies/cell]; Table I ). To corroborate the QC-RT-PCR data, the frequency of vRNA + cells was also determined by ISH analysis using digoxigenin-labeled riboprobes as previously described ( 10 , 11 ). As observed by others using 35 S-labeled riboprobes ( 5 , 6 , 17 – 19 ), HIV RNA is present in lymphoid tissue in two distinct patterns: within individual cells and between cells in the FDC network of germinal centers . The frequency of vRNA + cells we observed by ISH is comparable to the frequency of vRNA + cells determined by the independently performed LDA method on adjacent portions of the same tissue specimen (Table I ). In this series of patients, most with relatively advanced HIV disease, many of the lymph nodes demonstrated negligible amounts of ISH signal associated with germinal centers. Nevertheless, in each specimen, morphological germinal centers were identified by immunohistochemistry (data not shown). In several cases, the ISH procedure was repeated with 35 S-labeled HIV riboprobes and with proteinase K–digested tissue as described ( 6 , 17 ), with results equivalent to those of digoxigenin-labeled probes for both frequency of individual vRNA + cells and detection of FDC virions (data not shown). The relative intensity and extent of germinal center–associated virus was assessed on a semiquantitative visual scale (Table I ), but no attempt to precisely calculate numbers of vRNA molecules was made based on this image analysis. Although several of these tissue specimens from advanced HIV disease patients (CD4 <200 cells/μl) had negligible FDC-associated vRNA signal, in this analysis the quantification of vRNA was carried out exclusively using the standardized soluble vRNA assay by QC-RT-PCR. To cross-validate the HIV RNA mean copy number per cell by LDA, a further independent analysis of the vRNA content of homogenized tissue was performed on each biopsy specimen. Tissue homogenates from serial sections from the same tissue block used for the ISH analysis were made as described. The amount of vRNA present in tissue homogenates, representing both individual vRNA-expressing infected cells and free virions trapped in FDC structures, was then directly determined using the QC-RT-PCR procedure and is expressed in Table I as HIV RNA copies/million lymph node cells (see Bulk Tissue Analysis ). The total amount of tissue vRNA was then divided by the number of HIV-expressing cells detected by ISH and the results listed as HIV RNA Copies/Cell bulk/ ISH in Table I . Consistent with the LDA data, when FDC-associated virus was not detected (Neg), the HIV RNA Copies/ISH + cell hovered around 5,000 copies. When significant FDC-associated virus was detected by ISH, then the HIV RNA copies/ISH + cell was much higher (Table I ; see patients BABI, EVJE, and TRRA). Thus, the detection of FDC-associated virions is not simply dependent on the sensitivity of the ISH analysis. Direct measurement of total tissue vRNA detects significantly more vRNA than can be accounted for by the frequency of individual vRNA + cells only in those cases where FDC signal is detected by qualitative ISH methodology. The relatively constant mean vRNA content per cell implies a constant instantaneous production of HIV virions (instantaneous burst size). Although the actual burst size represents the total virus production over the lifetime of the cell and depends upon several factors (see below), one prediction of a constant burst size would be a direct correlation between the number of infected cells and the plasma viral load over a broad range of values. To investigate this possibility, we compared the frequency of vRNA + cells per million lymph node cells determined by ISH with the plasma viral load of the patient at the time of the biopsy . As predicted, there was a direct and highly significant correlation between the logarithm of the vRNA + cell frequency in lymph node tissue and the logarithm of the plasma vRNA. The slope of this relationship is relatively constant, whether the data derives from different patients (slope = 1.6; Pearson product moment correlation coefficient ( r ) = 0.89; P < 0.0005) with widely varying viral loads (a 6 log 10 range) before protease inhibitor therapy or from patients undergoing serial biopsies before and after potent therapy . The overall relationship among the logarithms of these values is highly significant (slope = 1.6; r = 0.95; P < 10 −8 ). Although the relationship is consistent over a wide range of viral load values, the direct correlation is not 1:1 (that is, the slope of the log–log plot ≠ 1). This result differs from predictions based on standard mathematical models of viral dynamics ( 20 , 21 ). In other words, a 1 log 10 drop in the number of vRNA + cells in lymph node tissue is associated with a 1.6 log 10 drop in vRNA in plasma. The interpretation of this observation remains open for speculation, but the consistency of this direct relationship over such a broad range of viral load values is a novel observation that must be accounted for in quantitative models of viral dynamics. In each of the three biopsy specimens obtained from subjects on stable highly active antiretroviral therapy (HAART) with a plasma viral value <50 copies/ml, we continued to detect individual vRNA + cells by ISH, albeit at extremely low frequency. This analysis required examination of 50–100 sections spaced at least 12 μm apart to detect the few positive cells. The quantification of the total vRNA from the interspersed sections used for ISH analysis divided by the frequency of vRNA + cells detected by ISH indicated a copy number per cell similar to that obtained during episodes of high viral replication (Table I ). Although this estimate is not highly precise due to the low number of total cells detected, these data indicate that the majority of detectable vRNA molecules in lymph node tissue of subjects on HAART is contained within a few individual cells. Because the copy number estimate for these residual cells is similar to that measured by both LDA and bulk tissue analysis in subjects not treated with antiretroviral drugs, it is likely that these very few cells represent fully replication-active cells, rather than some form of abortive transcriptional activity. This coordinated analysis of HIV vRNA by three interlocking but independent methods (ISH, QC-RT-PCR of bulk tissue, and LDA) provides cross-validation of the results. First, we found that the frequency of positive cells determined by ISH is equivalent to the frequency determined independently by LDA (Table I ). Thus, the sensitivity of ISH for detecting individual vRNA + cells was directly validated by the completely independent method of LDA, which has not been performed when using other ISH methods. Second, when there was negligible FDC-associated virus detected by ISH, the copy number of HIV RNA per cell determined by LDA is equivalent to the vRNA copies present in bulk tissue divided by the frequency of cells. In contrast, when significant FDC-associated vRNA was detected by ISH, this ratio was higher than the independent estimate of copy number determined by LDA (Table I ). These data imply that the ISH procedure using digoxigenin-labeled riboprobes is not missing significant amounts of FDC-associated virus when it is present. If ISH was underestimating the extent of FDC-associated virus, then the copy number calculated based on the bulk tissue method would not match the result obtained by LDA. As the quantification of vRNA copies is not performed by image analysis of the ISH signal but rather by the standardized soluble QC-RT-PCR assay, this quantification does not directly depend on the sensitivity of detection by ISH. Even if the intensity of the FDC-associated ISH signal is weaker using this ISH procedure, this potential problem does not affect the analysis based on cross-validation of independent methods. For example, the first biopsy of a subject with advanced disease (Modified Card Sorting Test [MCST], CD4 count = 27; viral load = 1.1 × 10 6 ; Table I ) showed negligible FDC signal. The frequency of individual positive cells was essentially equivalent by two independent methods (140/10 6 cells by ISH and 125/10 6 cells by LDA). The copy number per positive cell was also equivalent by two independent methods (3,700 copies/cell by LDA and 4,100 copies/cell by the ratio of total tissue RNA divided by the frequency of identified cells). If there was significant FDC virus in this tissue specimen, then this cross-validation would not have been observed. Indeed, when significant qualitative FDC signal was observed, there was significantly more total vRNA in tissue than could be accounted for by the frequency of individual positive cells (Table I ; see subjects BABI, EVJE, and TRRA before HAART). Thus, this analysis is internally validated by independent methods, with the quantitation depending on the QC-RT-PCR analysis of soluble RNA rather than image analysis. The measurement of soluble RNA has been standardized using both internal and external standards and is comparable to the Roche Amplicor HIV Monitor ® assay ( 15 ) . This analytical approach demonstrates that each vRNA- expressing cell in patient lymph node tissue contains, on average, ∼3.6 log 10 copies of HIV RNA. This result is independent of the frequency of infected cells, the plasma viral load or CD4 count, or the form or potency of the antiretroviral being administered at the time of the biopsy. Our determination of the HIV RNA copies per cell is significantly higher than that of previous results based on image analysis using 35 S-labeled probes ( 17 , 18 , 22 , 23 ). Furthermore, in a recent report, image analysis of lymph node specimens from patients with acute and early HIV infection did not demonstrate a relationship between infected cell frequency in tissue and plasma viral load ( 24 ). Although previous investigators have emphasized the major predominance of FDC-associated virions over intracellular virus, in the current analysis the degree of trapping of virions in lymph node germinal centers is less than previously reported ( 17 , 22 ). It is likely that this discrepancy in absolute quantification of copies per detectable vRNA + cell is due to the standardization of the method used to determine copy number. Molar quantification using image analysis of 35 S-based ISH relies on complete efficiency of RNA preservation and probe access in fixed, paraffin-embedded tissue and 100% efficiency of grain production in the emulsion. These assumptions have not be independently validated. In separate experiments not reported here, both of these potential problems appear to be significant. To examine the efficiency of grain production in the emulsion for 35 S-labeled probe ISH, we measured the frequency of infected cells and counted grains over each positive cell in populations of HIV-1 infected PBMC. Calculation of copy number by grain count, using the specific activity of the probe and time of emulsion exposure as described ( 17 ), generated a significantly lower estimate of copy number per cell than direct QC-RT-PCR analysis of 10,000 such cells (corrected for the fraction that was vRNA + ). By this analytical approach, detection of silver grains is only about 5% efficient; in other words, determination of the probe specific activity in a well scintillation counter is more efficient than silver grain production in a very thin emulsion. To examine the potential loss of signal by tissue block fixation and paraffin embedding, the expression of murine Ig μ heavy chain mRNA was compared in fresh-frozen tissue versus formalin-fixed, paraffin-embedded tissue. Both small B cells in primary follicles and sinusoidal plasma cells were detected by digoxigenin-labeled riboprobes in frozen tissue, whereas only the plasma cells were clearly detected in formalin-fixed, paraffin-embedded tissue. Thus, previous reports that used the image analysis method of RNA quantitation may have two systematic errors that lead to an underestimation of the amount of cell-associated virus. Another aspect of the precision and sensitivity of ISH analysis is the discrimination of cells with positive ISH signal from cellular debris that nonspecifically binds the labeled riboprobe. Although debris of irregular shape and size larger than a cell can be discriminated with both techniques, the immunoenzymatic signal is detected directly in the same focal plane as the individual cell in thin section, unlike detection of grains deposited in a photographic emulsion above the tissue section. This additional criterion of morphological analysis allows discounting of rare bits of debris that are similar in size and shape to individual cells but localized out of the plane of section using the digoxigenin system. Perhaps previous reports of significant decreases in the grain count per cell with therapy ( 17 ) may be due to inclusion of a higher fraction of artifactual localized accumulations of grains with lower grain density in the setting of a very low absolute frequency of HIV positive cells. Although interpretation of the relative sensitivity of various histochemical techniques is controversial and has not been cross-validated by multiple independent laboratories, the analysis presented in this report is not fundamentally dependent upon the quantitative sensitivity of the ISH procedure. Rather than simply assume that the frequency of cells detected by ISH was correct, we directly confirmed this frequency by an independent LDA method. Furthermore, the paucity of FDC signal intensity in some of these specimens was directly confirmed by analysis of a bulk quantity of RNA in these tissues. Thus, unlike other analyses of HIV RNA in tissue, this analysis depends on quantitative agreement between several independent analytical methods rather than sole reliance on ISH methodology. In addition to the novel methodological approach, the biological implications of the findings presented here are significant. The invariant mean HIV RNA copy number per cell regardless of treatment history or stage of disease implies that infected cells in tissue maintain a constant instantaneous burst size. The actual burst size is dependant on the life span of the replication-active cell and rate of virion budding in addition to the cell-associated vRNA pool size. We hypothesize that viral production at the single-cell level may be an all-or-none phenomenon, analogous to the situation described for T cell cytokine gene expression ( 10 , 13 , 25 ). The consistency of vRNA amount in productively infected cells during treatment with antiretroviral drugs is consistent with the mechanism of action of these agents, which inhibit either reverse transcription or maturation of viral particles rather than transcriptional activity. Although the precision of the estimate of copy number per identified vRNA + cell in the few cases with very low cellular frequency is not sufficient to rule out a modest change after therapy, a substantial change of >10-fold as reported previously ( 17 ) is not supported by this analysis. Independent of the measurement of absolute copies of vRNA present in each cell, the frequency of such cells is highly correlated with plasma viral load. The surprising observation that this relationship is nonlinear (slope of log–log plot ≠ 1) requires some modification of the standard models of viral dynamics ( 20 , 21 ). One potential explanation for this observation is that the production rate of plasma virus is significantly modified by the presence of antiretroviral drugs. Several lines of evidence argue against this possibility. First, the mechanism of action of these agents is a blockade of de novo infection, either by inhibition of reverse transcription or extracellular maturation of virion particles rather than transcriptional inhibition or virion budding. Second, our demonstration of a roughly constant amount of vRNA in individual cells argues that the amount of intracellular vRNA awaiting assembly and budding is not altered by these agents. Finally, the same nonlinear relationship is found when comparing different individual subjects before therapy but with differing viral load set points. Thus, the nonlinear character of this relationship cannot arise from alteration of viral production rate per cell induced by antiretroviral therapy. Another potential explanation is that clearance of viral particles is a direct function of viral load. To account for the extent of nonlinearity over the range of viral load values examined, the clearance rate must vary by ∼100-fold in a consistent relationship to viral load. Although antibody-mediated clearance rate may be more efficient for low virus load and may be saturated at high virus load, it seems unlikely that such a tremendous variation in clearance rate could account for these observations. Alternatively, we favor a hypothesis that focuses on the different in vivo tissue compartments containing replication-active cells and their relative contribution to the systemic plasma viral load. It is possible that most of the infected cells are present in spleen and gut rather than lymph nodes due to the sheer volume of these compartments. As the T cell zones in lymph nodes are single, relatively contiguous areas unlike the larger spleen and gut tissue environments, the density of infection in these areas may be consistently higher at any given steady state level. The effect of decreased efficiency of cell-to-cell transfer of infectious virus induced by antiretroviral drugs may thus have a lesser effect on the lymph node microenvironment than on the bulk of the lymphoid tissue present in spleen and gut. A comprehensive, quantitative analysis of various tissue compartments containing infected cells in rhesus macaques infected with SIV variants may provide a direct experimental test of this model. A clinically relevant prediction based on the observed relationship between vRNA + cell frequency and plasma viral load is that cells actively replicating viral proteins persist after the substantial decline in plasma viremia observed during combination antiretroviral therapy. The correlation of plasma virus load with the frequency of vRNA + cells over the six log range of quantifiable viral load suggests that this relationship is quite robust. The intercept of this relationship at “undetectable” plasma viral load is ∼1 vRNA + cell/million lymph node tissue cells. Given the large lymphoid tissue mass present in the entire body (estimated at 10 11 cells), a reasonable interpretation of these data is that ∼100,000 cells with viral replication potential could persist in an infected subject, but the virions produced by these few cells would be cleared too quickly to achieve a detectable steady state concentration in plasma. The extrapolation of this relationship to plasma viral load values of <50 copies/ml is validated by our direct observation of cells containing full length viral transcripts within tissue specimens from three patients with concomitant “undetectable” plasma viremia. The origin and fate of the few residual vRNA + cells in patients with substantial drug-induced suppression of viral replication is of fundamental importance for understanding HIV disease pathogenesis and developing further therapeutic alternatives for HIV-infected individuals. If these cells represent new rounds of de novo cellular infection, the implications concerning the eventual emergence of drug- resistant mutants are significant. Alternatively, these few cells could represent in vivo activation of latently infected cells, a process not blocked by HAART. If the virions produced by such activated, latently infected cells do not complete the viral life cycle due to effective antiretroviral therapy, new mutations will not be fixed in the viral population. The apparent extremely low clearance rate of such latently infected cells in subjects on prolonged HAART ( 26 – 28 ) may be attributable to a decreased antigen-driven immune clearance mechanism of these cells ( 29 ) rather than a complete failure of these cells to ever become transcriptionally active. Whatever the biological origin of these few residual vRNA + cells, their persistence may result in rapid reignition of high level viral replication after discontinuation of antiretroviral drugs. Consistent with such a conceptual model are anecdotal reports that patients who discontinue combination antiretroviral therapy, even after many months of apparently complete viral suppression in the blood, experience a rapid return of viremia. Attempts to accurately determine the status of HIV infection in aggressively treated hosts must take into account both the residual pool of latently infected cells and the presence of persistent active viral replication in lymphoid tissue cells. Although combination antiretroviral regimens can reduce plasma HIV RNA levels below current detection limits, our data demonstrate that this parameter is inadequate to measure the extent to which potentially infectious virus remains in other sites (such as lymphoid tissue). Therapeutic strategies that attempt to completely eradicate HIV must be based on firm, quantitative information concerning both blood and tissue sources and the biological nature of residual HIV-1 virus. | Study | biomedical | en | 0.999996 |
10330434 | C57BL/6 and BALB/c mice were purchased from The Jackson Laboratory or from our breeding colony at The Scripps Research Institute. LCMV strain Armstrong (ARM) 53B, a clone triple plaque purified from ARM CA 1371 ( 15 ), was used in this study. Viral stocks were prepared by growth in BHK-21 cells. Viral titers were measured by plaque assay as described ( 15 ). In brief, monolayers of Vero cells were infected with different dilutions of mouse sera or tissue homogenates, and plaques were counted 6 d later. To establish a persistent infection, C57BL/6 and BALB/c mice were infected within 24 h of birth by intracardiac inoculation of 10 3 PFU of LCMV ARM. LCMV-immune mice were obtained by injecting 8–10-wk-old mice intraperitoneally with 2 × 10 5 PFU of LCMV ARM. Immune mice were used at >60 d after infection. BALB/c-derived LCMV-immune splenocytes (5 × 10 7 cells) were injected intraperitoneally into persistently infected BALB/c mice that were irradiated (350 rads) a few hours before transfer and killed at multiple time points thereafter. A recombinant, replication-deficient adenovirus, designated Ad.CBlacZ, provided by Dr. James Wilson (University of Pennsylvania Medical Center, Philadelphia, PA ), was also used to infect LCMV-carrier BALB/c mice. Stocks of Ad.CBlacZ were grown in 293 cells ( 17 ), and were purified by two rounds of CsCl density centrifugation, as described previously ( 18 ). Viral titers were determined by plaque assay on 293 cells, and a single stock was used throughout this study. Mice were injected intravenously with 1.5 × 10 9 PFU/mouse, a dose of Ad.CBlacZ known to infect 100% of the hepatocytes and to cause a CD8-dependent liver disease ( 4 ). Control mice were injected with the same volume of saline. Animals were killed at multiple time points after infection. Recombinant murine IL-12 was provided by Dr. Maurice Gately (Hoffmann-La Roche, Nutley, NJ). C57BL/6 mice were injected intraperitoneally with IL-12 (1 μg/d/mouse). Control animals were injected with saline diluent (saline containing 1% serum) only. Animals were killed 24 h after the last injection of IL-12, and their sera, livers, and spleens were harvested for subsequent analyses. Frozen tissues were mechanically pulverized, and RNA was extracted by the acid-guanidium phenol-chloroform method ( 19 ). Total RNA (20 μg) was analyzed for 2′,5′-oligoadenylate synthetase (2′5′ OAS) and glyceraldehyde-3-phosphate (GAPDH) expression by Northern blot as described previously ( 3 ). The RNase protection assay for quantitation of mRNA was performed exactly as described ( 20 ). The mouse IL-1α(B), mIL-1β(A), mIL-2(A), mIL-3(B), mIL-4(B), mIL-5(C), mIL-6(B), mIFNγ(B), mTNFα(A), mTNFβ(A), and mL32(A) subclones in the pGEM-4 transcription vector were described in a previous report ( 20 ). The mCD4(IC), mCD3γ(IC), mCD8α(DM), and F480 subclones in the pGEM-4 vector were described previously ( 1 ). This procedure was carried out exactly as described ( 21 ). The 33 P-labeled RNA probe used in this study was prepared by transcription from the T7 promoter of plasmid nucleoprotein (NP) Bluescript, a plasmid created by cloning the 1,164-bp BglII fragment from a cDNA of the LCMV ARM S RNA segment ( 22 ) into the plasmid Bluescript KS (Stratagene, Inc.). Transcription from the T7 promoter of pNP Bluescript generates a single-stranded RNA probe complementary to the viral NP mRNA and antigenomic sequence. Total liver RNA (1 μg) was reverse transcribed into cDNA and amplified by PCR using LCMV glycoprotein– specific primers exactly as described ( 23 ). Quantitation of LCMV ARM and clone 13 RNA was carried out by densitometric analysis (NIH Image software) of the amplified PCR products after MnlI digestion, gel electrophoresis, and ethidium bromide staining, exactly as described ( 23 ). Hepatocellular injury was monitored by measuring serum alanine aminotransferase (sALT) activity ( 1 ). Results were expressed as mean sALT activity ± SEM. Tissue samples were fixed in 10% zinc-buffered formalin (Anatek, Ltd.), embedded in paraffin, sectioned (3 μm), and stained with hematoxylin and eosin as described ( 1 ). The intracellular distribution of LCMV NP was analyzed by immunohistochemical analysis based on a method described by Surh et al. ( 24 ). 3-amino-9-ethyl carbazole (red) was used as coloring substrate for LCMV NP, exactly as described ( 3 ). The in vivo expression of β-galactosidase in the livers of Ad.CBlacZ-infected animals was quantitated by 5-bromo-4-chloro-3-indolyl-β- d -galactosidase (X-gal) histochemistry exactly as described ( 4 ). C57BL/6 and BALB/c mice were infected at birth with LCMV ARM as described in Materials and Methods. At 7–8 wk of age, all animals were bled and serum viral titers were analyzed by plaque assay. Mice with a serum titer between 10 4 and 10 5 PFU/ml were selected, and 2–4 wk later were used as recipients of saline, LCMV-immune “memory” cells, adenovirus, or IL-12. Table I shows the viral titers in serum, liver, and spleen of control BALB/c and C57BL/6 mice that were killed at the indicated time points after saline injection. The hepatic content of LCMV RNA in the saline-injected controls was monitored by Northern blot analysis and by in situ hybridization analysis in which hepatic LCMV RNA was detected in ∼30–40% of the hepatocytes and in nonparenchymal cells in the hepatic sinusoids . Next, we monitored the hepatic content of mRNA for CD3, CD4, CD8, various cytokines, and 2′5′ OAS (a marker of IFN-α/β induction) in these control animals. In keeping with the massive LCMV infection, high levels of 2′5′ OAS mRNA were detected in the liver, illustrating that type I IFN is not sufficient to clear LCMV from the organ. Furthermore, all T cell markers and T cell–dependent cytokines were absent, and only low levels of mRNA for the monokines TNF-α, IL-1α, and IL-1β were detected , reflecting the absence of histological or biochemical (Table I ) evidence of liver disease in these persistently infected animals. Clearance of persistent LCMV infection after adoptive transfer of memory cells occurs very rapidly in the liver without massive liver cell injury. Splenocytes derived from mice that have cleared an acute LCMV infection (i.e., memory cells) are known to clear LCMV when injected into persistently infected animals ( 5 ). To better understand the mechanisms responsible for this effect, 2 × 10 7 BALB/c LCMV-immune memory splenocytes were adoptively transferred into 12 BALB/c mice, and groups of 3 mice were killed on days 3, 7, 14, and 24 after transfer (Table I ). Predictably, LCMV was undetectable on day 24 in serum, liver, and spleen by plaque assay (Table I ). Interestingly, the viral titer dropped very rapidly in the liver (>6 logs in 4 d), becoming undetectable by day 7 after memory cell transfer (Table I ). At this time point, viral RNA was also undetectable by Northern blot analysis of total liver RNA . However, traces of viral RNA remained detectable at day 7 in a few nonparenchymal cells that resemble endothelial cells and biliary duct epithelial cells in the portal tracts . Interestingly, these cells remained LCMV RNA–positive even at the latest time point examined (day 24; not shown), suggesting that they are relatively resistant to viral clearance. Along with LCMV RNA, LCMV NP was undetectable immunohistochemically in the hepatocytes by day 7 (not shown). Importantly, LCMV titer dropped much more slowly (<2 logs in 14 d) in serum and spleen, although LCMV was eventually cleared from these compartments between days 14 and 24 (Table I ). sALT activity (a marker of hepatocellular necrosis ) reached values of ∼600, ∼700, and ∼900 U/l at days 3, 5, and 7 (Table I ), respectively. After its peak at day 7, sALT activity returned to normal levels (∼60 U/l) between days 14 and 24 (Table I ). sALT activity was only modestly elevated in these mice, compared with previously reported mouse models of liver disease ( 26 ). In particular, by comparing these ALT profiles with known standards ( 1 ), we estimate that no more than 5–10% of the hepatocytes were killed during the first week after adoptive transfer, despite the fact that at least 30–40% of hepatocytes were infected . This notion is also supported by histological analysis of these livers, which shows that 3 and 7 d after transfer of memory cells, the vast majority of the hepatic parenchyma is cytologically normal . At day 3, few mononuclear cells are seen in portal tracts (PT), and at day 7 the hepatic parenchyma contains small, scattered necroinflammatory foci ; and, in keeping with the relatively mild liver disease, no liver cell regeneration was observed at the peak of sALT activity , at which time LCMV RNA had disappeared from the hepatocytes . The kinetics of liver disease and the kinetics of viral clearance coincided with the appearance of T cell (especially CD8), macrophage, and cytokine (2′5′ OAS, IFN-γ, TNF-α, IL-1α, and IL-1β) markers in the liver . It is noteworthy that the virus was cleared much more slowly in the spleen despite the fact that the same cytokines were induced with the same kinetics observed in the liver . Collectively, these results indicate that hepatocytes have the capacity to clear LCMV very rapidly, and this occurs in the presence of antiviral cytokines and in the absence of a commensurate degree of cell death. In contrast, the slower kinetics of clearance of LCMV from nonparenchymal liver cells and from the spleen indicate that these cells are relatively resistant to memory cell– induced clearance. To monitor the antiviral effect of the induction of these cytokines in the liver of mice persistently infected with LCMV, 20 BALB/c mice were infected intravenously with a dose (1.5 × 10 9 PFU) of a recombinant, replication-deficient adenovirus (Ad.CBlacZ) that we have previously shown to infect virtually all of the hepatocytes ( 4 ), and groups of four mice were killed on days 3, 7, 14, 21, and 24 after infection (Table I ). Additionally, 13 C57BL/6 mice were injected intraperitoneally with recombinant murine IL-12 (1 μg/d) for either 3 or 10 d, and all animals were killed 24 h after the last injection (Table I ). Results were compared with saline-injected controls as indicated in Table I . These treatments were chosen because they are known to induce IFN type I, IFN-γ, and TNF-α in the liver either with (adenovirus) or without (IL-12) destroying hepatocytes ( 2 , 4 ). As expected, the adenovirus-infected animals developed a necroinflammatory liver disease that was detectable histologically (not shown) and biochemically as elevated sALT activity (Table I ) starting between 3 and 7 d after inoculation and lasting ∼3 wk until lacZ -positive hepatocytes were no longer detectable (not shown). Despite the relatively severe liver disease (compared with that induced by transfer of memory cells), the viral titers in the serum, liver, and spleen did not change at any of the time points examined (Table I ). Similarly, no change in viral titer was observed in the IL-12–treated animals in which we observed no biochemical (Table I ) and/or histological evidence of liver cell injury (not shown). The absence of viral clearance in the livers of adenovirus-infected and IL-12–injected mice was also confirmed by Northern blot analysis of LCMV RNA, as shown in Figs. 5 and 7 . Because these results appeared to contradict the results of the memory cell transfer experiments, we performed in situ hybridization analyses on the adenovirus-infected and IL-12– injected livers to examine the hepatic distribution of LCMV RNA. Surprisingly, LCMV RNA was dramatically reduced in the hepatocytes (but not in nonparenchymal cells, which include resident nonparenchymal cells and infiltrating inflammatory cells) by days 7 and 14 (not shown) after adenovirus infection, concomitant with the induction of cytokines in the liver . In contrast, the intensity and distribution of grains in the hepatocytes on day 24 after adenovirus infection were similar to those of saline-injected controls , and this occurred at a time point when the liver disease and the adenoviral infection had resolved (not shown) and the cytokines were no longer induced in the liver . Similarly, LCMV RNA disappeared from the hepatocyte (but not from nonparenchymal cells) after 3 or 10 (not shown) IL-12 injections, and this occurred in livers in which there was inflammatory cell infiltration but little or no histological evidence of hepatocyte injury (not shown) or sALT elevation (Table I ). Collectively, these results indicate that LCMV is rapidly cleared from the hepatocyte in a bystander manner concomitant with intrahepatic cytokine induction, and this effect is not dependent on the destruction of hepatocytes. As shown in Figs. 6 and 8 , the local induction of cytokines observed after adenovirus infection and IL-12 administration was not associated with clearance of LCMV RNA from nonparenchymal liver cells or the spleen. This probably explains why the virus was not eliminated from these organs or the serum as measured by plaque assay (Table I ). Indeed, the number of splenocytes positive for LCMV RNA by in situ hybridization was similar in the saline-injected controls and in mice injected with IL-12 (not shown), despite the fact that the cytokines were strongly induced in these organs (not shown). Collectively, these results indicate that LCMV clearance from nonparenchymal cells does not occur in the absence of a LCMV-specific immune response, suggesting that other mechanisms are probably required to clear the virus from these cells. After neonatal infection with LCMV ARM, variants such as clone 13, which are known to be more resistant to control by IFNs ( 11 ), have been isolated from persistently infected livers ( 23 ). To test whether these variants are present in the livers of control mice (in which most viral RNA is in the hepatocytes) and IL-12–treated or adenovirus-infected mice (in which most viral RNA is in the nonparenchymal cells), we performed a PCR assay for the detection of a point mutation in the viral RNA that results in a phenylalanine versus a leucine change at position 260 in the viral glycoprotein ( 23 ). The phenylalanine/leucine change is diagnostic for the ARM strain of LCMV versus variants such as clone 13. Roughly equimolar amounts of RNA encoding a phenylalanine versus a leucine residue were detected in the liver of saline-injected controls and IL-12–treated or adenovirus-infected mice (not shown). This suggests that the ARM as well as the variant strains of LCMV are resistant to the antiviral effects induced by IL-12 or adenovirus when they replicate in the nonparenchymal cells. Whether the hepatocytes were infected with ARM or the variant strains or both, and whether these viruses would be differently sensitive to clearance by IL-12 injection or adenovirus infection, remain to be determined. In this study, we demonstrate that LCMV can be cleared from hepatocytes by noncytopathic, cytokine-associated antiviral mechanisms that are not operative in splenocytes or in nonparenchymal cells (which include resident nonparenchymal cells and infiltrating inflammatory cells) in the liver. Several lines of evidence support these conclusions. After transfer of memory cells, viral clearance from the hepatocytes occurs very rapidly, between 3 and 7 d after transfer and 4 d (or less) after the peak of LCMV replication (Table I ). This is associated with a relatively mild liver disease, and it occurs in the context of intrahepatic cytokine induction. Indeed, the liver disease in these animals would probably be much more severe if viral clearance was due primarily to the destruction of the infected cells, based on comparison with previously reported mouse models of liver disease ( 1 , 26 ). Conversely, viral clearance from the intrahepatic nonparenchymal cells and from the spleen of the same animals is a much slower process that follows the peak of local cytokine induction by >10 d, suggesting either that these cells are less responsive to cytokines or that other events, including killing, are required to clear the virus from these cell types. Furthermore, LCMV clearance from the hepatocytes (but not from the intrahepatic nonparenchymal cells and from the spleen) can also occur when cytokines are locally induced after adenovirus infection or IL-12 administration. Again, this effect occurs rapidly (by day 3 for IL-12 and by day 7 for adenovirus), concomitant with the peak of intrahepatic cytokine induction and, in the case of IL-12, independently of hepatocyte destruction. This strongly suggests that cytokines can activate intracellular antiviral pathways in the hepatocytes (but not in the intrahepatic nonparenchymal cells or in the spleen). In support of this hypothesis, LCMV RNA reappears in the hepatocytes of adenovirus-infected and IL-12–injected animals when the intrahepatic cytokine induction subsided, in contrast to the memory cell recipients. This is presumably due to the absence of anti-LCMV antibodies and LCMV-specific T cells in the adenovirus-infected and IL-12–injected animals, since both B and T cells are required to clear a persistent LCMV infection ( 27 ). These results indicate that cytokine-dependent activation of hepatocytes triggers intrahepatocellular antiviral events that are absent or not operative in certain other cell types. This is reminiscent of the unique role that hepatocytes play in host defense mechanisms after tissue damage. Indeed, during the acute phase response, the interaction of a wide variety of cytokines (including IFN-γ, IL-1α, IL-1β, IL-6, TNF-α, and TNF-β), glucocorticoids, and growth factors with hepatocytes results in a complex response characterized by activation of the acute phase plasma proteins (APPs) and profound changes in most metabolic pathways ( 28 ). The regulation of APP gene expression depends on transcriptional and posttranscriptional events ( 28 ) that are most likely operative in hepatocytes but not in nonhepatic cells. This study indicates that cytokines may activate hepatic cells not only to regulate APP genes but also to induce cell-specific antiviral pathways. It has been suggested that specific destruction of infected cells (including hepatocytes) is required to control LCMV replication in the liver and other organs, since perforin- deficient mice fail to clear acute LCMV infection ( 6 , 7 ). However, it is important to note that LCMV clearance was monitored by plaque assay in those studies which might not have detected the selective clearance of LCMV from the hepatocytes. Experiments designed to monitor the cytokine profile and the cellular distribution of viral RNA in the liver of these animals are needed to examine the possibility that LCMV clearance may have occurred from the hepatocytes but not from nonparenchymal cells in the perforin-deficient mice, as we observed after adenoviral infection and IL-12 administration. Indeed, we have previously shown that 2 d after acute LCMV infection, most of the virus replicates within nonparenchymal cells rather than hepatocytes ( 3 ). In addition, passive transfer of IFN-γ–deficient memory cells into persistently LCMV ARM–infected animals fails to clear the virus in liver and spleen ( 14 ), and mice genetically deficient for IFN-γ or its receptor have been shown to control LCMV replication less efficiently in various organs, including the liver, despite normal immune responses ( 8 ). Finally, in vivo treatment of neutralizing antibodies to this cytokine results in higher levels of LCMV replication during acute infection ( 11 ). In conclusion, the results of this report show that LCMV, like HBV, is susceptible to noncytopathic antiviral control mechanisms that most likely depend on local cytokine induction, provided that the virus replicates within the hepatocyte. We have recently shown that HBV replication is completely abolished in the hepatocytes of HBV transgenic mice by cytokine-dependent pathways that do not require cell death ( 29 ), and that intrahepatic induction of the IFNs and TNF-α mediates the antiviral effect ( 1 ), irrespective of the stimuli that trigger their induction. Whether similar intracellular events may interfere with the life cycle of additional viruses remains to be determined. Nevertheless, viral clearance of these and other viruses might rely very heavily on noncytopathic curative mechanisms, especially for viruses that infect a large number of parenchymal cells in vital organs, like the liver. Indeed, it has been recently suggested that the control of murine CMV infection in the liver may depend on IFN-γ–dependent noncytopathic mechanisms ( 30 ). Conversely, viral clearance from nonparenchymal cells might rely mostly on immune-mediated cytodestructive mechanisms. | Other | biomedical | en | 0.999997 |
10330435 | The single targeting vector consisted of 6 kb of the IL-13 gene providing the 5′ arm of homology and 4.0 kb of the IL-4 gene comprising the 3′ homology. The replacement vector was constructed to insert the neomycin resistance gene into an engineered SalI site in exon 3 of the IL-13 gene. Stop codons in all three frames were inserted 5′ of the selectable marker. The IL-4 region was a HindIII fragment containing exon 4. The targeting vector was linearized and electroporated into E14.1 embryonic stem (ES) 1 cells ( 7 ). Of 500 G418-resistant clones screened by Southern blot analysis, using a probe made with PCR primers (TGACCACAGGCAGTTTCACCTGC and TTATCATCTCAGCCTCATATACAG), one was found to be correctly targeted. Hybridization with a probe to the neomycin sequence and IL-13 cDNA sequences confirmed the predicted size of the targeted fragment and that only a single integration had occurred. The targeted ES cell clone was microinjected into 3.5-d C57BL/6 blastocysts to generate chimeras. These mice were mated with C57BL/6 mice and transmitted the ES cell genotype through the germline. Mice homozygous for the disrupted IL-4 and IL-13 genes were obtained by interbreeding the heterozygotes. The IL-4/13 gene–targeted, IL-13 gene–targeted ( 7 ), and wild-type animals used in the experiments reported below were maintained on a 129 × C57BL/6 (F 2 ) background in a specific pathogen–free environment. IL-4 −/− mice ( 1 ) had been backcrossed 10 times onto C57BL/6. Wild-type C57BL/6 mice were included as controls in the schistosome egg experiments and N . brasiliensis experiments, and showed similar phenotypes to the wild-type 129 × C57BL/6 (F 2 ) controls. Therefore, for the sake of clarity only the wild-type 129 × C57BL/6 (F 2 ) controls are presented. Splenocytes were cultured on plastic tissue culture plates for 1 h at 37°C to remove macrophages. Nonadherent cells were incubated with biotinylated anti–I-A b antibody (clone AF6-120.1; Becton Dickinson ), biotinylated anti-CD8 antibody (clone 53-6.7; Becton Dickinson ) and biotinylated anti-B220 antibody (clone RA3-6B2; Becton Dickinson ), and streptavidin magnetic beads (MACS ® ; Miltenyi Biotec) followed by magnetic field separation to remove MHC class II, CD8, and B220-expressing cells. Cell purity was determined using FITC-labeled anti-CD4 and PE-labeled anti-CD8 antibodies and was generally 90–95% CD4 + cells. Purified cells were cultured on anti-CD3ε antibody–coated plates (10 μg/ml of clone 2C11; Becton Dickinson ) plus anti-CD28 antibody (1 μg/ml of clone 37.51; Becton Dickinson ) in the presence of exogenous cytokines or anticytokine antibody as indicated. IL-2 (10 ng/ml; R&D Systems) was added to all cultures. Th2 cell differentiation was promoted in the presence of 100 ng/ml IL-4 (R&D Systems) and anti–IFN-γ antibody (10 μg/ml of clone XMG1.2; Becton Dickinson ). Cells were cultured for 5 d, washed, and resuspended at 10 6 cells/ml for 24 h in the presence of anti-CD3. Supernatants were analyzed by cytokine ELISA performed as above. Synchronous pulmonary granulomas were induced by intravenous injection of mice with Schistosoma mansoni eggs. S . mansoni eggs were isolated from the livers of infected mice as described ( 25 ). Mice were sensitized to schistosome eggs by intraperitoneal injection of 5,000 live eggs. 2 wk later, sensitized and naive mice, six mice per group, were injected intravenously with 5,000 eggs to induce synchronous pulmonary granulomas. 15 d after intravenous egg injection, mice were killed, serum was recovered, and the draining mediastinal lymph nodes were removed. The lungs were inflated with formol saline and processed for histology. The size (diameter on hematoxylin and eosin [H&E]-stained sections) and cell composition (percentage of eosinophils on Giemsa-stained sections) of the granuloma surrounding individual eggs were measured with an ocular micrometer using a double blind protocol by an investigator not involved in the study; >100 individual granulomas were analyzed per group. Cells were prepared from pooled mediastinal lymph nodes from each group and processed for cell culture as described ( 25 ). 3 × 10 6 cells/ml were cultured and restimulated with 10 μg/ml of soluble egg antigen. IL-4, IL-5, IL-10, and IFN-γ were assayed using ELISA. Egg antigen–specific IgE, IgG1, and IgG2a isotype responses were measured using ELISA as described ( 26 ). Statistical analysis was performed using analysis of variance (ANOVA) and Dunnett's test; P < 0.05 was considered significant. Individual mice were inoculated subcutaneously with 400 viable third-stage N . brasiliensis larvae. 8–10-wk-old mice were immunized intraperitoneally with 100 μg of OVA adsorbed to aluminium hydroxide (OVA/alum) with subsequent boost injections with 100 μg of OVA/alum after 10 and 20 d. Serum samples were assayed for Ig isotypes. Serum Igs were assayed using sandwich ELISA. 96-well plates were coated with anti-Ig isotype capture mAbs, and bound Ig of diluted serum samples was detected using biotinylated anti-Ig isotype detection mAbs ( Becton Dickinson ). Concentrations were calculated using purified Ig isotypes as standards ( Becton Dickinson ). OVA-specific ELISAs were performed by coating 96-well plates with OVA at 2.5 μg/ml; bound Ig of diluted serum samples was detected using biotinylated anti-Ig isotype detection mAbs ( Becton Dickinson ). Cytokine ELISA also used the sandwich format, with capture and detection antibodies purchased from Becton Dickinson . ELISAs were performed according to Becton Dickinson 's ELISA protocol. The IL-13 ELISA was purchased from R&D Systems. Since the IL-4 and IL-13 genes are closely linked on mouse chromosome 11 (∼11 kb apart), it would be impractical to attempt to mate IL-13–deficient mice with IL-4–deficient mice in order to generate a crossover event between these two genes. Therefore, we have used a single targeting construct to exploit their juxtaposition and simultaneously target both genes. The mouse IL-4 and IL-13 genes are transcribed in the same orientation, with the IL-13 gene lying upstream of the IL-4 gene (A.N.J. McKenzie, unpublished data). The targeting vector comprised a 5′ arm of homology derived from the IL-13 gene, and a 3′ arm of homology derived from the IL-4 gene positioned on either side of the neomycin resistance cassette . The resulting homologous recombination event excises ∼15 kb of intervening sequence. Genotyping of wild-type (IL-4 +/+ IL-13 +/+ ), heterozygous (IL-4 +/− IL-13 +/− ), and homozygous null (IL-4 −/− IL-13 −/− ) mice is shown in Fig. 1 B. The IL-4 −/− IL-13 −/− mice were healthy and displayed no overt phenotypic abnormalities. Analysis of the IL-4 −/− IL-13 −/− mice failed to detect IL-4 or IL-13 RNA transcripts from activated lymphocytes using reverse transcriptase PCR assays (data not shown), and ELISAs also failed to identify IL-4 or IL-13 protein in supernatants from spleen-derived CD4 + T cells cultured under Th2 cell differentiation conditions . To determine the relative in vivo roles of IL-4 and IL-13, we have assessed the immune responses of the IL-4 −/− IL-13 −/− animals, in combination with IL-4–deficient mice (IL-4 −/− ) and IL-13–deficient mice (IL-13 −/− ), to a range of immunological challenges that normally provoke a Th2 phenotype. To determine the relative in vivo contribution of IL-4 and IL-13 in a Th2 cytokine–mediated inflammatory response, we used a model system in which synchronous pulmonary granuloma formation is induced around S . mansoni eggs ( 27 ). In this model, a cellular granulomatous response develops around parasite eggs that lodge in the lungs after their intravenous injection into mice. This inflammatory response is characterized by the high-level expression of Th2 cytokines ( 28 ). We observed a profound inability of IL-4 −/− IL-13 −/− mice to develop the Th2 cell–mediated inflammatory response normally generated during synchronous pulmonary granuloma formation in response to schistosome egg immunization . This was in marked contrast to the response generated by wild-type mice in which large eosinophil-rich granulomas formed around eggs lodged in the lung alveoli . Furthermore, this wild-type response was associated with expression of IL-4, IL-5, IL-10, and IL-13 by cells recovered from the draining mediastinal lymph nodes of the lung and the generation of increased titers of egg antigen–specific IgE and IgG1 . By contrast, the IL-4 −/− IL-13 −/− mice did not develop granulomas , and eosinophil infiltration was virtually absent, although some monocyte infiltration was evident . Correlating with this, schistosome egg–challenged IL-4/13–deficient mice also expressed very low levels of IL-5 , the primary cytokine in the induction of eosinophil differentiation ( 29 ). IL-10 production was also impaired, but there was a significant increase in the expression of IFN-γ . Antigen-specific IgE was also not detected in the serum of IL-4 −/− IL-13 −/− mice, and antigen-specific IgG1 was virtually absent . We also assessed how IL-4–deficient mice and IL-13– deficient mice responded to schistosome egg challenge. Although granuloma size and eosinophil infiltration were impaired in both single cytokine–deficient IL-4 −/− and IL-13 −/− mice, compared with wild-type , they continued to develop a Th2 response with the expression of IL-5 and the infiltration of eosinophils . Significantly, the granuloma response observed in both of the single cytokine gene–disruption mouse lines was substantially greater than that formed in the doubly targeted mice . It is also noteworthy that both the IL-4 −/− and IL-13 −/− mice continued to produce egg antigen– specific IgE and IgG1, although the levels of these Ig isotypes were reduced relative to wild-type mice . Thus, the pulmonary granulomatous model demonstrates that mice deficient in either IL-4 or IL-13 are still capable of mounting a Th2-like response, albeit reduced compared with wild-type mice, but that simultaneous disruption of IL-4 and IL-13 results in abrogation of the granulomatous response. This demonstrates that IL-4 and IL-13 perform compensatory roles that are essential in combination for the successful development of a Th2 cell–driven inflammatory response, and that in their absence the response becomes dominated by the Th1 cell cytokine IFN-γ . Immunological responses to gastrointestinal parasitic worm infections are also characterized by the expression of Th2 cytokines ( 30 ). Using N . brasiliensis as a model, recent studies have shown that although IL-4–deficient mice expel these worms almost as efficiently as wild-type animals ( 16 , 17 ), IL-13–deficient mice display impaired expulsion kinetics ( 16 ). We have infected the IL-4 −/− IL-13 −/− mice with N . brasiliensis and compared the kinetics of worm expulsion with that of wild-type and IL-13 −/− animals . As expected, the wild-type mice expelled their worms rapidly, with complete expulsion by day 10 post- infection (p.i.), whereas the expulsion of worms from the IL-13 −/− mice was delayed beyond 10 d . Significantly, the combined ablation of both cytokines further delayed the expulsion of N . brasiliensis , with substantially more worms present at day 14 p.i., even when compared with IL-13 −/− animals . Thus, although IL-13 is apparently the primary cytokine regulating N . brasiliensis expulsion, IL-4 does play an additional role in this process. Interestingly, we have also found that treatment of IL-13 −/− mice with recombinant IL-4 results in the rapid expulsion of worms from these animals (data not shown). Thus, once again there is a dual effect of removing both IL-4 and IL-13, indicating that these cytokines act in combination to initiate a potent Th2 response. The normal immune response to N . brasiliensis is characterized by Th2 cytokine expression, elevated levels of IgE expression, and eosinophilia . Indeed, wild-type animals developed a profound eosinophilia by day 10 p.i. , and total serum IgE had increased by ∼100-fold by day 14 p.i. . Unexpectedly, we found that IL-5 expression and eosinophilia were still induced in the IL-4 −/− IL-13 −/− mice p.i. with N . brasiliensis , although their production was significantly delayed . However, we failed to detect IgE expression in the serum of the IL-4 −/− IL-13 −/− mice , and levels of worm antigen–specific IgG1 were also undetectable (data not shown). Thus, we conclude that although IL-4 and IL-13 are required for the rapid initiation of Th2-like responses, alternative IL-4/IL-13–independent processes compensate for their loss and facilitate the expression of IL-5 and the coordinate development of eosinophilia. Analysis of total serum Ig isotypes demonstrated that like the IL-4–deficient animals, mice lacking both IL-4 and IL-13 had 10–50-fold lower levels of serum IgG1 and undetectable levels of IgE, whereas other serum isotypes remained similar to wild-type (data not shown). However, due to the specific roles of Th2 cell cytokines in regulating humoral immune responses, we have also analyzed the antigen-specific Ig responses of wild-type, IL-4 −/− , IL-13 −/− , and IL-4 −/− IL-13 −/− mice immunized with the protein antigen OVA complexed to alum. As shown in Fig. 6 A, the IL-4 −/− IL-13 −/− animals are severely impaired in their ability to mount an IgG1 response, a deficiency also apparent in IL-4–deficient animals . By contrast, IL-13 −/− animals develop a normal IgG1 response to antigen challenge . Thus, the regulation of antigen-specific IgG1 responses appears to require IL-4, but not IL-13. However, cumulative roles for IL-4 and IL-13 in the generation of the antibody responses are suggested by the highly biased IgG2a and IgG2b responses evoked upon immunization of the IL-4 −/− IL-13 −/− animals . These isotype profiles are typical of a Th1 response and are significantly elevated. The generation and analysis of IL-4/13–deficient mice has enabled us to demonstrate conclusively that these cytokines cooperate in the development of Th2 cell–mediated immune responses. Due to the close proximity of the IL-4 and IL-13 genes, we used a single vector targeting strategy to disrupt the expression of both cytokine genes, thereby allowing us to dissect the potential compensatory roles played by these cytokines. To define their functional importance, we have used several model antigenic challenges that are normally characterized by Th2-like responses. We have found that the Th2-like characteristics of synchronous pulmonary granuloma formation, including eosinophil infiltration and IgE production, are only abolished when both IL-4 and IL-13 are removed, whereas disruption of each individual cytokine resulted in only partial abrogation of the response. Thus, our data demonstrate for the first time that IL-13 also plays a significant role in granuloma formation and, since neither cytokine is fully able to compensate for the absence of the other, they illustrate a clear functional specificity for IL-4 and IL-13 in the development of the response. Even more significantly, the combined disruption of IL-4 and IL-13 results in the almost complete abolition of the Th2-driven granuloma response, demonstrating the additive roles of these two cytokines in the generation of Th2 responses. In the double-deficient mice, the virtual absence of eosinophil infiltration, antigen-specific IgE, and antigen-specific IgG1 is replaced by enhanced expression of IFN-γ and the upregulation of IgG2a (data not shown), both indicative of Th1-like responses. These data generated using the ligand-deficient mice also clarify any ambiguity raised by the results that have been generated using signal transducer and activator of transcription (Stat)6-deficient and IL-4Rα–deficient mice in which pulmonary granuloma responses were reported to be more significantly reduced than in the IL-4–deficient mice ( 31 – 34 ). While previous studies have demonstrated that IL-13 plays a unique and dominant role in the efficient expulsion of N . brasiliensis , the double-deficient mice have enabled us to identify an additive role for IL-4 in this process. These data are supportive of studies in which the exogenous administration of IL-4 induced worm expulsion ( 35 ) and our own experiments showing that exogenous IL-4 can induce rapid expulsion of N . brasiliensis from the intestines of IL-13–deficient mice (data not shown). However, since IL-4–deficient mice expel N . brasiliensis worms normally ( 16 , 17 ), the most significant defect in the clearance of these parasites is the absence of IL-13. Interestingly, although Th2 responses to N . brasiliensis infection are significantly delayed in the IL-4/13–deficient mice, our experiments have also identified alternative, IL-4– and IL-13–independent mechanisms for IL-5 production and eosinophilia in IL-4/13–deficient mice. Thus, even in the absence of IL-4 and IL-13, a response can develop Th2-like characteristics. The mechanism underlying the development of IL-5–producing cells is unclear. Such cells have been recognized in IL-4–deficient mice ( 2 ) and IL-4Rα– deficient mice ( 3 ) infected with N . brasiliensis , but normally only constitute a minor population when evaluated at 7 d p.i. This is also the case in the IL-4/13–deficient mice, where IL-5 expression at day 6 p.i. is very low. However, IL-5 levels in the double-deficient mice become highly elevated by day 10 p.i., indicating that, although significantly delayed, IL-5–producing cells can receive sufficient costimulation to expand in the absence of IL-4 and IL-13 signals. Since this phenomenon was not observed in the lung response to schistosome eggs, it may indicate a unique feature of the intestinal response to nematode infection. To date, IgE responses by IL-4/13–deficient mice to a range of antigenic challenges have remained below the level of detection in ELISA assays, although IgE responses were detected in individual cytokine–deficient mice. These findings support previous studies which have identified IL-13 and IL-4 as alternative cytokines in the regulation of IgE expression ( 1 , 2 , 7 , 36 ). Furthermore, in the combined absence of IL-4 and IL-13, the Ig response becomes more characteristic of a Th1 cell response, illustrating the additive roles played by these cytokines in the regulation of Ig expression. This was clearly demonstrated by assessing the antigen-specific Ig isotype response generated against protein antigen OVA immunization in the presence of the Th2- inducing adjuvant alum. Although the expression of IgG2a and IgG2b was only minimally enhanced after the individual disruption of IL-13, there was a significant increase when both IL-13 and IL-4 are cotargeted, indicating that at least in the regulation of Ig expression IL-4 appears able to compensate more effectively for the loss of IL-13 than IL-13 does after the disruption of IL-4. The development and analysis of mice with a combined deficiency for IL-4 and IL-13 expression have clearly demonstrated that IL-4 does not act in isolation in the development of Th2 cell responses. It is noteworthy that IL-4 and IL-13 share the α chain of the IL-4 receptor ( 37 ) and consequently signal through related pathways, including Stat6 ( 38 ). Our results now explain why IL-4Rα–deficient mice and Stat6-deficient mice display more severe phenotypic differences than were reported for the IL-4–deficient mice ( 3 – 5 , 17 ). It is apparent that IL-4 and IL-13 act in conjunction to ensure the rapid onset of a Th2-like response and that in their combined absence the vestiges of the Th2 response are abolished or significantly delayed. Th2 cell–driven responses, particularly IgE and eosinophilia, are instrumental in disease processes, including allergies, asthma, and helminth infections ( 29 , 30 , 39 , 40 ). Indeed, recent studies have identified that IL-13 is also a major mediator in experimental models of allergic asthma ( 41 , 42 ). Our findings have obvious implications for the development of therapeutic strategies for cytokine/anticytokine modulation of immune reactions. Thus, to control the initiation of Th2 cell responses and the abolition of IgE production, it appears that it will be necessary to inactivate both IL-4 and IL-13, and that even then default mechanisms may allow the development of eosinophilia. The IL-4/ 13–deficient mice will prove an important tool for dissecting the intimate interaction of these cytokines in numerous disease states. | Study | biomedical | en | 0.999995 |
10330436 | Rat anti–mouse FcγRIIB/III (2.4G2; PharMingen ) and mouse anti-TNP IgE (IGELa2; American Type Culture Collection) and anti-TNP IgG1 (G1; 15) were purified from the ascites of hybridomas by ion exchange chromatography on DEAE– cellulose (Merck) ( 16 ) and by affinity isolation with protein G column ( 17 ), followed by removal of aggregated materials by ultracentrifugation at 130,000 g for 90 min at 20°C. All experiments were performed on 6–12-wk-old mice. Male and female FcγRIIB −/− ( 11 ) or FcγRIII −/− mice (Y. Ishikawa, J.V. Ravetch, and T. Takai, unpublished results) were generated by breeding the F2 offspring of crosses between chimeras and C57BL/6 mice, and the wild-type mice generated by the same breeding protocol were used as wild-type animals. FcγR −/− mice were generated as described previously ( 3 ) and back-crossed to C57BL/6 background over six generations. FcγRIII −/− mice were generated using RW4 embryonic stem cells (GenomeSystems Inc.) as described previously ( 3 , 11 ). Mice were housed in cages in cabinets supplied with high efficiency particulate-free air and were monitored monthly as specific pathogen free. Mouse IgG1 or IgE anti-TNP mAbs were administered intravenously through the tail vein in volumes of ∼200 μl/mouse. 30 min after injection of anti-TNP IgG1 or 24 h after injection of IgE, mice were injected with 1.0 mg i.v. TNP 4 -OVA in PBS. Control mice received OVA in PBS instead. The concentration of IgG1 and IgE mAbs used for passive sensitization and the amount of TNP-OVA used for challenge was determined based on preliminary dose–response experiments required to produce significant drops in body temperature in wild-type and FcγRIIB −/− or FcγRIII −/− mice. Alternatively, systemic anaphylaxis was induced by the intravenous injection of 10 μg 2.4G2 in 200 μl PBS. The amount was determined based on the preliminary dose–response experiment in the same way described above. In a blocking experiment in FcγRIII −/− mice, 100 μg 2.4G2 was administered. Changes in core body temperature associated with systemic anaphylaxis were monitored by measuring changes in rectal temperature using a rectal probe coupled to a digital thermometer (Natsume Seisakusyo Co.) as described ( 4 , 9 , 10 ). Heart rate was recorded as electrocardiograms (Nihon Kohden) of mice under 2,2,2-tribromoethanol (0.25 mg/g body weight, i.p.) anesthesia. Bone marrow–derived cultured mast cells (BMMC) were prepared as described previously ( 3 ). For monitoring of upregulation of FcεRI protein on BMMC membrane, cells were cultured in the presence of 0.1 or 5 μg/ml biotinylated IgE or 5 μg/ml biotinylated 2.4G2 for 4 d before final staining with biotinylated IgE (5 μg/ml) plus PE-conjugated streptavidin. Peritoneal resident cells were collected by washing with Tyrode's buffered solution and incubated with 5 μg/ml IgE for 20 min at 4°C to saturate IgE binding to FcεRI, followed by staining with FITC-conjugated rat anti–mouse IgE (Serotec Ltd.) for 20 min at 4°C. Flow cytometric analyses were performed with FACSCalibur™ ( Becton Dickinson ), and peritoneal mast cells were sorted as c-kit and IgE-positive cells as described ( 18 ). Blood was collected from subocular plexus of mice into microcentrifuge tubes containing EDTA on ice at 5 min after antigen challenge, and plasma was prepared. Histamine in the plasma samples was quantified using ELISA plates (ICN Pharmaceuticals, Inc.) according to the manufacturer's instructions. Mice were killed by cervical dislocation. Their tissues were removed and fixed in 10% (vol/vol) neutral buffered formalin and then embedded in paraffin. The specimens were sectioned at 3 μm and stained with toluidine blue at pH 4.0. The number of mast cells/mm 2 was determined under a light microscope. A ‘degranulated' mast cell was defined as a cell showing extrusion of >10% cell granules. Statistical differences were calculated using Student's t test or Fisher's test. P < 0.05 was considered significant. Bocek et al. ( 7 ) reported that coclustering of FcγRIIB and FcγRIII on RBL-2H3 cells did not lead to stimulation of the cells, suggesting a possible inhibitory role of FcγRIIB in this process. In addition, in vitro observations by Daëron et al. ( 12 ) demonstrated that mast cell secretory responses triggered by FcεRI may be controlled by FcγRIIB/III. Moreover, the regulatory role of FcγRIIB was also observed in the cellular activation process via B cell receptors ( 19 – 21 ) and T cell receptors (13; for review see reference 14 ). Our previous studies using gene-targeted mice had demonstrated the role of FcγRIIB in modulating IgG1-mediated passive cutaneous anaphylaxis ( 11 ). To establish the generality of those in vivo observations, we investigated IgG1-mediated passive systemic anaphylaxis in FcγRIIB −/− and FcγRIII −/− mice. We chose to evaluate a passive rather than active model in our studies because FcγRIIB − / − mice display enhanced humoral immune responses ( 11 ) that could complicate the comparison and interpretation of the anaphylactic responses. To elicit the anaphylactic response, mice were injected intravenously with IgG1 specific for TNP, followed by intravenous administration of TNP-OVA 30 min later. Fig. 1 A shows that FcγRIIB − / − mice developed an enhanced IgG1-dependent passive systemic anaphylactic response as compared with passively sensitized wild-type controls challenged with TNP-OVA. In wild-type mice, the decrease in core temperature was also transient, reaching a nadir ∼15 min after induction, whereas the drop in temperature of FcγRIIB − / − mice persisted for more than 30 min without returning to baseline. The mAb 2.4G2 is specific for the extracellular domains of murine FcγRIIB and FcγRIII ( 22 ). 2.4G2 induces a degranulative response in BMMC, which is enhanced in cells derived from FcγRIIB −/− mice ( 11 ). This enhancement is apparent in vivo as well as shown in Fig. 1 B, where the decrease in core temperature after administration of 2.4G2 was more pronounced in FcγRIIB −/− mice than in control mice. These results indicate that FcγRIIB on effector cells, such as mast cells, inhibits the systemic anaphylaxis elicited via FcγRIII. In contrast to the enhanced responses in FcγRIIB −/− mice described above , both FcγRIII −/− mice and FcRγ −/− mice failed to develop IgG1-mediated passive systemic anaphylaxis , directly establishing that IgG1-mediated anaphylaxis is triggered through FcγRIII, as was indirectly suggested by others ( 9 , 10 ). As IgE immune complexes can bind with low affinity to FcγRII and III in vitro, we next induced passive systemic anaphylaxis upon anti-TNP IgE adoptive transfer and TNP-OVA administration into FcγRIIB −/− mice. IgE-mediated systemic anaphylaxis was significantly enhanced in FcγRIIB −/− mice, as assessed by changes in core temperature , heart rate , and augmented hemorrhage in the ileum villi . These results indicate that IgE/FcεRI-mediated anaphylaxis is facilitated by the deletion of FcγRIIB in vivo without any apparent involvement of IgG-immune complexes. Systemic anaphylaxis can result in a fatal outcome. In mice, this mortality has been shown to be associated with IgG1 and FcγRIII ( 9 ). As shown in Table I , we observed mortality as a consequence of the anaphylactic response only in FcγRIIB −/− mice upon administration of either IgG1 or IgE and the corresponding antigen, or 2.4G2. These results confirm that either IgE- or IgG-induced systemic anaphylaxis is indeed augmented in FcγRIIB −/− mice, as assessed by mortality during anaphylaxis. These unexpected observations for IgE-mediated anaphylaxis prompted us to examine whether deletion of FcγRIIB influenced FcεRI expression levels on effector cells. We confirmed by flow cytometric analysis that the expression level of FcεRI on BMMC from FcγRIIB − / − mice was comparable to the level on wild-type BMMC (data not shown). In addition, we could not demonstrate any significant difference in the expression levels of FcεRI on mast cells after IgE-induced upregulation in vitro or in vivo . As shown in Fig. 3 A, BMMC derived from either from FcγRIIB − / − or wild-type mice displayed the same level of upregulation of FcεRI in response to IgE ( 18 ). Similarly, peritoneal mast cells isolated from FcγRIIB − / − and wild-type mice 24 h after intravenous administration of 20 μg IgE had equivalent levels of FcεRI . Histopathological examinations indicated that the density and morphology of mast cells in ear, abdominal skin, and trachea from the mutant mice were not significantly different from those in wild-type mice (data not shown). The mechanism by which FcγRIIB −/− mice augmented IgE-mediated anaphylaxis was examined by determining the activation of effector cells in these animals as compared with their wild-type counterparts. Blood histamine levels were measured after the induction of anaphylaxis in FcγRIIB −/− and wild-type mice. As shown in Fig. 4 A, blood obtained both from wild-type or FcγRIIB −/− -sensitized animals 5 min after challenge with antigen or 2.4G2 revealed increased histamine concentrations. The histamine levels seen in FcγRIIB −/− -challenged mice were consistently higher in response to IgE, IgG1, or 2.4G2 stimulation than in control mice, suggesting that the enhanced anaphylaxis in FcγRIIB −/− mice could be interpreted in part by accelerated activation of mast cells in the mutant animals. To directly demonstrate enhanced degranulation, lung samples from FcγRIIB −/− or wild-type mice were removed before and 30 min after the induction of IgG-mediated passive systemic anaphylaxis and examined histopathologically. As shown in Fig. 4 B and E, mast cells around bronchi in FcγRIIB −/− mice displayed quantitatively more degranulation than comparable samples taken from wild-type mice subjected to similar treatment. Although Takizawa et al. ( 6 ) demonstrated that FcγRIIB and FcγRIII act as low-affinity receptors for IgE on cultured mast cells and macrophages in vitro, the physiological significance of this interaction between IgE and FcγRIIB/III has not been established. The consequence of a low-affinity interaction between IgE and FcγRs in vivo would result in IgE immune complexes binding not only to FcεRI but also to FcγRIIB/III on those cells and potentially modulating mediator release. Dombrowicz et al. ( 4 ) have shown that although BMMC from FcεRI −/− mice can bind IgE immune complexes via FcγRIIB/III in vitro, the abrogation of IgE-mediated systemic anaphylaxis in vivo by deletion of FcεRI would indicate that the interaction of IgE with FcγRs is not significant. However, an alternative explanation for their data is suggested by the present studies, as the FcεRI −/− strain retains FcγRIIB as well as FcγRIII on its mast cells ( 4 ). Based on our data, we propose that the IgE immune complex–mediated response would represent the sum of three components, i.e., an FcεRI-mediated major positive factor, an FcγRIIB negative response, and an FcγRIII-mediated positive component, respectively. When the FcεRI component had been lost, the sum of the remaining FcγRIIB and FcγRIII components would be negligible. Our present results predict that a sum of the components of FcεRI and FcγRIIB would be a positive, although diminished, response. This prediction is supported by the IgE-mediated anaphylactic response in FcγRIII −/− mice. As shown in Fig. 5 A, FcγRIII −/− mice indeed show a decreased response in IgE-mediated systemic anaphylaxis. Moreover, we found that blocking of FcγRIIB by preadministration of 2.4G2 resulted in an enhanced response in IgE-mediated systemic anaphylaxis in FcγRIII −/− mice . Taken together, these results support the conclusion that FcγRIIB attenuates IgE-mediated anaphylactic responses triggered by FcεRI or FcγRIII. Further support for the role of FcγRIIB in modulating the IgE-mediated response comes from studies in Src homology 2–containing inositol phosphatase (SHIP)-deficient mice ( 23 ). This inositol polyphosphate phosphatase is recruited to FcγRIIB upon cross-linking with an immunoreceptor tyrosine-based activation motif (ITAM)-containing activation receptor through its SH2 (Src homology 2) domain and leads to the hydrolysis of phosphatidylinositol 3,4,5-trisphosphate, with release of Bruton's tyrosine kinase and phospholipase Cγ from the inner leaflet of the cell membrane ( 24 ). The net result of this pathway is the termination of calcium influx, with subsequent inhibition of activation responses ( 20 , 21 , 25 ). Mast cells derived from SHIP-deficient mice display a hyperresponsive IgE phenotype similar to the response seen in FcγRIIB −/− mice ( 26 ). Thus, functional uncoupling of FcγRIIB from its signaling pathway results in similar phenotype deletion of the receptor itself. The observations presented here support the hypothesis that IgE-mediated activation is modulated by inhibitory receptors like FcγRIIB. Perturbation of an inhibitory pathway would be predicted to render mast cells more sensitive to IgE activation and could account for some atopic phenotypes. Upregulation of FcγRIIB or its constitutive engagement would result in desensitization of mast cells to IgE triggering and reversal of the atopic state. | Study | biomedical | en | 0.999997 |
10330437 | The T cell hybridomas 3DO, 2B4, and the human cell line Jurkat were grown in RPMI 1640, containing 2 mM glutamine, 25 nM β-mercaptoethanol, 10 mg/ml streptomycin, 10 mg/ml gentamicin. Anti-CD3ε was purified on a protein A–Sepharose column from culture supernatants of the 2C11 hybridoma cell line. FITC-conjugated 2C11, anti–mouse TCR-β H57, anti–human TCR HIT3a, anti–mouse Fas Jo2, and anti–hamster IgG Abs were purchased from PharMingen . Anti–mouse CD28 37.51 was diluted from ascites fluids ( 23 ). Anti–mouse CD45R B220 Ab conjugated to microbeads was from Miltenyi Biotech GmbH. Anti–mouse class II mAb, clone M5/114 ( 24 ), was biotinylated. FB1, CM, and bacterial SMase (bSMase) were purchased from Biomol, and daunorubicin and cyclosporin A (CsA) were from Sigma . Splenic T cells from Balb/c mice were isolated by negative depletion of B cells and class II + cells. B lymphocytes and macrophages were coated with B220 conjugated to microbeads and biotinylated rat M5/114 Abs, followed by incubation with microbeads conjugated to streptavidin. The cells were passed through a MACS column in the MACS separator, and the flow through was recovered. The purity of the T cell preparation was 85–92% as determined by staining of the recovered cells with FITC-conjugated 2C11 Ab. For TCR stimulation, 3DO and 2B4 cells were seeded on plates coated overnight at 4°C with 1 μg/ml of 2C11 Ab. For Fas stimulation, cells were preincubated for 30 min at room temperature with 1 μg/ml of anti– mouse Fas Ab Jo2, washed twice with medium, and seeded on plates coated overnight at 4°C with 5 μg/ml of anti–hamster IgG. Splenic T cells were stimulated using plates coated overnight with 1 μg/ml of H57 Ab. For costimulation, the anti– mouse CD28 37.51 Ab was added in solution at a 1:5,000 dilution from ascites fluids. Jurkat cells were cultured on plates coated overnight at 4°C with 5 μg/ml of anti–human TCR Ab HIT3a. FB1 was added to the cell cultures 30 min before stimulation. Daunorubicin was used at 10 μM. In the experiments shown in Fig. 4 , exogenous CM (10 μM), nSMase (0.1 U/ml), and CsA (100 nM) were added to the cell cultures at the time of TCR stimulation. The percentage of cell death was assessed by measuring the DNA content of isolated nuclei stained with propidium iodide ( 25 ). IL-2 production was determined by ELISA ( Genzyme ). Expression of FasL and Nur77 mRNAs was quantitated by Northern blot analysis. Each lane was loaded with 20 μg of total RNA isolated from cells treated with 2C11 for the indicated time. The amount of mRNA loaded was normalized by using cyclophilin expression as an internal control. CM was quantified by the diacylglycerol kinase assay. After incubation with the indicated stimuli, cells were pelleted by centrifugation, washed twice with ice-cold PBS, and extracted with chloroform/methanol/1 N HCl (100:100:1, vol/vol/vol). The dried samples were resuspended in a 40-μl reaction mixture containing 5 mM of cardiolipin (Avanti Polar Lipids), 1 mM diethylenetriaminepentaacetic acid (DTPA; Sigma ), 7.5% octyl-β- d -glucopyranoside ( Calbiochem ), 10 mM imidazole. After five cycles of freeze–thaw, the reaction was started by adding to the lipids suspension 100 μl of reaction buffer (100 mM imidazole-HCl, pH 6.6, 100 mM NaCl, 25 mM MgCl 2 , and 2 mM EGTA), 20 μl of 20 mM dithiothreitol, 19 μl cold 10 mM ATP, 10 μCi of [γ- 32 P]ATP (3,000 Ci/mmol; DuPont-NEN ), and 10 μl Escherichia coli diacylglycerol kinase ( Calbiochem ). After 1 h at room temperature, the reaction was stopped by adding 0.5 ml of CHCl 3 /CH 3 OH/1 N HCl (100:100:1) and 85 μl of PBS, pH 7.4. 100 μl of the lower organic phase was resolved by thin-layer chromatography on silica gel 60 plates (Whatman) using a solvent system of chloroform/acetone/methanol/acetic acid/water (10:4:3:1) and visualized by autoradiography. Incorporated 32 P was quantified by scintillation counting. The level of CM was determined by comparison with a standard curve generated concomitantly with known amounts of CM (CM type III; Sigma ). CM synthase activity was determined as described previously ( 26 ). In brief, 75 × 10 6 cells were pelleted, washed once with cold PBS, and resuspended in 300 μl of homogenization buffer (25 mM Hepes, pH 7.4, 5 mM EGTA, 50 mM NaF, and 10 μg/ml each of leupeptin and soybean trypsin inhibitor). Cells were homogenized, and the lysates were centrifuged at 800 g for 5 min. The postnuclear supernatant was centrifuged at 250,000 g for 30 min. The microsomal membrane pellet was resuspended in 1 ml of homogenization buffer. Microsomal membrane protein (75 μg) was incubated in 1 ml reaction mixture containing 2 mM MgCl 2 , 20 mM Hepes (pH 7.4), 20 μM defatted BSA ( Sigma ), varying concentrations (0.2–20 μM) of dihydrosphingosine (Biomol), 70 μM unlabeled palmitoyl-coenzyme A (palmitoyl-CoA; Sigma ), and 3.6 μM (0.2 μCi) [1- 14 C] palmitoyl-CoA (55 mCi/mmol; NEN Life Science Products). The reaction was started by addition of palmitoyl-CoA, incubated at 37°C for 1 h, and then stopped by extraction of lipids using 2 ml of chloroform/methanol (1:2). Lower phase was removed, concentrated, and applied to a silica gel 60 thin-layer chromatography plate. Dihydroceramide was resolved from free radiolabeled fatty acid using a solvent system of chloroform/ methanol/3.5 N ammonium hydroxide (85:15:1), identified by iodine vapor staining based on comigration with CM standards, and quantified by liquid scintillation counting. The cells were harvested and washed three times with ice-cold PBS, 100 μM Na 3 VO 4 at 4°C. Cells were disrupted by five cycles of freezing–thawing (in methanol/dry ice) in 100 μl of lysis buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM EGTA, 1 mM sodium vanadate, 10 mM β-glycerol phosphate, 1 mM PMSF, 5 mM dithiothreitol, 20 μg/ml each of chymostatin, leupeptin, antipain, and pepstatin. The lysate was centrifuged for 10 min at 1,000 g at 4°C, and the supernatant (post-nuclear homogenate) was centrifuged for 60 min at 100,000 g at 4°C. The resulting pellet (membrane fraction) was resuspended in 50 μl of lysis buffer. The membrane preparation was incubated for 30 min at 37°C with 14 C-SM (1,000,000 dpm, 10 nmol) in a mixed micelle assay containing 100 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , and 0.1% Triton X-100 (final volume 100 μl). The reaction was stopped by adding 800 μl of CHCl 3 /CH 3 COOH (2:1, vol/vol) and 250 μl of water. The radioactivity was determined by liquid scintillation counting. To determine the aSMase activity, membranes were prepared from cells using lysis buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM EGTA, and 1 mM PMSF. The micelle assay used contained 100 mM sodium acetate, pH 5, and 0.1% Triton X-100. 3DO cells were grown for 48 h in the presence of 0.5 μCi/ml (80 Ci/mmol) [ 3 H]choline chloride. Post-labeling cells were washed with PBS, reseeded at 0.5 × 10 6 cells/ ml in RPMI, and rested for 2–4 h. Cells were then subjected to a variety of treatments. After treatment, cells were harvested and cell pellets were resuspended in 3 ml of chloroform/methanol (1:2). Standard Bligh and Dyer extraction was used to recover lipids. Lipids dried under vacuum were resuspended in 50–100 μl of chloroform and spotted on thin-layer chromatography plates, and plates were developed in chloroform/methanol/acetic acid/water (50:30: 8:5). Plates were sprayed with En 3 Hance and exposed to film for 24–48 h. The labeled SM spots were scraped into scintillation fluid and counted in a scintillation counter. Human nSMase cDNA was pulled out by PCR from a cDNA library derived from human fetal liver (Invitrogen) and cloned in pcDNA3.1 vector (Invitrogen). The library was screened using primers designed from the human nSMase sequence recently published ( 27 ). Transient transfections in Jurkat T cells were performed by electroporating 50 μg of the indicated cDNAs together with 1 μg of En 3 Hance Green Fluorescent Protein-N1 (EGFP; Clontech ). The efficiency of transfections was monitored by analyzing the percentage of EGFP + cells by flow cytometry. EGFP + cells were sorted using a Becton Dickinson FACStar™. Cells were lysed in a buffer comprised of 60 mM Tris-HCl, pH 7.8, containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, and phosphatase and protease inhibitors as described previously ( 28 ). Post-nuclear fractions were precleared with protein A–trisacryl beads (Pierce) and subjected to immunoprecipitation with a mixed mAb preparation directed against phospholipase Cγ1 (PLCγ1; Upstate Biotechnology) bound to protein A/G–agarose beads (Pierce). Proteins were eluted with sample buffer, resolved by SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes (Hybond-C super; Amersham Pharmacia Biotech ). Protein detection was via an antiphosphotyrosine primary Ab (4G10; Upstate Biotechnology) with a second Ab (rabbit anti– mouse IgG; Cappel) followed by 125 I–protein A (ICN Biomedicals). Immunoblots were stripped according to the membrane manufacturer's instructions and reprobed with other Abs. Immunoblots were scanned on a PhosphorImager (Molecular Dynamics) to produce the images shown, with no manipulation except for the adjustment of the exposure range. Densitometry was performed using ImageQuant™ software (Molecular Dynamics). To test the hypothesis that the sphingolipid pathway might be implicated in the apoptotic process initiated by TCR triggering, we used an inhibitor of sphingolipid synthesis, FB1 ( 29 ). As shown in Fig. 1 A, this compound protected the T cell hybridoma 3DO from TCR-induced cell death. Stimulation of the TCR on a T cell hybridoma induces FasL upregulation, and the engagement of Fas by FasL activates PCD ( 30 – 32 ). Considering that activation of the sphingolipid pathway has been described to occur after Fas triggering in several cellular systems ( 33 , 34 ), we also tested the effect of FB1 on Fas-induced cell death. This toxin did not affect Fas-triggered apoptosis , indicating that FB1 interfered with the TCR-induced death pathway upstream of Fas. A key event of TCR-induced apoptosis is upregulation of FasL. Our results raised the possibility that FB1 inhibited FasL induction after TCR triggering, which indeed turned out to be the case. FasL mRNA expression, detectable 4 h after TCR stimulation, was blocked by FB1 . This inhibition was specific for FasL, since expression of Nur77 mRNA, another gene known to be induced upon receptor triggering ( 35 , 36 ), was not affected . FB1 could either specifically interfere with PCD or compromise early events in TCR signaling. To differentiate between these two possibilities, we measured IL-2 levels in culture supernatants after TCR triggering. Pretreatment with FB1 inhibited IL-2 production in 3DO cells , indicating that this compound interfered with signaling events that are common to the activation and cell death pathways. To determine if this observation was true for normal T cells, the clone A.E7 was tested as well as freshly isolated splenic T lymphocytes . FB1 reduced production of IL-2 after simultaneous stimulation of either T cell population with anti-TCR and anti-CD28 Ab . Inhibition of IL-2 production and cell death by FB1, which is known to interfere with the de novo synthesis of CM by inhibiting CM synthase, suggested that CM may participate in early events in TCR signaling. Therefore, we tested whether TCR triggering induced production of CM. As shown in Fig. 2 A, intracellular CM concentration increased rapidly upon TCR stimulation. CM is produced by two distinct routes: de novo synthesis and SM hydrolysis by SMases ( 37 ). The de novo synthesis of CM via stimulation of CM synthase has been implicated in the apoptotic pathway ( 38 ). Since FB1 has been shown to inhibit this enzyme ( 38 , 29 ), we first tested whether CM production upon TCR triggering resulted from activation of CM synthase. As shown in Fig. 2 B, CM synthase activity was not increased upon TCR stimulation. Next, we analyzed whether TCR engagement induced activation of SMases, enzymes responsible for CM generation induced by stimulation of several membrane receptors, including nerve growth factor receptor (NGFR), TNFR, and Fas ( 39 ). SMases are known to exist in at least two forms, a Mg 2+ -dependent membrane-bound form with a neutral pH optimum (nSMase) and a lysosomal acidic form (aSMase) ( 37 ). Triggering of the TCR resulted in activation of nSMase . This activation was selective since the aSMase, which is readily stimulated upon Fas triggering, was not induced by the TCR . The time course of nSMase activation paralleled CM production as well as hydrolysis of the nSMase substrate, SM . Thus, the rapid increase in intracellular CM upon TCR stimulation depends on activation of nSMase. To determine whether nSMase activation was a general and specific consequence of TCR engagement, we analyzed nSMase activity after triggering of the TCR and CD28 molecules in other T cell types. Purified splenic mouse T cells , mouse T cell hybridoma 2B4 , and human T cell clone Jurkat , all showed activation of nSMase in response to stimulation of the TCR. An isotype-matched Ab specific for the costimulatory molecule, CD28, did not activate nSMase or alter the TCR-dependent induction of this enzyme in purified splenic T cells . Thus, nSMase is specifically activated by the TCR in all T cell types analyzed. Although FB1 is considered to be an inhibitor of CM synthase ( 38 , 29 ), our data suggest that the toxin interferes with the TCR-dependent nSMase activation. Therefore, we measured nSMase activity, and CM and SM levels after TCR triggering. We found that FB1 inhibits activation of this enzyme and, consequently, SM hydrolysis and CM production . In contrast, Fas-induced aSMase activation was not effected by FB1 (data not shown). Thus, FB1 inhibits TCR-induced CM production by interfering, either directly or indirectly, with nSMase activation. Altogether, these findings indicate that FB1 impairs TCR signaling by inhibiting CM production. However, this toxin could also inhibit other pathways that are essential for the TCR to cause IL-2 production, FasL upregulation, and, hence, cell death. To distinguish between these two possibilities, we used bSMase or a cell-permeable CM analogue to reconstitute intracellular CM levels. Both treatments reversed the inhibitory effect of FB1 and restored TCR-induced IL-2 production . However, neither compound reconstituted IL-2 production when TCR signaling was inhibited by CsA, an immunosuppressant that blocks dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT)-c1 ( 40 ). Furthermore, C2 CM restored TCR-induced PCD only when signaling was blocked by FB1 and not by CsA . To directly test the involvement of nSMase in TCR signaling, we cloned by PCR the cDNA coding for the recently identified and cloned human nSMase ( 27 ). Northern blot analyses showed that nSMase is expressed in all human lymphoid tissues and cell lines tested, including spleen, lymph node, thymus, peripheral blood lymphocytes, bone marrow, fetal liver, and Jurkat T cells (data not shown). The nSMase cDNA was then cloned into the mammalian expression vector pcDNA3.1, in both sense and antisense orientation. Jurkat T cells were transfected with either sense or antisense constructs together with a plasmid coding for EGFP. The efficiency of transfection, determined by measuring the percentage of EGFP + cells, was between 60 and 70% (data not shown). Consistent with our hypothesis, overexpression of nSMase by transfection of the sense construct resulted in a significant increase in IL-2 production upon antigen receptor triggering . In contrast, expression of the antisense construct reduced IL-2 production by ∼50% compared with mock-transfected cells . To accurately measure the inhibitory effect of the antisense vector and to determine whether expression of the nSMase antisense RNA efficiently depleted the cellular pool of nSMase protein, we isolated transfected cells by sorting EGFP + cells. Sorted cells were then assayed for TCR-induced IL-2 and CM production . In this experimental setting, antisense nSMase almost completely blocked both IL-2 and CM production. Taken together, these results demonstrate that CM is an essential second messenger for the TCR to adequately signal for IL-2 production and PCD. In an attempt to localize where in the TCR signaling pathway CM production plays its most important role, we studied the effect of nSMase inhibition on the activation of known biochemical signals that are induced upon TCR stimulation. A critical early event in TCR signal transduction is the induction of protein tyrosine kinase activity with ensuing phosphorylation of multiple substrates ( 41 ). However, inhibition of nSMase activity by transient transfection with an antisense vector had no effect on the overall level of protein tyrosine phosphorylation or the pattern of phosphorylated proteins . To confirm this finding, we analyzed directly the phosphorylation status of PLCγ1, one of the targets of tyrosine kinases during TCR stimulation ( 42 ). No detectable inhibition of TCR-induced PLCγ1 tyrosine phosphorylation was observed in cells transfected with antisense nSMase . Taken together, these data suggest that CM production is not required for the early events leading to the activation of this pathway. TCR ligation activates the guanine nucleotide regulatory protein, Ras ( 43 ). GTP-bound Ras triggers a cascade of events that culminate in the phosphorylation and consequent activation of the MAP kinases, extracellular signal regulatory kinase (Erk)1 and Erk2. Inhibition of nSMase CM synthesis resulted in diminished MAP kinase activation , suggesting that this pathway may be regulated by CM production. Pharmacological activation of PKC resulting from treatment of T lymphocytes with the phorbol ester, PMA, activates MAP kinase in a Ras- dependent manner. This effect of PMA was not affected by blockade of CM synthesis . Together with the observation that early tyrosine kinase activation is insensitive to inhibition of nSMase activity, these data suggest that CM plays a role in MAP kinase activation upstream of Ras or on a parallel pathway. In this study, we show that ligation of the TCR induces production of CM, a molecule that acts as a second messenger in TCR signal transduction. This increase in intracellular CM results from specific activation of nSMase and SM hydrolysis. Recently, an important role for molecules of sphingoid nature in the modulation of cell response to different extracellular signals has been uncovered. CM has been shown to act as a key molecule in a new signal transduction pathway ( 3 ). This phospholipid has been implicated in signaling through several membrane receptors, including nerve growth factor receptor (NGFR), TNFR, and Fas. Exogenous CM analogues have been shown to regulate processes such as PCD ( 33 ), IL-2 production ( 44 ), and FasL expression ( 45 ). Some downstream targets of this molecule have also been identified. The action of a 97-kD plasma membrane–bound serine/threonine protein kinase, CM-activated protein kinase (CAPK), is enhanced by elevation of cellular CM. The CM-activated protein phosphatase (CAPP), which is a member of the protein phosphatase 2A class of serine/threonine protein phosphates, is also a target of CM. CM may also stimulate the guanine nucleotide exchange factor, Vav, a putative activator of Ras-like guanine nucleotide regulatory proteins regulating cytoskeletal assembly in hematopoietic cells. Finally, PKCζ, an atypical PKC that is insensitive to phorbol esters and diacylglycerol, may also be a direct target for CM ( 37 , 46 ). An early critical event in TCR signaling is the phosphorylation of CD3/ζ–ζ chains, docking sites for the T cell– specific tyrosine kinase, ZAP-70. Activation of ZAP-70 kinase, which is regulated via tyrosine phosphorylation, is essential for many of the early events in TCR signaling. One of the targets of tyrosine kinases during TCR stimulation is PLCγ1 ( 42 ), whose enzymatic activity is enhanced by tyrosine phosphorylation ( 47 ). PLCγ1 catalyzes the formation of inositol (1,4,5)-trisphosphate and 1,2-diacylglycerol. These second messengers induce elevation of intracellular calcium and activation of PKC, respectively. The increase in calcium ultimately results in nuclear translocation and activation of the transcription factor, NFAT ( 40 ). PKC and adapter proteins, some of which are also phosphorylated on tyrosines, participate in the activation of Ras and the MAP kinase cascade that ultimately controls the activation of other transcription factors ( 43 , 48 ). Here, we present evidence that CM production is required for TCR-induced MAP kinase activation, and that CM may act independently or upstream of Ras. However, CM production is not required for TCR-induced tyrosine kinase activation. In fact, like other biochemical messengers of the TCR signal, activation of nSMase requires protein tyrosine kinase activity (data not shown). Our data indicate that CM is a second messenger molecule essential for the antigen receptor to signal for both activation and cell death. Further investigation into the downstream targets of CM action and on how this pathway is integrated with others to give biological outcomes such as differentiation, activation, anergy, cell death, and effector function will help to elucidate the physiological role of CM in TCR signaling. | Study | biomedical | en | 0.999996 |
10330438 | Male 8-wk-old DBA/2 (Ly 5.2) mice were purchased from IFFA-Credo. B6–Ly 5.1 mice were obtained from the Centre de Développement des Techniques Avancées pour L'Expérimentation Animale (Orleans, France). DBA/2–Ly 5.1 mice were generated by crossing B6–Ly 5.1 to DBA/2. After 10 backcrosses to DBA/2, an Ly 5.1 homozygote line was established. Immunizations were performed by intraperitoneal injection of 10 7 P815-CW3 transfectant tumor cells ( 18 ). Hemisplenectomy was performed 1 wk before immunization. CyChrome-conjugated anti-CD8 (53-6.7) and FITC-conjugated anti-BV10 mAbs were purchased from PharMingen ; FITC-conjugated anti-CD8, and biotinylated anti-B220 and anti-CD4 mAbs were purchased from Caltag. Biotinylated anti–Ly 5.1 (clone A20.1.7) was provided by Dr. A. Cumano (Pasteur Institute, Paris). K d -CW3 tetrameric complexes were prepared as described previously ( 12 ). Cell samples were incubated for 1 h with PE-labeled K d -CW3 tetramers, washed, and incubated with the indicated antibodies. Flow cytometry was performed on a FACScan™ and cell sorting on a FACStar PLUS ™ ( Becton Dickinson ). RNA was extracted using Trizol reagent ( GIBCO BRL ) following the manufacturer's instruction, with the addition of 20 μg/ml of glycogen as carrier ( Boehringer Mannheim ). cDNA was synthesized using (dT) 17 oligonucleotide and Moloney murine leukemia virus reverse transcriptase ( GIBCO BRL ). Immunoscope analysis and BV10-, BC-, and BJ-specific primers have been described previously ( 19 ). In brief, PCR was performed on the indicated cDNA using a BV10- and a BC-specific primer. The PCR product was then subjected to run-off reactions using nested fluorescent primers specific either for BC or one BJ segment. Run-off products were resolved on an automated 373A sequencer ( Perkin-Elmer ). The size and the intensity of each band were recorded and then analyzed using Immunoscope software ( 19 ). Analysis of the TCR-β rearrangements before immunization was performed as follows. Half of the spleen of a naive DBA/2 mouse was removed from the animal and prepared as a single-cell suspension. These splenocytes were depleted of CD4 + cells using biotinylated anti-CD4 antibody and streptavidin beads ( Dynal ). The cDNA was prepared and amplified using BV10- and BJ1.2-specific primers. TCR-β rearrangements displaying a 6 amino acid (aa) 1 –long CDR3β were separated on an 8% polyacrylamide 7 M urea gel as described ( 12 ) and cloned using the Topo TA cloning kit (Invitrogen). TCR-β rearrangements from immune animals were cloned after amplifying the indicated cDNA using a BV10-specific primer and either a BC- or a BJ1.2-specific primer. DBA/2 mice, when injected intraperitoneally with P815-CW3 tumor cells, develop a massive CD8 + T cell response against the 170-179 epitope derived from HLA-Cw3 and presented by K d ( 20 ). Peculiar features are observed in TCR usage among CW3-specific T cells, including exclusive usage of BV10, 6 aa–long CDR3β, preferential usage of BJ1.2, and a serine and a glycine at position 1 and 3 of the CDR3β (referred to as a SXG motif) ( 21 ). To determine whether the abundance of each immune T cell clone correlates with its frequency before immunization, we used hemisplenectomized mice and analyzed TCR-β chains in the immune population and in its naive counterpart. Four naive DBA/2 mice, raised in specific pathogen– free conditions, were hemisplenectomized and immunized 1 wk later by intraperitoneal injection of 10 7 P815-CW3 tumor cells. After 13 d, PBLs were analyzed by flow cytometry using an anti-CD8 antibody and K d -CW3 tetramers. As shown in Fig. 1 A, hemisplenectomized animals mount a strong CW3-specific T cell response: more than half of the CD8 + PBLs were stained with the tetramers, whereas no staining was detected on the PBLs from a naive animal. The magnitude of the response was comparable to that observed in normal DBA/2 mice. On day 19, mice were killed and the second half of the spleen was recovered. Splenocytes were triple stained with K d -CW3 tetramers, anti-CD8 and anti-BV10 antibodies. Spleens were infiltrated with CW3-specific T cells since ∼30% of the CD8 + splenocytes were stained with the K d -CW3 tetramers . In addition, the response of hemisplenectomized animals was restricted to BV10 usage as usually observed for normal DBA/2 mice. Taken together, these observations demonstrate that hemisplenectomy does not alter the CW3-specific response and that the immune half of the spleen is a large source of specific T cells. For two of these mice (mice A and B), we sorted the specific T cells on the basis of K d -CW3 tetramer staining. Cell purity after sorting was ∼98% . The cDNA was prepared and amplified using a BV10- and a BC-specific primer. The PCR product was cloned and sequenced. 10 and 6 distinct TCR-β rearrangements were identified in mice A and B, respectively (Table I ). As expected from previous studies, all of these sequences displayed a 6 aa–long CDR3β and the SXG motif within their CDR3β. Moreover, the majority of the rearrangements (6/10 for mouse A, 3/6 for mouse B) used the BJ1.2 segment. Nucleotide and deduced aa sequences are listed in Table I . In addition, based on tetramer staining and sequence occurrence, we estimated the number of immune T cells bearing the indicated CDR3β sequences (Table I ). This analysis revealed extensive differences in the abundance of the various specific T cell clones, varying from 4 × 10 4 to 3.7 × 10 5 cells. The majority of the CW3-specific T cells (79 and 50% in mice A and B, respectively) display BV10 and BJ1.2 usage together with a 6 aa–long CDR3β containing the SXG motif. We took advantage of these hallmarks to identify T cells which bear such TCR-β chains in the half of the spleen removed before immunization. We cloned and sequenced the TCR-β chains of CD8 + lymphocytes using the BV10–BJ1.2 combination and displaying a 6 aa–long CDR3β as described in Materials and Methods. On mouse A, 196 sequences were performed: 72 distinct nucleotide sequences were identified, among which only 11 contained the SXG motif. On mouse B, we found 10 distinct sequences containing the SXG motif (out of 86 sequences performed). These sequences are listed in Table II , along with the estimated number of T cells calculated from the sequence occurrence, and the percentage of BV10, BJ1.2, and 6 aa–long CDR3β usage in naive DBA/2 mice ( 12 ). Comparison of the abundance of the various TCR-β chains before and after immunization is shown in Fig. 2 . There is no correlation between the abundance of a given TCR-β sequence in the immune animal and its frequency in the naive repertoire (Kendall rank correlation test, P < 0.05). These results demonstrate that even the most abundant clones in the immune response preexist at very low frequency in the naive repertoire. As already mentioned, a large part of the CW3 response is comprised of T cells bearing the BV10, BJ1.2 gene segment and a 6 aa–long CDR3β. Other BJ segments may be used as well with a conserved 6 aa–long CDR3β (see Table II and reference 21 ). We performed CDR3 size distribution analyses (Immunoscope) on splenocytes from immunized DBA/2 animals using BV10- and BJ-specific primers. For several BV10–BJ combinations, we observed a clonal expansion corresponding to T cells bearing a 6 aa–long CDR3β . We have previously shown that K d -CW3 tetramers identify all CW3-specific T cells ( 12 ). Therefore, using K d -CW3 tetramers, we sorted the CW3-specific T cells from the same animal and analyzed their CDR3β length. All the clonal expansions detected before sorting and corresponding to T cells bearing a 6 aa–long CDR3β were purified in the CW3-specific population, indicating that all of these expanded T cells were antigen specific . The same experiment performed on other animals yielded the same conclusion although different BJs were involved, revealing individual variability in the response. To follow antigen-specific T cell expansions occurring after P815-CW3 intraperitoneal injection, DBA/2 mice were immunized and bled at various time points. PBLs were double stained with K d -CW3 tetramers and an anti-CD8 antibody. Fig. 4 A shows the kinetics of appearance of CW3-specific T cells from days 0 to 16. As described previously using BV10 staining, the peak of the response is reached at day 11, where >50% of the peripheral CD8 + are CW3 specific ( 20 ). Background level of staining was in the order of 0.5%, preventing detection of the specific subset before day 8. We performed an Immunoscope analysis on these blood samples using a BV10-specific primer and either a BC- or BJ1.2-specific primer. As shown in Fig. 4 B, the clonal expansion of T cells bearing 6 aa–long CDR3β could be detected on the BV10–BC profile from day 7. Moreover, CW3-specific T cells could be detected as soon as day 6 by using a BJ1.2-specific primer. Thus, Immunoscope analysis allowed us to detect CW3-specific T cells 5 d before the peak of the response. As shown in Fig. 3 , CDR3β size distribution analysis allowed us to monitor the CW3-specific repertoire. We took advantage of this observation to compare the outcome of various CW3-specific T cell populations bearing different BJ segments. Longitudinal analyses of the response were performed on three DBA/2 mice immunized intraperitoneally with P815-CW3 (referred to as mouse 1, 2, and 3) and bled at various time points. CDR3β size distribution was analyzed using all BV10–BJ combinations. Shown in Fig. 5 are the clonal expansions detected on day 6 for one mouse: they correspond to TCR-β chains bearing a 6 aa–long CDR3β . All of these subsets went on proliferating, as revealed by the increase of the 6 aa peak in the BV10–BJ profiles on day 10. In mouse 1, five CW3-specific subpopulations were detected corresponding to BJ1.1, BJ1.2, BJ1.4, BJ2.3, and BJ2.4 usage. To compare the proliferative capacity of these subpopulations, we measured their expansion between days 8 and 10. We estimated that after day 7, the number of CW3-specific T cells in the blood samples was sufficient to allow accurate quantitation by PCR (on day 8, ∼200–300 CW3-specific T cells were collected in 100 μl of blood). Expansion index (EI) was calculated as the ratio between the surface area of the 6 aa peak to the surface area of the other peaks. Since only specific T cells modify the CDR3β size distribution, evolution of the EI directly reflects the expansion of the CW3-specific T cells. The EI of the total specific population can be calculated by analyzing BV10– BC profiles . We also calculated the EI of the five CW3-specific subpopulations by analyzing BV10–BJ profiles. This approach allowed us to compare the proliferation of the CW3-specific T cells bearing different BJ segments. Remarkably, all five specific subsets are proliferating at the same rate . Similar results were obtained analyzing CW3-specific subpopulations from mouse 2 (bled at days 7 and 10) and mouse 3 (bled at days 7, 9, and 11). In these two animals, four expanded subpopulations were identified corresponding to BJ1.2, BJ1.4, BJ2.3, and BJ2.5 (on day 11, BV10–BJ1.2 profile from mouse 3 displayed a single peak, preventing the calculation of the EI for this particular point). Taken together, these results demonstrate that the relative contribution of the various CW3-specific subpopulations to the response is conserved during the expansion. To evaluate the expansion of various CW3-specific T cells at the clonal level, we analyzed the repertoire of TCR-β–specific sequences during the expansion. A P815-CW3–immunized DBA/2 mouse was bled at days 7 and 11. Tetramer staining indicated that between days 7 and 11, the absolute number of CW3-specific T cells had increased by ∼100-fold . As shown in Fig. 4 B, Immunoscope analysis revealed that, on day 7, the vast majority of T cells bearing the BV10 and BJ1.2 segments were CW3 specific. Therefore, we amplified the cDNA prepared from blood samples using BV10- and BJ1.2-specific primers, then cloned and sequenced the PCR products. As expected, all sequences obtained on day 11 displayed the hallmarks of CW3 response, i.e., 6 aa–long CDR3β containing the SXG motif. This was also the case for almost all sequences (31/33) obtained on day 7; the two exceptions are likely due to the residual T cells displaying BV10–BJ1.2 usage without being CW3 specific . Of the 31 sequences from either day 7 or day 11, we identified 10 distinct nucleotide sequences in both samples (Table III ). Therefore, the expanding repertoire on day 7 displays the same complexity as the repertoire at the peak (day 11) of the response (Table III ). In addition, 25 out of 31 nucleotide sequences obtained from day 7 were identical to 20 out of 31 sequences from day 11. Taken together, these results demonstrate that the complexity as well as the clonal composition of the immune response are conserved during the expansion phase. We next determined to what extent timing of initial activation affects the expansion of individual T cell clones. For that purpose, we monitored the expansion of naive T cells after transfer into immunized recipients. As a preliminary experiment, a naive Ly 5.1 DBA/2 mouse was injected intravenously with naive splenocytes (10 8 ) from Ly 5.2 DBA/2 mice and then immunized with P815-CW3. 2 wk later, splenocytes were analyzed by flow cytometry in order to determine the percentage of CW3-specific T cells among Ly 5.1 + and Ly 5.2 + populations. We found that 14 and 16% of Ly 5.1 + and Ly 5.2 + CD8 + cells, respectively, were stained with K d -CW3 tetramers. This observation indicates that transferred T cells responded equivalently to endogenous T cells. We then compared the effect of transferring the naive T cells at various time points after immunization. DBA/2 (Ly 5.2) naive splenocytes (10 8 ) were injected intravenously into DBA/2 (Ly 5.1) mice immunized either 2 or 4 d before. 12 d after transfer, PBLs were triple stained with K d -CW3 tetramers and anti-CD8 and anti–Ly 5.1 mAbs, and the contribution of T cells from donor origin to the response was determined. As shown in Fig. 7 , when Ly 5.2 + splenocytes are transferred into a recipient immunized 2 d before, antigen-specific T cells from donor origin expand to 0.3–1.3% of the total T cells. In contrast, no specific expansion could be detected when donor cells were transferred into recipients immunized 4 d before. Therefore, a 48-h delay in antigen encounter dramatically affects the extent of clonal expansions. Numerous studies have focused on the composition of immune repertoires at or near the peak of T cell responses. A feature that has been extensively analyzed is the complexity of TCR usage ( 22 ). Some responses were found to be diverse, while others were strongly constrained ( 23 ). More scarce are the data on the complexity of the response in a given individual, i.e., the number of antigen-recruited T cell clones. Interestingly, when we and others analyzed the extent of antigen-specific CD8 + T cell response, large variations (20–50-fold differences) were observed in the relative abundance of the various recruited T cell clones ( 10 – 12 ). In fact, at least in these systems, a small number of clones are very efficiently expanded and contribute to the vast majority of the effectors. In contrast, many of the elicited T cells are poorly expanded and only account for a minor part of the response. The present work was undertaken in order to understand why so few clones are expanded and to identify the parameters that shape the clonal hierarchy of T cell response. Why does a given clone become dominant in the immune response? One hypothesis might be that the composition of the immune repertoire is a magnified image of the naive repertoire. In other words, a dominant T cell clone in the response would be highly represented in the naive repertoire, and a large number of naive cells corresponding to the same T cell clone would be primed after immunization. In contrast, T cells derived from a single precursor could not contribute significantly to the immune response. We tested this hypothesis by using hemisplenectomized animals and compared TCR-β chains found in the CW3-specific immune repertoire with the TCR-β chains present before immunization. Clearly, our data do not support this hypothesis. In the hemisplenectomized animals, the immune T cell repertoire was isolated using K d -CW3 tetrameric complexes and characterized by TCR sequencing. In the immune repertoire of mice A and B, we identified 10 and 6 CDR3β sequences, respectively. Their abundance was highly variable, indicating that some immune CTL clones are much more numerous than others (Table I ). In the same mice, before immunization, we found that 11 and 10 TCR-β chain sequences (in mouse A and B, respectively) displayed the hallmark of CW3 specificity (BV10 and BJ1.2 usage, a 6 aa–long CDR3β, and the SXG motif). Most of these sequences (9/11 for mouse A, 9/10 for mouse B) are not found in the immune response. The fact that some CTL precursors bearing an appropriate β chain are not expanded during the response could be explained by a pairing with an α chain that does not provide CW3 specificity. It is also possible that some of the CW3-specific T cells are not expanded because they did not encounter the antigen. In the immune repertoire, several distinct nucleotide sequences displaying BV10, BJ1.2, and 6 aa–long CDR3β with the SXG motif were identified. Most of them (4/6 for mouse A, 2/3 for mouse B) were not observed in the naive spleen. This could be explained by the arrival of new T cell precursors from the thymus after the hemisplenectomy. However, immunization was performed only 1 wk after the hemisplenectomy, and the release of new T cells from the thymus has been shown to be low ( 24 ). Therefore, it is unlikely that most of the clones have emerged from the thymus in 1 wk. We favor the hypothesis that these cells were represented at extremely low frequencies (one to six cells in the entire animal) before immunization, preventing their detection by our approach. In contrast, some of these sequences are displayed by 2 × 10 5 cells in the immune animal . The remaining immune sequences were also found in the half of the spleen removed before immunization. We estimated that about five to six cells bore the indicated TCR-β chains in the removed part of the spleen . In the immune spleen, they have expanded to 4 × 10 4 and 2.8 × 10 5 cells (mouse A, sequences a5 and a6, respectively) and to 6 × 10 4 cells (mouse B, sequence b7). If the abundance of T cell clones in the immune repertoire was proportional to their frequencies before immunization, the TCR-β chains of the most abundant clones should be found at high frequency in the naive repertoire. This was not the case, and no correlation was observed between naive and immune TCR-β sequence frequencies. In conclusion, this analysis shows that abundant immune T cell clones are not of particularly high frequency before immunization. Variations in the proliferative capacity of individual T cell clones could generate large differences in their contribution to the immune repertoire. If so, the fastest proliferating T cell clones would dominate the immune response. It is unknown whether a competition between the various T lymphocytes occurs during T cell expansion. In a recent study, it was proposed that during a secondary response, expansion of memory T cells is selective, leading to a narrowing of the T cell repertoire ( 15 ). Whether such a selection occurs during the primary expansion has not been addressed until now because of the lack of means to detect and characterize T cell repertoires at an early stage. In our experimental system, the sensitivity of Immunoscope analyses allows us to detect clonal expansions 5 d before the peak of the response. Conservation of the antigen-specific repertoire during the expansion was observed at two levels of resolution. First, by analyzing the CDR3β size profiles using various BJ primers, we showed that the pattern of TCR usage is conserved between days 6 and 10. Second, analysis of the clonal composition of the CW3 response revealed a remarkable conservation during the T cell expansion (Table III ). If large differences exist in the proliferative capacity of the early detected clones, one would expect a narrowing of the repertoire as the T cells expand. In contrast, we showed that the complexity is conserved during the expansion (Table III ). In good agreement with these results, we have quantified the expansion of various CW3-specific T cell subpopulations in the same animal and showed that all of them displayed a concurrent expansion, independent of their TCR sequences. We cannot formally exclude that all T cells specific for K d -CW3 have the same avidity; however, this is unlikely, since CW3-specific clones use various TCR-α chains and to a lesser extent different TCR-β chains ( 21 ). In addition, differences in antigen recognition efficiency have been reported for various CW3-specific T cell clones ( 25 ). Therefore, in our system, TCR affinities do not play a major role once T cells have begun to proliferate. It has been suggested that individual differences in the orientation of the CTL response against P815 could be explained by the stochastic timing of recruitment of the various epitope-specific T cells ( 26 ). We tested directly the impact of the timing of T cell recruitment on antigen-specific expansions. Using transfer experiments, we showed that a 48-h difference in timing of entry in the immune repertoire dramatically affects the extent of clonal expansion . In addition, we found that each specific T cell clone preexists at very low frequency in the naive repertoire (generally less than five cells). Accordingly, it is unlikely that these rare T cells encounter the antigen simultaneously. Therefore, only the first elicited T cells could account for a significant part of the immune repertoire. Due to the short doubling time of proliferating T cells (estimated to be down to 6–8 h), a 1-d difference in T cell recruitment could lead to a 16-fold difference in the extent of expansion ( 2 ). Several parameters could affect the clonal composition of the immune repertoires. One is the frequency of the various specific T cell precursors before immunization. Our analysis of naive and immune TCR-β chains in an hemisplenectomized mouse ruled out this possibility, since the most abundant clones in the immune animals were not the largest ones before immunization. An alternative hypothesis would be that various T cell precursors proliferate at different rates, depending for instance on their avidity for the MHC–peptide complex. Here, we showed a remarkable stability of the T cell repertoire during the expansion. Therefore, it appears that, once T cell clones have entered the immune repertoire, their respective contribution is conserved. Finally, we demonstrate that timing of T cell recruitment has a major impact on T cell expansion. Therefore, the major parameter that determines the hierarchy of the various elicited clones is their time of entry in the immune repertoire, i.e., the time of first cell division. Whether high-affinity T cells are first recruited because of a faster activation time or whether timing of recruitment is a purely stochastic event remains to be elucidated. | Study | biomedical | en | 0.999996 |
10330439 | CB samples were collected with the informed consent of the mothers involved in our study. Low density CB mononuclear cells were first subjected to a standard CD34 immunomagnetic bead separation using the miniMACS ® system (Miltenyi Biotec). Bead-separated CD34 + cells (purity >75%) were either injected into NOD-SCID mice (10 5 cells/mouse) or further fractionated in CD34 + CD19 − CD38 − or CD34 + CD19 − Thy1 + fractions by cell sorting using a FACS Vantage™ equipped with an argon ion laser (Innova 70-4-coherent radiation) tuned to 488 nm and operating at 500 Mw. A morphological gate including all of the CD34 + cells was first defined on two-parameter histograms, side scatter versus forward scatter. Positivity or negativity for CD19, Thy1, and CD38 among the CD34 + cells was determined using cells labeled with CD34-PE-Cy5 (Immunotech) and an irrelevant IgG1 mAb. The Thy1 + and CD38 − subsets of CD34 + cells were obtained in two steps. CD34 + CD19 − cells were sorted and relabeled with CD34-PE-Cy5 (Immunotech) and either CD38-PE ( Becton Dickinson ), or Thy1 (CD90)-PE ( PharMingen ). Limits of the sorting gates for Thy1 + and CD38 − cells were set as illustrated . Single cell cultures were initiated by directly sorting cells into 96-well tissue culture plates precoated with MS5 murine stromal cells using an automatic cloning design unit ( Becton Dickinson ). The standardized distribution of one cell per well was controlled by examining over 4,000 wells individually in parallel experiments initiated with human cells. For in vivo experiments, 10 5 CB CD34 + cells were intravenously injected into irradiated (3–3.5 Gy; cobalt-60 Eldorado S irradiator; AECL Medical) NOD-LtSz- scid/scid (hereafter called NOD-SCID) mice anesthesized briefly with ether. 10–16 wk later, marrow cells were flushed from the femurs and tibias of recipient mice as previously described ( 12 ), and human CD34 + cells were isolated and fractionated into CD34 + CD19 − , CD34 + CD19 − Thy1 + , or CD34 + CD19 − CD38 − as indicated above . Isolation of murine embryonic thymic lobes, incubation with human cells after the hanging drop procedure, and organotypic cultures were performed following standard procedures initially described to analyze mouse T lymphoid differentiation and adapted to the identification of human T cell potential ( 22 , 29 ). Our slight modifications of the technique included the use of NOD-SCID embryonic (day 14) thymic lobes and the addition of recombinant human (rhu) IL-2 (5 ng/ml; Diaclone), 20 ng/ml rhu-IL-7 (Diaclone), and 50 ng/ml rhu-stem cell factor (SCF; provided by Amgen) during the hanging drop procedure. Thymic lobes were incubated onto floating filters (Isopore membrane, 25-mm diameter, 8-μm pore size; Millipore SA) at 37°C and 5% CO 2 in medium without cytokines. Cells recovered from the thymic lobes after 28–35 d were labeled with anti–human CD4-PE and CD8-FITC ( Becton Dickinson ), TCR-α/β-PE, and CD3-FITC (Immunotech). Lack of reactivity of the mouse anti–human mAbs with NOD-SCID murine cells was verified in pilot experiments ( 12 , 24 ). The different cell fractions were incubated in 24- or 96-well plates precoated with confluent murine marrow–derived MS5 cells ( 30 ) in RPMI supplemented with 10% human serum, 5% FCS, and the following six rhu cytokines: rhu-SCF (50 ng/ml; Amgen), rhu-Flt3-ligand (50 ng/ml; Diaclone), pegylated (PEG)- rhu-megakaryocyte growth and differentiation factor (MGDF) (50 ng/ml; Amgen), rhu-IL-2 (5 ng/ml; Diaclone), rhu-IL-15 (10 ng/ ml; Diaclone), and rhu-IL-7 (20 ng/ml; Diaclone). Wells with significant cell proliferation (usually >500 cells after 3–6 wk) were selected, and cells were collected and their phenotype assessed by flow cytometry after labeling with the following mAbs: CD19-PE ( Becton Dickinson ), CD15-FITC (Dako Corp.), and CD56-PE-Cy5, CD34-PE-Cy5, CD1a-PE, and HLA-DR-FITC (all from Immunotech). In some experiments, the intracellular expression of myeloperoxidase (MPO; Caltag Labs.) was determined using a cell permeabilization kit (Harlan, Sera-Lab Ltd.). Analysis was performed on a FACScan™ ( Becton Dickinson ) using CellQuest software. Individual CD34 + CD19 − , CD34 + CD19 − Thy1 + , or CD34 + CD19 − CD38 − cells from either fresh CB or the marrow of chimeric NOD-SCID mice 4 mo posttransplant were induced to proliferate in 96-well plates precoated with MS5 cells in the presence of the six growth factors listed above. Wells were carefully monitored for 7–21 d, and clones containing ≥1,000 cells were divided: for each clone, half of the cells were cultured on new MS5 feeders as described above and the other half were incubated in NOD-SCID FTOC together with 5,000 irradiated (15 Gy) CD34 + CD38 + CB cells, as accessory cells have been shown to help engraftment in FTOC seeded with small numbers of cells ( 24 ). At the end of the FTOC, cells collected from each lobe were analyzed individually for the presence of CD4 bright and/or CD4 + CD8 + human cells. In three control experiments, 25 NOD-SCID fetal thymic lobes were reconstituted with 10,000 irradiated, fresh CD34 + CD38 + cells/lobe, but none of them yielded human T cells. In contrast, all lobes incubated with unirradiated CD34 + CD38 + cells produced CD4 bright and CD4 + CD8 + T cells (not shown). Because primitive lymphomyeloid cells most likely represent only a minor fraction of human CD34 + cells collected from both fresh CB and NOD-SCID marrow, we performed an enrichment step. We first removed CD34 + CD19 + committed pre-B cells, which represented 13 ± 8% ( n = 6) of fresh CB cells but 83 ± 6% ( n = 11) of NOD-SCID–derived CD34 + cells ( 12 ). CD34 + CD19 − cells were further fractionated into Thy1 + or CD38 − cells, as both cell types exhibit primitive functions. Thy1 + cells represented 10 ± 4% ( n = 5) of fresh CD34 + CD19 − CB cells , whereas Thy1 + and CD38 − cells accounted only for 2.5 ± 1.8% and 2.3 ± 0.6% of CD34 + CD19 − cells from NOD-SCID mice ( n = 5), respectively. We have previously shown that murine MS5 stromal cells support myeloid ( 30 ) and B cell differentiation ( 20 ) from CD34 + CB cells when studied in separate assays but also from single bipotent B/myeloid progenitors in a switch system ( 20 ). Because MS5 cells also promote the production of NK cells from CD34 + cells in the presence of human IL-15 ( 31 ), we reasoned that slight modifications of these culture conditions should allow the simultaneous development of all three lineages (B, NK, and Gr/M). To this end, we supplemented the medium with rhu-SCF, IL-2, IL-15 (three-cytokine condition), and, in later experiments, PEG-rhu-MGDF, Flt3-ligand, and IL-7 were also added (six-cytokine condition). PEG-rhuMGDF and Flt3-ligand were chosen to trigger active proliferation of immature progenitors that may not respond to lineage-specific cytokines ( 32 ), and IL-7 was chosen to minimize the loss of B and/or T cell potential ( 33 ). Thus, CD34 + CD19 − and CD34 + CD19 − Thy1 + fresh CB cells (2–10,000 cells/well), when cocultured on MS5 cell feeder layers in three- or six-cytokine conditions, reproducibly generated CD19 + B, CD56 + NK, and CD15 + / MPO + Gr/M cells in 2–3 wk (Table I ). In the presence of six cytokines, up to 70% of cells recovered after 2 wk of culture were still CD34 + (not shown). Table I and Fig. 2 A–C also show that CD34 + CD19 − cells collected from the marrow of NOD-SCID mice ( n = 4) and cultured for 3 wk in the presence of six cytokines generated 3.1 ± 0.7% CD19 + B cells, 9.5 ± 4.1% CD56 + NK cells, and 22 ± 13% CD15 + Gr/M cells (mean ± SEM of nucleated cells). Interestingly, all three cell types also exhibited almost nonoverlapping forward scatter versus side scatter profiles. Similar differentiation profiles and lineage yields were observed in three experiments initiated with CD34 + CD19 − CD38 − cells (1 ± 0.4, 44 ± 3, and 28 ± 5% B, NK, and Gr/M cells, respectively) and CD34 + CD19 − Thy1 + cells (6.4 ± 6, 47 ± 9, and 20 ± 5% B, NK, and Gr/M cells, respectively) (Table I ). Cells coexpressing CD1a and HLA-DR, most likely of dendritic origin, were detected in a few experiments in which these markers were analyzed (data not shown). As previously described, cultured CD19 + cells coexpressed CD38 and CD10 but lacked CD20, CD22, and sIgM, whereas NK cells were mature and functional, as shown by their spontaneous cytotoxic activity against K562 and Daudi cells (reference 31 ; data not shown). The culture conditions described above were not permissive to T cell differentiation, which requires the presence of thymic stromal elements. We have recently reported the successful use of NOD-SCID embryonic thymus to identify T cell potential of CB and adult marrow CD34 + , CD34 + CD38 + , and CD34 + CD38 − cells ( 24 ). Here, we applied this strategy to demonstrate that human CD34 + CD19 − cells selected from the bone marrow of NOD-SCID mice generated CD4 bright and double positive (DP) CD4 + CD8 + T cells in NOD-SCID FTOC. As illustrated in Fig. 2 B–G, in 5/5 experiments, human CD4 + cells (which represented 69 ± 13% of nucleated cells collected from thymi) were produced, and 82 ± 5% of those were CD4 bright , 46 ± 12% were DP CD4 + CD8 + , 31 ± 5% were CD4 + TCR-α/β + ( n = 3), and 32 ± 7% were CD4 + CD3 + ( n = 3). As described for fresh CB cells, very few CD4 − CD8 + cells were produced ( 22 ). Taken together, these experiments demonstrated that CD34 + CD19 − human cells detected in NOD-SCID recipients 4 mo after intravenous injection of CD34 + CB cells had retained the ability to differentiate into B, NK, and Gr/M lineages as well as T cells in vitro, even though T and NK terminal maturation did not occur in vivo. Before investigating the full lymphomyeloid differentiative potential of single cells, we first determined the frequency of single cells that produce high numbers of B, NK, and Gr/M cells when cocultured on MS5 cells with six cytokines. In two experiments, 180 single CD34 + CD19 − cells from fresh CB were cultured in the presence of six cytokines and MS5 cell feeder layers. Cells from 55 wells were phenotyped and found to contain multiple combinations of B, NK, and Gr/M cells (Table II ). Nine clones (16%) contained cells from all three lineages, and 54% of the clones yielded cells of only one lineage. When CD34 + CD19 − Thy1 + cells were used, the frequency of the three-lineage clones increased to 28% (28 clones analyzed) and the frequency of one-lineage clones decreased (21%), in agreement with the preferential expression of Thy1 by immature progenitors. Lineages were similarly distributed in clones grown from single CD34 + CD19 − Thy1 + or CD38 − cells isolated from the marrow of transplanted NOD-SCID mice. Analysis, for instance, of cells from 240 wells, each seeded with a single CD34 + CD19 − Thy1 + (120 wells) or CD34 + CD19 − CD38 − (120 wells) cell, indicated that >50% of the clones contained cells of two lineages and 15–33% cells of three lineages but only 13–23% cells of one lineage (Table II ). In contrast and as noted above, 69% of the more mature CD34 + CD19 − cells generated cells of only one lineage. Importantly, individual clones grown as described above in six cytokines on stromal feeders contained high numbers of nucleated cells (from 10,000 to 400,000 total cells per well at week 2–3), which made it possible to remove part of the clone to separately assay T cell differentiation in FTOC. Thus, in a second set of experiments, multiple wells were seeded with CD34 + CD19 − Thy1 + from fresh CB (420 wells) and with CD34 + CD19 − Thy1 + and CD34 + CD19 − CD38 − cells from the marrow of transplanted NOD-SCID mice (each population, 240 wells). After 2–3 wk, clones that had proliferated (150, 76, and 92, respectively) were divided to initiate NK-B-Gr/M and FTOC assays (Table III ). The most remarkable result was that 10/150 clones grown from fresh CD34 + CD19 − Thy1 + CB cells and 14/ 68 clones grown from human CD34 + CD19 − Thy1 + (or CD38 − ) cells from NOD-SCID chimeras generated not only B, NK, and Gr/M cells but also T cells (CD4 bright cells usually associated with DP CD4 + CD8 + cells) in FTOC assays (Table III ). As illustrated in Fig. 3 , the proportions of lymphoid (CD19 + , CD56 + ) and Gr/M (CD15 + ) cells varied between clones. However, the distribution of clones containing one, two, three, or four lineages in experiments initiated with either fresh CB cells or cells from NOD-SCID mice was almost identical (Table III ). Interestingly, the distribution of lineages was not strictly identical in clones grown from CD34 + CD19 − Thy1 + and CD34 + CD19 − CD38 − NOD-SCID–derived human cells. The biological significance of this observation is unclear, and this result must be confirmed in experiments initiated with fresh CB cells of both phenotypes. In addition to totipotent clones, multiple combinations of lineages were detected: 30% of the clones (45/150 wells seeded with CD34 + CD19 − Thy1 + fresh CB cells and 27/92 wells seeded with CD34 + CD19 − CD38 − cells from transplanted NOD-SCID mice) generated combinations of three lineages. Among these three-lineage clones, 30% included T cells, and 4% combined B, NK, and T cells but no CD15 + cells and were most likely derived from lymphoid-restricted progenitors . None of the clones contained B cells alone and <2% exclusively T cells (Table III ). Both T (52/54) and B (151/154) cells were almost always combined with NK cells. This was the predominant phenotype in our culture conditions: 128/150 clones from fresh CB and 145/168 clones from marrow of transplanted NOD-SCID mice contained human NK cells. In this study we provide, for the first time in humans, direct evidence that a significant proportion of CD34 + CD19 − Thy1 + (and/or CD38 − ) fresh CB cells are totipotent and that similar totipotent cells can be isolated from the marrow of NOD-SCID mice transplanted several months earlier with fresh human CD34 + CB cells. The ability of a single cell to generate T, B, and NK lymphocytes and Gr/M cells defines totipotency and was demonstrated in vitro by combining FTOCs and cocultures on competent murine stromal cell feeder layers. A prerequisite for the success of these experiments was to first overcome previous limitations in the assay system for B and T lymphoid potential of human cells in vitro, and a decisive step was to use the murine stromal cell line MS5 ( 34 ). Feeder layers from MS5 and other cell stromal lines ( 17 , 19 , 35 ), in contrast to those from human stromal cells, support the differentiation of CD34 + cells into pro-B cells; although spontaneous terminal B cell maturation remains compromised, probably because T helper function is lacking, it can be induced ( 36 ). However, despite recent suggestions that extra-thymic differentiation may occur in the marrow environment ( 37 ) or in the gut and the unique finding of CD3 + human cells in the marrow of bgnuXid mice engrafted with CD34 + marrow cells ( 7 ), human T cells never developed in vitro in MS5 cocultures. The presence of an intact thymic structure remains essential, although recent results indicate that thymic stromal feeders can replace the intact thymus ( 27 , 38 ). The observation that cultures of embryonic thymic lobes, initially used to follow mouse T cell development, could be successfully applied to the study of human T cell differentiation represents an improvement over the use of human embryonic thymus. Thus, we ( 24 ) and others ( 22 , 23 ) recently described that FTOC initiated with thymic lobes from immunodeficient mice efficiently supported the first steps of human T cell differentiation up to the DP CD4 + CD8 + stage and are sensitive enough to detect the production of CD4 + CD8 + T cells from as few as 100 CD34 + CB cells ( 24 ). To prove cell totipotency, it was essential to work at the clonal level, which imposed the need to initiate FTOC and cocultures on MS5 feeders with cells issued from the same clone. This required induction of the proliferation of input single cells to allow subdivision of the clone without loss of potentials. This was accomplished by adding early-acting proliferative cytokines, including IL-7, to avoid the loss of early T cell progenitors ( 33 ) and by plating the cells directly on MS5 cells, because we have shown that these stromal cells are important to retain the primitive potential of actively proliferating adult CD34 + CD38 − marrow long-term culture–initiating cells ( 39 ). Our results showed that 6.7% of CD34 + CD19 − Thy1 + CB cells were totipotent, which leads to an estimate of one totipotent progenitor per 200 CD34 + CB cells. This number is surprisingly high and may even be underestimated, as we selected only actively proliferating clones and also because the hanging drop procedure that we used may limit the homing of T cell progenitors into thymic lobes. Although it is hazardous to draw any firm conclusion about stem cell hierarchy based on the distribution and combination of lineages observed in single clones grown in vitro, both were very similar to those reported from the analysis of lymphomyeloid clones grown in vitro from murine fetal liver ( 26 ) or embryonic splanchnopleura ( 40 ) and in vivo from recipients of genetically marked donor cells ( 1 ). Particularly interesting was the observation in our study that some clones, derived from fresh CB cells and the marrow of transplanted NOD-SCID mice, produced T, B, and NK but no Gr/M cells. Although we cannot exclude that other conditions could have unmasked additional myeloid potentials, erythroid or megakaryocytic, the total lack of CD15 + cells makes this hypothesis very unlikely. The parental progenitors most probably represented the human counterpart of the murine lymphoid-restricted progenitors identified among IL-7Rα + c-kit + Lin − Sca + adult mouse marrow cells ( 28 ). As reported in the mouse ( 26 ), we also failed to detect bipotent T/B progenitors, an observation that contrasted with the high frequency, in our study, of clones with both NK and B cells. It is important to stress that NK cells were the most frequent cell type in our cultures and that their outgrowth might have interfered (in a positive or negative way) with the development/survival of other lineages. Indeed, there were very few if any clones containing only B or T cells. A possible explanation for the lack of B cell clones was the lack of signals released by T cells, which was not compensated for by the action of MS5 cells. A second major observation was that human totipotent cells were detected in the marrow of NOD-SCID recipients several months after transplantation of CD34 + CB cells. Tracing individual stem cell fate and function in vivo is complicated in the NOD-SCID chimeras, as opposed to the syngenic murine situation, as terminal T and NK cell differentiation, in contrast to B cell differentiation, does not take place in vivo. However, human CD34 + cells isolated from NOD-SCID chimeras reproducibly produced CD4 + CD8 + T cells and NK cells in vitro, indicating that human cells have not lost their intrinsic T and NK potential after their engraftment in the NOD-SCID environment. A more likely hypothesis is that regulatory steps involved in the development of T and NK pathways do not take place in this xenogenic model. For T cells, the defect most likely involves molecules regulating cell trafficking, whereas the maturation of human NK cells was blocked because human cells do not respond to murine IL-2 and IL-15. A close frequency of totipotent clones (7%) was found in experiments initiated with fresh CD34 + CD19 − Thy1 + CB cells and CD34 + CD19 − CD38 − cells from transplanted NOD-SCID mice (14%). Calculations yielded an absolute number of ∼50 totipotent cells in four long bones of a NOD-SCID 4-mo posttransplant. Considering that 500 totipotent cells were present among the 10 5 CD34 + cells injected, this suggests loss rather than amplification of totipotent cells. However, totipotent clones may in fact actively proliferate in vivo. A strong argument in favor of this hypothesis is that, in our experience as well as in others' ( 8 ), <1% of the injected CD34 + human cells are detected in the marrow of NOD-SCID mice 48 h after their injection (range 0.1 to 4% in six experiments; our unpublished data). Therefore, it is conceivable that <10 totipotent cells eventually colonize the marrow of NOD-SCID mice and contribute to the reconstitution of all compartments, a hypothesis that is in agreement with murine studies ( 1 , 4 , 41 , 42 ). Definite determination of the number of human clones contributing to the reconstitution of hematopoiesis in NOD-SCID mice will await results from transplantation experiments using genetically marked human cells. These studies will be facilitated by the availability of conditions that allow the expression of the full differentiative potentials of individual clones, as demonstrated in this study, but also by improved procedures that will allow efficient transduction of NOD-SCID-CRU competitive repopulating unit ( 43 , 44 ). | Study | biomedical | en | 0.999999 |
10330440 | The 410-kb HuIgλYAC, accommodating a 380-kb region (Vλ-JCλ) of the human λ L chain locus with V, J, and C genes in germline configuration, was constructed as previously described ( 29 ). To allow selection, two copies of the neomycin resistance gene ( NEO r ) were site-specifically integrated into the ampicillin gene on the left (centromeric) YAC arm. YAC-containing yeast cells were fused with HM-1 embryonic stem (ES) cells (a gift from D. Melton, Department of Pathology, University Medical School, Edinburgh, UK), as previously described ( 30 ), and G418-resistant colonies were picked and analyzed 2–3 wk after protoplast fusion. ES cells containing a complete HuIgλYAC copy, confirmed by Southern hybridization, were used for blastocyst injection to produce chimeric animals ( 31 ). Breeding of chimeric animals with BALB/c mice resulted in germline transmission. Further breeding with κ −/− mice ( 32 ) established the lines for analysis. Conventional DNA was obtained ( 33 ) or high molecular weight DNA was prepared in agarose blocks ( 34 ). For the preparation of testis DNA, tissues were homogenized and passed through 70 μM nylon mesh. Pulsed-field gel electrophoresis (PFGE) conditions to separate in the 50–900 kb range were 1% agarose, 180 V, 70 s switch time, and 30 h running time at 3.5°C. Hybridization probes were Cλ2+3 and the left YAC arm probe (LA) comprising LYS 2 ( 29 ). Hybridomas were obtained from 3-mo-old HuIgλYAC/κ +/− animals by fusion of splenocytes with NS0 myeloma cells ( 35 ). After fusion, cells were plated on 96-well plates such as to obtain single clones. Human and mouse antibody production was determined in sandwich ELISA assays ( 36 ) on MaxiSorp plates (Nalge Nunc, Denmark). For the detection of human or mouse Igλ, coating reagents were a 1:500 dilution of anti–human λ L chain mAb HP-6054 or a 1:500 dilution of the 2.3 mg/ml rat anti–mouse λ mAB , respectively. Respective binding was detected with biotinylated antibodies: polyclonal anti–human λ , a 1:1,000 dilution of polyclonal anti–mouse λ or rat anti–mouse Igλ followed by streptavidin-conjugated horseradish peroxidase ( Amersham International ). Mouse IgG2aλ myeloma protein from HOPC1 and human serum IgGλ were used to standardize the assays. To determine mouse κ L chain levels, plates were coated with a 1:1,000 dilution of rat anti–mouse κ, clone EM34.1 , and bound Ig was detected using biotinylated rat mAb anti–mouse Igκ . Mouse myeloma proteins IgG2aκ and IgG1κ were used as standards. For detection of mouse IgM, plates were coated with polyclonal anti–mouse μ (The Binding Site, UK) and bound Ig was detected with biotinylated goat anti–mouse μ followed by streptavidin-conjugated horseradish peroxidase. Mouse plasmacytoma TEPC183, IgMκ was used as a standard. Cell suspensions were obtained from bone marrow, spleen, and Peyer's patches (PPs). Multicolor staining was then carried out with the following reagents in combinations : FITC-conjugated anti–human λ , PE-conjugated anti–mouse c-kit (CD117) receptor , PE-conjugated anti–mouse CD25 (IL-2 receptor) , biotin-conjugated anti– human κ , biotin-conjugated anti-mouse CD19 , followed by Streptavidin-Quantum red or Streptavidin-PerCP and rat monoclonal anti–mouse κ L chain (clone MRC-OX-20, cat. MCA152; Serotec, UK) coupled according to the manufacturer's recommendations with allophycocyanin (APC) (PJ25C; ProZyme). Data were collected from 10 6 stained cells on a FACScalibur ® flow cytometer ( Becton Dickinson ) as previously described ( 32 ). Cells were first gated on forward and side scatter to exclude dead cells. To obtain accurate percentage distribution for comparison, cells from normal mice were stained in parallel. In addition, human peripheral blood lymphocytes were purified on Ficoll gradients (1.077 g/ml) and stained with PE-conjugated anti–human CD19 antibody , biotinylated anti–human κ followed by Streptavidin-Quantum red, and FITC-conjugated anti–human λ antibodies as above. For reverse transcriptase (RT)-PCR cloning of Vλ genes, PP cells were stained with FITC-conjugated peanut agglutinin (PNA) and PE-conjugated anti–mouse B220 antibodies . Double-positive cells were sorted on the FACStar Plus flow cytometer ( Becton Dickinson ) as previously described ( 32 ) and 5 × 10 3 cells were lysed in denaturing solution ( 37 ). 5′RACE was carried out as described below with one modification: 2 μg of carrier RNA was added to the cell lysates before RNA extraction and precipitation. Spleen RNA was prepared as previously described ( 37 ) and for cDNA preparation 2–3 μg of RNA was ethanol precipitated and air dried. For rapid amplification of 5′ cDNA ends (5′RACE) ( 38 ) first strand cDNA was primed with oligo(dT)22, and 100 U of Super Script II reverse transcriptase ( GIBCO BRL ) was used at 46°C according to the manufacturer's instructions with 20 U of placental RNAse inhibitor ( Promega ). The DNA/RNA duplex was passed through 1 ml G-50 equilibrated with TE (10 mM Tris-HCl, pH 7.8, and 1 mM EDTA) in a hypodermic syringe to remove excess oligo(dT). For G-tailing, 20 U of TdT (Cambio, UK) was used according to standard protocols ( 39 ). Double-stranded cDNA was obtained from G-tailed single-stranded cDNA by addition of oligonucleotide Pr1 (see below), 100 μM dNTP, and 2.5 U of Klenow fragment (Cambio), followed by incubation for 10 min at 40°C. After heating the reaction for 1 min at 94°C and extraction with phenol-chloroform, the double-stranded cDNA was passed through G-50 to remove primer Pr1. PCR amplifications, 35 cycles, were carried out in the RoboCycler Gradient 96 Thermal Cycler (Stratagene) using oligonucleotides Pr2 and Pr3. For PCR of PP cDNA 50 cycles were used: 40 cycles in the first amplification and 10 cycles in additional amplifications. Pfu Thermostable Polymerase (Stratagene) was used instead of Taq polymerase to reduce PCR error rates. The amplification products were purified using a GENECLEAN II kit (BIO 101) and reamplified for five cycles with primers Pr2 and Pr4 to allow cloning into EcoRI sites. Oligonucleotide for 5′RACE of Vλ genes were: Pr1, 5′-AATTCTAAAACTACAAACTGCCCCCCCCA/T/G-3′; Pr2, 5′-AATTCTAAAACTACAAACTGC-3′ (sense); Pr3, 5′-CTCCCGGGTAGAAGTCAC-3′ (reverse); and Pr4, 5'-AATTCGTGTGGCCTTGTTGGCT-3′ (reverse nested). Vλ PCR products of ∼500 bp were cut out from agarose gels and purified on GENECLEAN II. The DNA was incubated in 50 mM Tris-HCl, pH 7.4, and 10 mM MgCl 2 , with 100 μM dGTP/dCTP and 1 U of Klenow fragment for 10 min at room temperature. Under these conditions the Klenow fragment removed the 3′ ends of the PCR products (AATT) leaving ligatable EcoRI overhangs. DNA was ligated with EcoRI-restricted pUC19, transformed into competent E. coli XL1Blue, and colonies were selected on X-Gal/IPTG/amp plates. Plasmid DNA prepared from white colonies was used for sequencing. Sequencing of both strands was done on the ABI 373 automated sequencer (Applied Biosystems, Inc.) in the Babraham Institute Microchemical Facility. The human Igλ translocus was assembled as a YAC by recombining one YAC containing about half of the human Vλ gene segments with three overlapping cosmids containing Vλ and Jλ-Cλ gene segments and the 3′ enhancer ( 29 ). This produced a 410-kb YAC accommodating a 380-kb region of the human λ L chain locus containing 15 Vλ genes regarded as functional, 3 Vλs with open reading frames not found to be expressed, and 13 Vλ pseudogenes ( 40 ). This HuIgλYAC was introduced into ES cells by protoplast fusion ( 30 ) and chimeric mice were produced by blastocyst injection ( 31 ). The ES cell clone used for blastocyst injection showed a 450-kb NotI fragment corresponding to HuIgλYAC, as identified by PFGE and Southern hybridization with probes to the 3′ end of the construct, identifying the Cλ2+3 regions, and to the left centromeric YAC arm at the 5′ end, identifying the LYS 2 gene (data not shown). Germline transmission was obtained, and PFGE analysis of testis DNA from one animal is illustrated in Fig. 2 . A NotI fragment larger than 380 kb is necessary to accommodate this region of the HuIgλYAC, and the 450-kb band obtained indicates random integration involving the single NotI site 3′ of Jλ-Cλ and a NotI site in the mouse chromosome. Digests with EcoRI/HindIII and hybridization with the Cλ2+3 probe further confirmed the integrity of the transferred HuIgλYAC . The results indicated that one complete copy of the HuIgλYAC was integrated in the mouse genome. To assess the human λ L chain repertoire for the production of authentic human antibodies, the HuIgλYAC mice were bred with mice in which endogenous Igκ production was silenced by gene targeting ( 32 ). In these κ −/− mice, the mouse Igλ titers are elevated compared with κ +/+ strains ( 32 , 41 ). Serum titrations showed that human Igλ antibody titers in HuIgλYAC/κ −/− mice are between 1 and 2 mg/ml, which is up to 10-fold higher than average mouse Igλ levels. Interestingly, in the HuIgλYAC/κ −/− mice, the mouse Igλ production returned to levels similar to that found in normal mice. High numbers of human Igλ + cells were also identified in flow cytometric analysis of splenic B cells from HuIgλYAC/κ −/− mice , with human λ expressed on the surface of ∼84% of the B cells and mouse Igλ + expressed on <5% (data not shown). Assessment of human Igλ production in heterozygous HuIgλ YAC + /κ +/− mice allowed a detailed comparison of expression and activation of endogenous versus transgenic L chain loci present at equal functional numbers. Serum analysis of mice capable of expressing both human λ and mouse κ showed similar titers for human and mouse L chains. Human Igλ levels in HuIgλYAC/κ +/+ transgenic mice were similar to those in HuIgλYAC/κ +/− mice. Total Ig levels in HuIgλYAC + /κ +/− mice were 1–2 mg/ml, with an average contribution of ∼51% mouse Igκ, 43% human Igλ, and 6% mouse Igλ. As is also seen in human serum, the analysis of individual HuIgλYAC/κ +/− animals showed there were variations in the λ/κ ratios. Three of the HuIgλYAC/κ +/− mice produced somewhat higher κ levels, whereas in two mice the human λ levels were higher than the Igκ titers. In HuIgλYAC/κ +/− mice, high translocus expression was also found in B220 + B cells from different tissues, with 38% of spleen cells expressing human λ and 45% expressing mouse κ . As illustrated, these values closely resemble the levels in human volunteers with 34% Igλ + versus 51% Igκ + in CD19 + peripheral blood lymphocytes. In HuIgλYAC/ κ +/+ mice, which carry a wild-type κ locus, the levels of Igλ are ∼25% and endogenous κ levels are ∼60% . It is likely that these differences in expression levels are dependent on the number of active gene loci. To assess the developmental stage at which the high contribution of the human λ translocus becomes established, we examined surface L chain expression by bone marrow cells of HuIgλYAC/κ +/− mice. For this, B cell lineage marker CD19 and specific antibodies to human λ and mouse κ were used in four-color staining with the early B cell markers c-kit (CD117) and CD25. Fig. 4 B shows that surface L chain expression (human λ or mouse κ) was detectable on a similar small proportion of B cells at each of these stages of development, which suggests that human and mouse L chain rearrangements are simultaneous. The specificity of the staining detecting mouse κ and human λ on small numbers of early B cells, which has been reported independently ( 42 ), was verified by the absence of similar positive cells in the analysis of bone marrow from control mice (data not shown). To further clarify the potential of the L chain translocus to contribute to the antibody repertoire, we analyzed human λ and mouse κ L chain production using individual hybridoma clones from HuIgλYAC/ κ +/− mice. Results from two fusions suggest that human λ and mouse κ L chain–producing cells were present in the spleen of HuIgλYAC/κ −/+ mice at similar frequencies. Furthermore, in the hybridomas the amounts of human Igλ (2–20 μg/ml) or mouse Igκ (4–25 μg/ml) were very similar. To determine whether Igκ rearrangement precedes Igλ, as found in mice and humans ( 4 , 5 ), the configuration of the endogenous Igκ and the human λ translocus were analyzed in these hybridomas. Southern blot hybridization of randomly picked hybridoma clones showed that in 11 human Igλ expressers, 7 had the mouse κ locus in germline configuration, 1 clone had mouse Igκ rearranged, and 3 clones had the mouse κ locus deleted, whereas in 19 mouse Igκ expressers, all but 2 had the human Igλ locus in germline configuration. This result suggests that there is no locus activation bias and further emphasizes that the human λ translocus performs with similar efficiency as the endogenous κ locus. The capacity of the human λ locus to produce a diverse antibody repertoire is further documented by the V-J rearrangement. Sequences were isolated from spleen and PP cells by 5′RACE PCR amplification to avoid bias from specific V gene primers. The use of individual Vλ genes is indicated by the triangles in Fig. 1 , and shows that a substantial proportion of the Vλ genes on the translocus are being used in productive rearrangements, with Vλ3-1 and Vλ3-10 being most frequently expressed. In Vλ-Jλ rearrangements, Jλ2 was used preferentially and Jλ3 and 1 were used less frequently, whereas, as expected, Jλ4, 5, and 6 were not used as they are adjacent to ψCs. Extensive variability due to N or P sequence additions, which is found in human but not mouse L chain sequences ( 25 , 27 , 28 ), was not observed. Sequences obtained by RT-PCR from FACS ® -sorted PP germinal center B cells (B220 + /PNA + ) revealed that somatic hypermutation is operative in HuIgλ YAC mice . We identified 11 unique Vλ-Jλ rearrangements with two or more changes in the V region, excluding the CDR3, which may be affected by Vλ-Jλ recombination. The majority of mutations lead to amino acid replacements, but there was no preferential distribution in CDR1 and CDR2. The ratio of λ to κ L chain expression varies considerably between different species ( 1 – 3 , 43 , 44 ), and in mice the low λ L chain levels are believed to be a result of inefficient activation of the mouse λ locus during B cell differentiation (for review see reference 6 ). The Igλ (∼40%) to Igκ (∼60%) ratio in humans is more balanced and suggests that both λ and κ play an equally important role in immune responses. This is supported by the finding that the mouse Vλ genes are most similar to the less frequently used distal human Vλ gene families, whereas no genes comparable to the major contributors to the human Vλ repertoire are present in the mouse locus ( 40 ). With the HuIgλYAC, these Vλ genes are available, and are able to make a significant contribution to the antibody repertoire. The 410-kb HuIgλYAC translocus accommodates V gene region cluster A containing at least 15 functional Vλ genes . In humans, cluster A is the main contributor to the λ antibody repertoire, with Vλ 2-14 (2a2) expressed most frequently at 27% in blood lymphocytes ( 23 ). We also find expression of Vλ 2-14 in the translocus mice, but the main contributors to the λ L chain repertoire were 3-1 (the Vλ gene most proximal to the J-C region) and 3-10, both of which are expressed at ∼3% in humans. Although the validity of conclusions about the contribution of different genes is dependent on the numbers examined, the overexpression of Vλ3-1 (11 sequences) and Vλ3-10 (8 sequences) in the 31 sequences obtained may imply that rearrangement or selection preferences are different in mice and humans. Analysis of recombination signal sequences (RSS) in mouse L chains showed that κ and λ RSS differ significantly, and that those genes with the highest similarity to consensus RSS rearrange most frequently ( 45 ). The RSS of Vλ3-1 and Vλ3-10 show a 100% match with the mouse consensus sequence, which may explain their frequent expression in the translocus mice. In addition, most human Vλ RSS match the established consensus sequence significantly better than mouse Vλs ( 21 , 45 ). We found extensive somatic hypermutation of many rearranged human Igλ sequences, indicating that they are able to participate in normal immune responses. The levels of mutation in B220 + /PNA + PP cells from HuIgλYAC translocus mice were similar to what has been reported for mouse L chains ( 46 ). Rather unexpected was the pattern of somatic hypermutation with similar numbers of silent and replacement point mutations found in the complementarity-determining and framework regions. Somatic hypermutation is usually associated with a higher level of replacement mutations in CDRs and more silent mutations in the framework regions, and the distribution observed here may argue against efficient antigen selection having taken place. Interestingly, however, λ L chain sequences obtained from human peripheral blood lymphocytes also showed high numbers of mutations in framework 2 ( 23 ). Part of framework 2 lies at the interface of the V L and V H domains and it has been suggested that this region may be important for optimal H and λ L chain interaction and, in particular, interaction of the human λ L chain and the endogenous mouse H chain ( 26 ). In the mouse, unlike in humans, L chain diversification due to untemplated nucleotide addition is essentially absent, because TdT expression has been downregulated by the stage at which L chain rearrangement takes place ( 28 , 47 ). This concept is challenged by the observation that mouse L chain rearrangement can occur at the same time as V H to DJ H rearrangements ( 48 ) or even earlier ( 42 ). Our results also show L chain rearrangement at the pre-B cell stage with a similar number of human λ- or mouse κ-expressing B cells also expressing c-kit + or CD25 + . Although the cell numbers are small, the results suggest that there is no preferential activation of either the human λ translocus or the endogenous κ locus. However, despite this early activation, there is no accumulation of N or P nucleotide diversity in the rearranged human λ L chains, unlike rearranged λ L chains from human peripheral B cells ( 27 ). The small number (<1%) of human Igλ + /mouse Igκ + double positive spleen and bone marrow cells may indicate that haplotype exclusion at the L chain level is less strictly controlled than is IgH exclusion ( 49 ). In transgenic mice carrying Ig regions in germline configuration on minigene constructs, efficient DNA rearrangement and high antibody expression levels are rarely achieved. Competition with the endogenous locus can be eliminated using Ig knockout strains, in which transgene expression is usually improved ( 50 ). Poor transloci expression levels could be a result of the failure of human sequences to work efficiently in the mouse background or, alternatively, of the absence of locus-specific control regions that are more likely to be included on larger transgenic regions ( 51 – 53 ). Recently we addressed this question in transgenic mice by the introduction of different sized minigene- and YAC-based human κ L chain loci ( 53 ). The result showed that neither the size of the V gene cluster nor the V gene numbers present were relevant to achieving high translocus expression levels. The YAC-based loci contained downstream regions of the human κ locus, and it is possible that the presence of an undefined region with cis-controlled regulatory sequences may have been crucial in determining expressibility and subsequently L chain choice. The HuIgλYAC contains equivalent regions from the human Igλ locus, which may promote the use of the translocus in the L chain repertoire. Hybridomas from HuIgλYAC + / κ +/− mice show no evidence for a bias in L chain locus selection during development, as demonstrated by the absence of rearrangement of the nonexpressed locus. This is in contrast with what is seen in Ig expressing mouse and human B cell clones ( 4 , 5 ), and supports the model that λ and κ rearrangements are indeed independent ( 15 , 54 ) and that poor Igλ expression levels in mice may be the result of inefficient signals acting during recombination ( 16 ). A possible signal that initiates L chain recombination has been identified through gene targeting experiments where the 3′ κ enhancer was deleted ( 17 ). In these mice, the κ/λ ratio was reduced from 20:1 in normal mice down to 1:1, and the κ locus was largely in germline configuration in λ-expressing cells, as we also see in the HuIgλYAC + /κ +/− hybridoma clones. The high level of human Igλ expression in the HuIgλYAC + /κ +/− mice could be due to the strength of the downstream enhancer of the human λ locus. An analysis of human L chain enhancer activities identified three synergistic modules at the 3′ end of the λ locus which constitute a powerful pre-B cell specific enhancer that appears to be stronger than the corresponding κ enhancer ( 55 ). Analysis of the mouse λ 3′ enhancer suggests the biased κ/λ ratio in mice may be a direct result of the differences in locus specific regulation provided by the respective enhancers ( 19 , 56 ). The results suggest that strength and ability of the human 3′ λ enhancer to function in the mouse background may be the reason that λ and κ loci can compete equally at the pre-B cell stage to initiate L chain rearrangement, resulting in the similar levels of human Igλ and mouse Igκ seen in the HuIgλYAC + /κ +/− mice. | Study | biomedical | en | 0.999998 |
10330441 | Neuronal (NOS1-KO), endothelial (NOS3-KO), and double neuronal and endothelial KO mice (NOS1&3-KO) and matched wild-type (WT) controls were bred in a sterile pathogen-free (SPF) barrier facility. These three gene-targeted mutants were produced on a mixed SV129/C57BL/6 background ( 38 – 40 ). The iNOS KO mice (NOS2-KO) were purchased from The Jackson Laboratory and were backcrossed for >10 generations onto a C57BL6/J (B6) background, the controls for the NOS2 KO mice. To control for gender-induced differences in airway reactivity, only male offspring were used for these studies. All mice were 4–5 wk old at entry into the protocol. Mice were housed in isolation cages under SPF conditions. Blood from sentinel animals was routinely tested to ensure their SPF status. All mice were acclimatized for 7–10 d after arrival and were studied at 7–8 wk of age. In one set of experiments the whole body plethysmographic method (Buxco ® ) was used to assess airway responsiveness in a different cohort of iNOS KO mice ( 41 ). These iNOS KO mice were provided by Drs. J.S. Mudgett (Merck Research Labs., Rahway, NJ), J.D. MacMicking, and C. Nathan (both from Cornell University Medical College, New York, NY) and had been backcrossed into a B6 background. Sex- and age-matched B6 mice were used as controls for the NOS2 group. The gene targeted mutants type (NOS1-KO, NOS3-KO, NOS2-KO, and NOS1&3-KO) and matched WT control mice (on the appropriate genetic background) were all sensitized to chicken OVA (Grade III; Sigma Chemical Co. ). Sensitized mice were then randomized to repeated exposure either to an aerosol generated from an OVA solution or to PBS. Two protocols were used in this study. In the first set of experiments, all mice were immunized on day 0 via an intraperitoneal injection with 10 μg chicken OVA, mixed with 1 mg Al(OH) 3 (alum; J.T. Baker Chemical) in 0.2 ml of PBS as previously described ( 7 ). A booster injection was given on day 7 using the identical reagents. Starting 7 d later, mice were exposed either to aerosolized OVA (6% OVA) dissolved in PBS (pH = 7.4) or to PBS alone for 25 min per day for 7 d consecutively. All mice were studied 24 h after the last aerosol (day 21). For the aerosol exposures mice were placed in a plastic chamber (23 × 23 × 11 cm), and the OVA or PBS solution was delivered via an ultrasonic nebulizer attached to a port in the mouse chamber. For mice studied using the whole body plethysmographic method of assessing airway responsiveness (Buxco ® ), mice were immunized on day 0 via an intraperitoneal injection with 20 μg chicken OVA mixed with 2 mg Al(OH) 3 . A booster injection was given on day 7 using 10 μg chicken OVA mixed with 1 mg Al(OH) 3 . All mice were exposed either to aerosolized OVA (3% OVA) or PBS for 10 min on days 14, 15, and 16, studied on day 17 (airway responsiveness measured by whole body plethysmography), and killed on day 18 (for bronchoalveolar lavage [BAL] and harvesting of tissues). OVA-specific IgE levels were measured by capture ELISA as previously described ( 42 ). ELISA plates were coated with a purified anti– mouse IgE mAb ( PharMingen ) at a concentration of 2 μg/ml and blocked with PBS/10% FCS. Serum samples were diluted in PBS/10% FCS and incubated in the wells for 2 h. After washing with 0.05% PBS/Tween 20, biotinylated OVA (10 μg/ml) was added to the wells and incubated for 1 h. The plates were washed with PBS/Tween 20 followed by the addition of avidin alkaline phosphatase ( Sigma Chemical Co. ) for 1 h. The plates were then washed with PBS/Tween 20 and distilled water, before the addition of the phosphatase substrate. The plates were allowed to develop for 30 min and read in an ELISA plate reader at 405 nm. Pulmonary NOS activity was measured using a modification of an in vitro [ 3 H] l -arginine to [ 3 H] l -citrulline conversion assay that has been described previously ( 43 ). Mouse lungs were kept frozen (−80°C) until the day of assay when they were homogenized in 10 vol of 50 mM potassium phosphate (pH 7.4). A 50-μl aliquot was incubated at 37°C for 15 min in 100 μl of incubation buffer (50 mM potassium phosphate, 60 mM l -valine, 1 mg/ml BSA, 1 mM NADPH, 10 μM FAD, 10 μM tetrahydrobiopterin, 30 μM [2,3- 3 H] l -arginine [200 counts/min/pmol], and 1.2 mM MgCl 2 ; pH 7.4). In the quantification of calcium-dependent NOS activity, indicating both constitutive calcium-dependent forms (i.e., nNOS and eNOS) and therefore termed cNOS, calmodulin (100 nM) and CaCl 2 (1 mM) were added to the incubation buffer. In contrast, EDTA (1.2 mM) and ethylene glycol-bis(b-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA; 1 mM) were added to the incubation buffer when measuring calcium-independent (iNOS) NOS activity. A third incubation condition of EDTA/ EGTA and l - N G -nitro-arginine methyl ester ( l -NAME; 1 mM) was used to account for nonspecific radiation and nonspecific metabolism (i.e., non-NOS-mediated conversion) of [ 3 H] l -arginine. The reaction was terminated by the addition of 500 μl of ice-cold stop buffer (4°C; 100 mM Hepes and 12 mM EDTA, pH 5.5) and 2 ml of 50% Dowex 50W (200–400, 8% cross-linked, Na + form, pH 7.0) in water in order to remove any excess [ 3 H] l -arginine. Samples were centrifuged for 20 min at 600 g and 0.5 ml of the supernatant was added to 4.5 ml of scintillation fluid; radioactivity was measured by liquid scintillation counting (Beckman Scientific Instruments). cNOS activity was calculated as the difference between the calcium–calmodulin sample (total NOS activity) and the EDTA–EGTA sample. iNOS activity was defined as the l -NAME–inhibitable proportion of the activity found in the samples containing EDTA/EGTA. Airway responsiveness was measured by two different methods in our study. In the first set of experiments airway responsiveness was measured in anesthetized mice using a sealed constant mass plethysmograph as previously described ( 7 , 29 , 44 – 46 ). In brief, dose–response curves to methacholine ( Sigma Chemical Co. ) were obtained 24 h after the last aerosol exposure of either OVA or PBS by administering sequentially increasing doses of methacholine intravenously (33–3,300 μg/kg) in a 20–35-μl volume. From the relationship between the administered dose and pulmonary resistance (R L ), the effective dose required to increase R L to 200% of control values (ED 200 R L ), was determined by log-linear interpolation. ED 200 R L is an index of airway responsiveness. In a second set of experiments, an alternative method of measuring airway responsiveness was adopted. This was done to confirm the results of a specific experiment and to see if the results could be reproduced in unanesthetized mice. The whole body plethysmograph system was therefore used to measure airway responsiveness in these experiments ( 47 , 48 ). The day after the last allergen challenge, each mouse was placed in a chamber and box pressure/time wave form was analyzed to yield the indicator of airflow obstruction, Penh. PBS or methacholine was given by aerosol in increasing concentrations through an inlet of the chamber for 1 min and readings were taken for 9 min at each dose step. Penh values averaged for 5 min after each nebulization were evaluated. After the termination of the experiments, BAL was performed on mice in each of the four treatment groups for NOS gene KO and WT strains. 2 ml of PBS with 0.6 mM EDTA was instilled into the lungs and retrieved using gentle suction. The lavage was centrifuged at 2,000 g for 10 min, the supernatant was separated from the cell pellet, and aliquots were frozen at −70°C for cytokine analysis. The cell pellets were resuspended in Hank's balanced salt medium (JRH Biosciences) and slides were prepared by spinning samples at 800 rpm for 10 min (Cytospin 2; Shandon). Total cell counts were made in a hemocytometer and differentials were prepared by cytospin and stained with Wright-Giemsa stain. The investigator counting the cells was blinded to the treatment groups. Eosinophil peroxidase (EPO) levels in the lavage were measured colorimetrically as previously described ( 2 , 49 ). 100 μl of sample or standard, porcine EPO (ExOxEmis Corp.) were pipetted, in duplicate, into the wells of a 96-well plate (Cell Wells™; Corning) followed by 100 μl of assay reaction mixture containing 0.05 M Tris buffer [Tris(hydroxymethyl)aminomethane; Trizma ® ; Sigma Chemical Co. ], 0.1 μl 30% H 2 O 2 ( Fisher Scientific Co. ), 0.015% Triton X-100 ( Sigma Chemical Co. ), pH 8.0, and 0.05 M ortho-phenylenediamine ( Sigma Chemical Co. ). The plate was incubated in the dark for 30 min and the reaction was terminated with 50 μl of 4 M H 2 SO 4 per well and then read on a plate reader (Spectramax Model 340; Molecular Devices Corp.) at 490 nm. A BCA protein assay (Pierce Scientific) was used to quantitate lavage protein. All regression analysis was performed using the Softmax Pro software (Spectramax Plate Reader Model 340; Molecular Devices Corp.). Mice were removed from the plethysmograph while under surgical anesthesia and killed by cervical dislocation. Blood was collected by cardiac puncture and the lungs were removed from the thoracic cavity and inflated with pH balanced 4% formaldehyde fixative (pH 7.4). A sagittal block of the whole left lung was dehydrated and embedded in paraffin and 5-μm sections were stained with hematoxylin and eosin and examined by light microscopy. Sections were examined for the presence of perivascular and peribronchiolar infiltrates by an investigator blinded to the treatment exposure or genotype groups. Computations were performed with the JMP ® 3.1.5 (SAS Institute Inc.) statistical package. A Tukey-Kramer HSD multiple comparison test was used to assess differences among the four treatment groups. For nonparametric data, differences between groups were analyzed using the Wilcoxon rank sum test. When appropriate, results are expressed as means ± SEM, and unless otherwise stated were considered statistically significant at the P < 0.05 level. In naive WT mice exposed to neither PBS nor OVA, basally expressed total pulmonary NOS activity was detectable at a low level (0.45 ± 0.08 pmol citrulline/mg/min), of which 75 ± 9% was accounted for by iNOS activity. In WT mice sensitized to OVA, but only challenged with aerosolized PBS, there was no change in total NOS activity (0.44 ± 0.12 pmol citrulline/mg/min, P = NS versus naive WT) or iNOS activity (80 ± 12% of total NOS, P = NS versus naive WT) . Basally expressed levels of total NOS activity were not significantly different in NOS3-KO (eNOS knockout), NOS1-KO (nNOS knockout), NOS1&3-KO (nNOS and eNOS double knockout), or NOS2-KO (iNOS knockout) mice in comparison with WT mice (analysis of variance on ranks, P = 0.23). The absolute level of cNOS activity (i.e., activity that was attributable to eNOS and nNOS) was not affected in the single cNOS KO strains, NOS1-KO and NOS3-KO, in comparison with the WT strain, but was significantly reduced in the double cNOS knockout (NOS1&3-KO) mice (0.0 ± 0.0 versus 0.08 ± 0.02 pmol citrulline/mg/min in naive WT, P < 0.05). The proportion of total NOS activity characterized as iNOS activity was not significantly different among WT, NOS1-KO, and NOS3-KO mice, but was increased in NOS1&3-KO mice (100 ± 0% of total NOS, P < 0.05 versus WT), and was markedly reduced in NOS2-KO mice to a level not different from zero (11 ± 6% of total NOS, P < 0.01 versus WT). Aerosol OVA exposure in OVA-sensitized (OVA/OVA) WT mice was associated with markedly increased total NOS activity (1.76 ± 0.30 pmol citrulline/mg/min, P < 0.01 versus naive WT), due completely to an increase in iNOS activity (99 ± 1% of total NOS, P < 0.05 versus WT) . OVA sensitization and challenge was associated with similar increases in total NOS activity and iNOS activity in NOS1-KO, NOS3-KO, and NOS1&3-KO mice. In contrast, OVA sensitization and challenge in NOS2-KO mice was not associated with any increase in total NOS (0.17 ± 0.06 versus 0.33 ± 0.10 pmol citrulline/mg/min in naive NOS2-KO, P = NS) or iNOS activity (0.08 ± 0.03 versus 0.04 ± 0.02 pmol citrulline/mg/min in naive NOS2-KO, P = NS). Airway responsiveness was measured in anesthetized OVA/OVA or OVA/PBS-treated mice. Airway responsiveness, expressed as the logED 200 R L , was measured in OVA/OVA NOS2-KO ( n = 7), OVA/OVA WT (SV129/B6, n = 10), OVA/OVA NOS3-KO ( n = 12), OVA/OVA NOS1&3-KO ( n = 10), OVA/OVA NOS1-KO ( n = 11), and OVA/OVA WT (B6, n = 15 mice) . Analysis of airway reactivity (assessed by the logED 200 R L ) revealed no significant differences between the OVA/OVA NOS2-KO and OVA/OVA WT (on a B6 background) groups . There was a significant induction in allergen-induced airway hyperresponsiveness for both the NOS2-KO and WT (B6) groups when compared with their respective OVA/PBS control groups ( P < 0.05). The OVA/PBS NOS2-KO group was significantly less responsive than the OVA/PBS WT (B6) group ( P < 0.001, data not shown). When the methacholine-induced airway responses were analyzed in the OVA/OVA NOS1-KO and OVA/OVA NOS1&3-KO groups, there were no significant differences between the two groups ( P = 0.76). When the OVA/OVA NOS1-KO airway responses were compared with the OVA/OVA NOS3-KO mice, the OVA/OVA NOS1-KO mice were significantly less responsive to methacholine challenge . Similarly, when the OVA/OVA NOS1&3-KO airway responses were compared with the OVA/OVA NOS3-KO mice, the OVA/OVA NOS1&3-KO mice were significantly less responsive to methacholine challenge ( P = 0.016). Therefore, the OVA/OVA NOS1&3-KO and OVA/OVA NOS1-KO groups were the most hyporesponsive of the OVA/OVA groups, whereas the OVA/OVA WT (B6 or SV129/B6) and OVA/OVA NOS2-KO groups were the most responsive . There were no significant differences between the OVA/PBS control groups (on a B6 or SV129/B6 background) with the single exception that the OVA/PBS NOS1&3-KO group was significantly less responsive to methacholine challenge when compared with the OVA/PBS WT (SV129/B6) group ( P = 0.004) (data not shown). Inducible NOS (iNOS) is upregulated in cases of human allergic asthma ( 18 , 28 ) and is believed to represent the main source of increased expired NO, a molecule believed to modulate airway function ( 50 ). The lack of a difference between the OVA/OVA NOS2-KO and OVA/OVA WT (B6) groups was unexpected and prompted us to carry out a second set of experiments to confirm these results using a different methodology for measuring airway obstruction and a modified sensitization and challenge protocol. As there were no discernible differences in airway reactivity after 7 d of daily allergen exposures, we reasoned that a shorter more acute exposure may reveal a difference in airway responsiveness between the OVA/OVA NOS2-KO and OVA/OVA WT groups. In this second set of experiments, Penh, an index of bronchoconstriction, was measured using a whole body plethysmograph system in awake OVA/OVA NOS2-KO ( n = 14) and OVA/OVA WT ( n = 13) mice. Analysis of aerosol methacholine dose–response curves similarly revealed no significant differences in the degree of airway responsiveness between the OVA/OVA NOS2-KO and OVA/OVA WT groups, confirming the earlier data obtained by measuring R L . These results indicate that our observation of no difference in airway responsiveness between WT and NOS2-KO mice is not an artifact of our specific protocols. Aerosol OVA exposure in all OVA-sensitized mice was associated with a significant increase in total cell counts in BAL fluid compared with OVA-sensitized and PBS-challenged mice. The BAL total cell counts did not differ among the WT, NOS2-KO, NOS1-KO, NOS3-KO, and NOS1&3-KO OVA/PBS control groups (data not shown). The BAL total cell counts (× 10 3 cells ± SEM) for the WT (SV129/B6) ( n = 10), NOS2-KO ( n = 7), NOS1-KO ( n = 11), NOS3-KO ( n = 16), and NOS1&3-KO ( n = 9) OVA/OVA groups were 642 ± 99.7, 843 ± 125, 904 ± 149, 883 ± 104, 531 ± 228 × 10 3 cells, respectively. There were no significant differences in the BAL total cell counts among the OVA-sensitized and -challenged mice groups. There were no significant differences in the proportions of macrophages, neutrophils, lymphocytes, and eosinophils in the OVA/OVA exposed mice among the treatment groups (Table I ). Differential cell counts in the OVA/PBS groups revealed no significant differences in the proportion of macrophages, neutrophils, lymphocytes, and eosinophils (Table I ). Antigen sensitization and challenge induced a significant increase in serum levels of OVA-specific IgE in the WT, NOS2-KO, NOS1-KO, NOS3-KO, and NOS1&3-KO groups compared with OVA-sensitized and PBS-challenged groups. OVA-specific IgE levels reported as absorbance values (OD 405 nm) measured in the OVA/OVA WT, NOS2-KO, NOS1-KO, NOS3-KO, and NOS1&3-KO groups were 0.71 ± 0.10, 0.61 ± 0.14, 0.43 ± 0.16, 0.93 ± 0.25, and 0.79 ± 0.32, respectively. There were no significant differences among the OVA/ OVA groups. The values for the absorbance (OD 405 nm) measured in the OVA/PBS WT, NOS2-KO, NOS1-KO, NOS3-KO, and NOS1&3-KO groups were 0.03 ± 0.01, 0.14 ± 0.05, 0.05 ± 0.03, 0.11 ± 0.06, and 0.03 ± 0.03, respectively. Similarly, there were no significant differences among the OVA/PBS control groups. When the OVA/OVA groups were compared with their matched OVA/PBS control groups, there were significant within genotype differences attributable to sensitization ( P < 0.05) for all groups with the exception of the NOS1-KO group, which approached but did not achieve statistical significance ( P = 0.052). BAL EPO and total protein levels were evaluated as fluid phase indicators of the inflammatory response to allergen challenge. There were no significant differences in either EPO or total protein levels within the OVA/OVA or OVA/PBS groups (data not shown). Examination of fixed lung tissue from the OVA-sensitized and -challenged groups revealed peribronchial and perivascular accumulation of eosinophils, granulocytes, and mononuclear cells. No differences in the degree of airway inflammation or presence of inflammatory infiltrates were noted between the OVA/OVA WT and NOS-deficient mice (all NOS KOs). Similarly, the histological findings in the OVA-sensitized and PBS-challenged groups were unremarkable with no observable differences between the KO and WT mice. Systemic sensitization and aerosol challenge with allergen in mice reproduces many of the phenotypic features of human asthma, including increased airway hyperresponsiveness, airway inflammation, and increased antigen-specific IgE. The role of NO in human asthma or animal models thereof is unclear, as is the relative contribution of each of the NOS isoforms. NO has been reported to exhibit both beneficial and deleterious effects in asthma. It has been reported to be a weak bronchodilator ( 19 – 21 ); however, others have reported no effect on airway tone (22– 24). Still others have reported that the formation of NO may have deleterious effects due to its reported cytotoxic effects ( 18 ), and finally it has been reported that NO may serve as a marker of inflammation ( 16 , 17 , 51 ). To determine the specific contribution of each of the NOS isoforms in asthma, we studied mice with targeted deletions of the endothelial, neuronal, inducible, and double endothelial and neuronal NOS isoforms using an established model of allergic asthma in the mouse ( 7 , 52 ). The use of gene KO mice is advantageous in isolating the specific contribution of each NOS isoform. Indeed, the use of NOS inhibitors to dissect out the role of constitutive and inducible NOS is subject to problems of inhibitor specificity and pharmacokinetic concerns. Because iNOS has been shown to be upregulated in asthma and is believed to represent the major source of NO in the lung ( 13 , 53 , 54 ), we reasoned that iNOS would play a pivotal role in either relaxing or exacerbating the allergen induced airway hyperresponsiveness. Airway responsiveness is classically assessed by analyzing dose– response relationships in which a bronchoconstrictor is administered in increasing doses while monitoring the effects of the agonist on mechanical indices of pulmonary obstruction such as pulmonary resistance (R L ) ( 2 , 7 , 44 , 45 ). This index of airflow obstruction represents the pressure loss in phase with airflow and is analogous to measures of pulmonary resistance in human subjects. Our data show that iNOS is significantly upregulated in the lung tissue of the WT mice sensitized and challenged with OVA, a finding reported in asthmatics ( 9 , 18 ). Despite this upregulation, there were no significant differences in airway responsiveness, airway inflammation, or cellular recruitment into the airway space when compared with the NOS2-KO OVA-challenged treatment group. In this regard our data do not agree with the recent findings of Xiong et al., who demonstrated a significant decrease in eosinophilia and suppression of allergic inflammation in NOS2 KO mice immunized and challenged with OVA ( 55 ). The discrepancy in their results and our own is likely due to significant differences in the immunization and challenge protocols ( 55 ). The immunization and challenge protocol used by Xiong et al. resulted in ∼90% eosinophilia in the BAL in their WT OVA/OVA group, considerably higher than the proportion reported by us and others in models of allergen- induced airway inflammation in the mouse ( 7 , 52 , 56 – 59 ). The lack of a significant difference in the pulmonary resistance (R L ) response to methacholine between the NOS2-KO OVA/OVA and WT (B6) OVA/OVA mice prompted us to repeat these experiments using unanesthetized mice studied in a whole body plethysmograph. This method ( 47 , 60 ) confirmed our previous observation whereby the NOS2-KO OVA/OVA and WT OVA/ OVA mice exhibited increased, albeit similar degrees of airway hyperresponsiveness. In this regard our data are in agreement with the findings of Xiong et al., who also demonstrated that NOS2-KO mice did not have diminished airway hyperresponsiveness when compared with WT mice exposed to an OVA sensitization and challenge protocol ( 55 ). Thus, our data and those of others clearly indicate that allergen-induced airway hyperresponsiveness is fully expressed in the absence of iNOS. Analysis of markers of inflammation (total protein and EPO) in the BAL fluid revealed no significant differences in our data set between the NOS2-KO and WT OVA/ OVA groups. Additionally, there were no significant differences in the levels of OVA-specific IgE between any of the OVA-sensitized and -challenged treatment groups. This is not surprising given that NO has no reported effects on antigen presentation and processing. These findings indicate that the iNOS isoform is not important in the genesis of airway inflammation in this allergic model of asthma. When airway responsiveness was ascertained in the NOS3-KO, NOS1-KO, and NOS1&3-KO mice, the NOS1-KO and NOS1&3-KO OVA/OVA groups exhibited increased airway responsiveness when compared with their respective controls (OVA/PBS), yet there was significantly less enhancement of airway responses secondary to OVA exposure than in NOS2-KO and WT mice. The similar degree of diminished airway hyperresponsiveness among the NOS1-KO and NOS1&3-KO mice, and the observation of a more intermediate degree of airway hyperresponsiveness in the NOS3-KO OVA/OVA mice, indicate that the nNOS isoform is the one principally responsible for the OVA-induced enhancement of airway responsiveness. The reduction in allergen-induced airway hyperresponsiveness in NOS1-KO mice is interesting given that nNOS has previously been shown to contribute to baseline airway responsiveness ( 29 ). Although the results of our study do not determine the mechanism(s) by which nNOS is modulating airway responsiveness, the neuronal-derived NO may directly effect airway smooth muscle tone. NO has been implicated in nonadrenergic, noncholinergic (NANC)-mediated airway relaxation ( 30 , 31 ). Indeed, our data are intriguing given recent studies that have established linkage of the diagnosis of asthma to a region that maps near the human nNOS gene ( 35 – 37 ). Aside from the differences in airway responsiveness, the NOS1-KO and NOS1&3-KO OVA/OVA groups did not differ in their degree of airway inflammation, levels of OVA-specific IgE, or recruitment of inflammatory cells into the BAL. These findings suggest a noninflammatory link between the nNOS gene and airway hyperresponsiveness in this murine model of allergic asthma. | Study | biomedical | en | 0.999995 |
10330442 | C57BL/6J (Ly5.1) mice were obtained from The Jackson Laboratory . C57BL/6-Ly5.2 mice were obtained from Charles River Labs. through the National Cancer Institute animal program. The OT-I mouse line ( 28 ) was maintained as a C57BL/6-Ly5.2 line or on a C57BL/6-RAG-1 −/− background (The Jackson Laboratory ). OT-I-β7 −/− mice and OT-I-αE −/− mice were generated by intercross of the β7 −/− ( 27 ) or αE −/− mouse lines with OT-I and screening for αE or β7 and transgenic TCR expression by flow cytometric analysis of peripheral blood. The αE −/− mouse line was produced by standard techniques and is described in detail elsewhere ( 29 ). Vesicular stomatitis virus encoding ovalbumin (VSV-ova) was produced by ligation of a XhoI-XbaI cDNA fragment containing the entire ova coding sequence into the pVSV-XN2 vector restricted by XhoI and NheI ( 30 , 31 ). The ova gene–containing vector was transfected along with helper plasmids into BHK cells, and rVSV was recovered as previously described ( 30 , 31 ). Ovalbumin production was assessed by Western blot analysis of detergent lysates and culture supernatants of infected BHK cells as previously described ( 26 ). This method was adopted from Kearney et al. ( 21 ). An equal mixture of 2.5 × 10 6 pooled LN cells (1.25 × 10 6 OT-I T cells from OT-I or OT-I-β7 −/− or OT-I-αE −/− mice) were injected intravenously into C57BL/6J (Ly5.1), C57BL/6-Ly5.2, or C57BL/6-Ly5.1/5.2 mice, depending on the donor cell phenotype. 2 d later, mice were infected with 10 6 PFU wild-type Indiana serotype VSV (as control) or VSV-ova by intravenous injection. At the later times indicated, cells were isolated and analyzed for the presence of donor cells by flow cytometric analysis of Ly5.1/Ly5.2 expression. IEL and LP cells were isolated as described previously ( 32 , 33 ). Superficial inguinal, axial, and brachial LNs (peripheral [P]LNs) or MLNs were removed and single cell suspensions were prepared using a tissue homogenizer. The resulting preparation was filtered through Nitex (Tetko Industries) and the filtrate centrifuged to pellet the cells. The following mAbs were used in this study: 53-6.7, anti-CD8α ( 34 ); anti-Ly5.1 and anti-Ly5.2 ( 35 ); and 2E7, anti-αE integrin ( 18 ). mAbs specific for Vα2, Vβ5, and β7 integrin were obtained from PharMingen as fluorochrome or biotinylated conjugates. For staining, lymphocytes were resuspended in 0.2% PBS, 0.1% BSA, NaN 3 (PBS/BSA/ NaN 3 ) at a concentration of 10 6 –10 7 cells/ml, followed by incubation at 4°C for 20 min with 100 μl properly diluted mAb. The mAbs were either directly labeled with FITC, PE, Cy5 ( Amersham Life Science), or were biotinylated. For the latter, avidin– PE–Cy7 (Caltag Labs.) was used as a secondary reagent for detection. After staining, the cells were washed twice with PBS/BSA/ NaN 3 and fixed in 3% paraformaldehyde buffer. Four-color analysis of relative fluorescence intensities was then performed with a FACSCalibur ™ ( Becton Dickinson ). Data were analyzed using LYSYS II™ ( Becton Dickinson ) or WinMDI software. To develop a system for analyzing the role of β7 integrins in trafficking of naive and activated CD8 T cells, we introduced TCR transgenes into mouse lines with disruption of the β7 integrin gene or the αE integrin gene. T cells of the OT-I mouse line express the transgene-encoded Vα2 and Vβ5 chains, are predominantly CD8, and recognize the ova peptide, SIINFEKL, in the context of MHC class I H-2K b ( 28 ). LN cells from OT-I, OT-I-αE −/− , and OT-I-β7 −/− mice were analyzed for TCR usage and CD8 and β7 integrin expression . CD8 cells from the LN of all three strains of mice expressed Vα2 and Vβ5 (representative example shown in top panel). OT-I CD8 LN cells (either peripheral or mesenteric) expressed heterogenous levels of the β7 integrin, with a population of β7 high cells evident. This population was absent in OT-I-αE −/− LN cells, indicating that the high-level expression was due to expression of the αE integrin chain coupled to the β7 chain. This was confirmed by simultaneous staining for αE and β7 integrins (data not shown). These patterns of β7 staining are analogous to what has been observed for nontransgenic naive T cell populations ( 36 ), indicating that despite homogenous TCR transgene expression, tissue-specific and cell type–specific heterogeneity in β7 integrin expression remained. To allow a direct comparison of migration of normal and integrin-deficient OT-I cells, we developed a system in which normal and mutant OT-I cells were mixed in equal proportions and then transferred to normal mice. By virtue of Ly5.1/Ly5.2 expression, transferred as well as host populations could be distinguished by flow cytometry . In unimmunized mice, OT-I-β7 −/− and normal OT-I cells migrated to PLN (data not shown) and MLN equally well. 3 d after immunization with rVSV-ova ( 26 ), a substantial increase in OT-I-β7 −/− and OT-I cells had occurred in PLN. This result indicated that β7 integrins were not involved in primary activation of OT-I CD8 T cells and showed that β7 integrins were not necessary for migration of naive or activated CD8 T cells to PLN. In contrast, despite similar numbers of normal and mutant cells in the starting MLN population, 4.5-fold fewer OT-I-β7 −/− cells were present in MLN after infection. All OT-I cells were activated, as determined by an increase in cell size and upregulation of CD44 (reference 26 ; data not shown). The population of OT-I-β7 −/− cells present in MLN after infection was likely the result of expansion of those cells present before immunization. Thus, the difference between this value and the number of control cells may be used as an indicator of the degree of migration into MLN from the peripheral CD8 T cell pool. When PP lymphocytes were examined after infection, a barely detectable population of OT-I-β7 −/− cells was found, indicating a near absolute requirement for β7 integrins in migration of activated CD8 T cells to PP. The finding that migration to MLN was apparently different than that to PP (based on a substantial number of OT-I-β7 −/− cells in MLN of naive and immunized mice) suggested that migration of naive OT-I-β7 −/− cells to PP may be distinct as compared with MLN. We tested this possibility by examination of PP 2 d after transfer of a mixture of naive LN cells from OT-I or OT-I-β7 −/− mice . This population contains primarily CD8 T cells and B cells. Analysis of total PP CD8 T cells indicated that β7 integrins were in fact required for naive OT-I T cells to enter PP, as a distinct population of normal OT-I but not OT-I-β7 −/− cells was detected. Interestingly, examination of transferred B cells also demonstrated a requirement for β7 integrins in their migration to PP. These results indicated that migration to PP was much more stringent than migration to MLN, particularly for naive lymphocytes. The requirement for β7 integrins in homing to the LP and IEL compartment of small and large intestine was examined using the adoptive transfer system. After transfer of naive OT-I cells to normal mice, few if any transgenic T cells could be detected in LP or IEL . After immunization with VSV-ova, a large population of normal OT-I CD8 cells were present in IEL and LP. However, 7–10-fold fewer OT-I-β7 −/− cells were present in either site, indicating a stringent but not absolute requirement for β7 integrins in migration of activated CD8 T cells to small intestine effector sites. Similarly, migration of activated CD8 T cells to large intestine IEL was also dependent on β7 integrins . These results agree well with analysis of β7-deficient mice in which LP and IEL populations are reduced ( 27 ) and suggest that many mucosal effector cells are derived from cells activated outside of the LP and IEL compartments. Because the β7 integrin requirements for migration to peripheral and mucosal lymphoid tissue were distinct, we analyzed β7 integrin expression of adoptively transferred normal OT-I cells before and after immunization with VSV-ova. After transfer, naive OT-I cells in PLN and MLN retained low levels of β7 integrin expression . After immunization of mice with VSV-ova, OT-I cells in PLN were comprised of two major populations based on β7 integrin expression. One subset expressed β7 levels slightly higher than those of naive cells, and the other population (approximately half the cells) had high levels of β7 integrins. The majority of this expression was due to α4β7 expression, as determined by two-color flow cytometry and analysis of αE −/− OT-I cells (data not shown). In contrast to activated OT-I cells in PLN, OT-I cells in MLN and IEL expressed homogeneously high levels of β7 integrins. In these populations, the αEβ7 integrin contributed more significantly to overall β7 integrin expression than it did in OT-I cells in PLN (25; data not shown). These results indicated that activated, but not naive, CD8 cells expressing high levels of β7 integrins preferentially home to MLN and IEL, a finding which correlates with our demonstrated requirement for β7 integrins in migration of activated CD8 T cells to MLN and PP. The data thus far using β7 integrin–deficient OT-I cells does not allow us to distinguish the relative roles of α4β7 integrin and αEβ7 integrin in migration. Although the available literature shows a clear role for α4β7 in migration of lymphocytes to the mucosa, the role of αEβ7 in trafficking of lymphocytes remains unclear. Therefore, as described above for OT-I-β7 −/− cells, we transferred a mixture of naive normal OT-I and OT-I-αE −/− cells to normal mice and analyzed distribution of naive and activated populations. Trafficking of naive OT-I cells to PLN, MLN , or PP (data not shown) did not require αEβ7 expression. Similarly, after infection with VSV-ova, OT-I and OT-I-αE −/− cells were equally distributed in PLN and MLN , indicating that the requirement for β7 integrins in homing to MLN was solely attributable to α4β7. As with normal naive OT-I cells, naive OT-I-αE −/− cells did not migrate to LP or IEL . Moreover, activation via VSV-ova infection resulted in equivalent migration of OT-I-αE −/− and normal OT-I cells into LP and the IEL compartment. This result indicated that the α4β7 integrin was the primary β7 integrin participating in homing of activated CD8 T cells to mucosal effector sites. Normal IELs express high levels of αEβ7 and low levels of α4β7 ( 36 ), supporting the concept that αEβ7 may be involved in tethering of IELs in the epithelium. We tested whether OT-I cells modulated their β7 integrin expression following entry into the epithelium by analyzing long-lived mucosal OT-I cells . 3 wk after immunization, a small population of OT-I cells was detected in the epithelium as well as in peripheral lymphoid organs (data not shown). Analysis of the long-lived transferred IEL revealed that normal OT-I cells had high levels of β7 integrins, and this was nearly all attributable to αEβ7 expression identical to that of host IEL. The latter finding was supported by the lack of appreciable β7 integrin expression by long-lived OT-I-αE −/− IEL. These results indicated that as a consequence of entry into the epithelium, α4β7 was downregulated and αEβ7 was highly upregulated. This phenomenon was not observed with long-lived nonmucosal OT-I cells (data not shown). A comparison of the long-term retention of OT-I cells with or without expression of αEβ7 revealed no significant differences between the populations. In the experiment shown, OT-I-αE −/− IEL were reduced by half as compared with normal OT-I cells . However, this difference was also evident in PLN and MLN and was not consistently observed. Thus, at least within the time frame analyzed, αEβ7 and perhaps α4β7 were not required for retention of CD8 T cells in the intestinal epithelium. The modified adoptive transfer system described here allowed, for the first time, visualization of the role of β7 integrins during an ongoing antiviral immune response in vivo. By transferring equal numbers of trackable naive normal and β7-deficient antigen-specific CD8 T cells, a direct comparison of the relative requirement for β7 integrins in lymphocyte migration before and after in vivo activation could be made. The system showed clearly that naive CD8 LN cells do not enter the effector sites of the intestinal mucosa, the LP and IEL compartments, whereas naive cells entered the inductive sites, the MLN and PP. However, an interesting finding was that the integrin requirements for trafficking of naive lymphocytes to MLN and PP were distinct. Thus, whereas the α4β7 integrin was not required for migration of naive LN cells to MLN or PLN, there was a near absolute requirement for entry of naive CD8 T cells and B cells into PP. The results from our and other studies ( 37 , 38 ) indicate that lymphocyte entry into MLN can be mediated by l -selectin in combination with α4β7 or LFA-1, and entry into PP requires l -selectin in combination with α4β7 or α4β7 alone, whereas the combination of l -selectin/LFA-1 is not sufficient for lymphocyte entry into PP. These findings suggest that a functional distinction exists even within inductive sites of the mucosal immune system, in that MLN and PP exhibit distinct requirements for lymphocyte entry. In contrast to the lack of a requirement for β7 integrins for naive lymphocytes to enter MLN, activated CD8 T cells relied heavily on this integrin to migrate to MLN. In this case, our system allowed an estimation of the degree of CD8 T cell expansion in situ versus the degree of migration to the MLN. That all OT-I-β7 −/− cells in MLN after immunization were activated suggested that these cells originated from the population of OT-I-β7 −/− cells present in MLN before immunization and underwent in situ expansion, which is not dependent on β7 integrins. If in situ expansion was dependent on β7 integrins, then a significant proportion of nonactivated OT-I-β7 −/− cells should have been detected in the MLN after immunization. There was also a clear early preference for activated CD8 T cell migration to MLN versus PLN, because after immunization, the proportion of antigen-specific CD8 T cells was greater in MLN versus PLN. Immunization resulted in a relative ∼4-fold increase in OT-I-β7 −/− cells in MLN versus an ∼20-fold increase in normal OT-I cells in the same MLN. If migration accounts for the majority of this difference, then ∼80% of the increase in CD8 T cells in the MLN was the result of migration rather than in situ expansion. These results delineated a dichotomy in β7 integrin requirements for entry into MLN, because activated but not naive CD8 cells required α4β7 for this movement. The α4β7 integrin was also essential for migration of activated CD8 T cells to PP. This effect was dramatic, as few naive OT-I-β7 −/− cells were present in PP before immunization. In short-term homing assays (90 min), little migration of β7 −/− whole spleen cells to PP was detected ( 27 ), in agreement with the results shown here. In the case of MLN, migration was reduced by approximately half in the absence of β7 −/− ( 27 ). As naive β7 −/− and β7 +/+ CD8 cells homed equally well to MLN in the present system, these results in short-term migration may indicate supplantation of β7 by other molecules in the longer term ( 37 ) or could be due to decreased binding of non-CD8 + cells or poor binding of memory lymphocytes contained in the spleen populations. Nevertheless, these results indicated a near absolute requirement for α4β7 to direct migration of naive and activated lymphocytes to PP, whereas the requirements for entry of naive and activated cells to MLN were different. Although migration of CD8 T cells to PP was totally dependent on β7 integrins, some β7-independent migration of activated CD8 T cells to intestinal mucosal effector sites was noted. Naive OT-I cells did not enter the LP or IEL compartments, whether they expressed β7 integrins or not. After immunization, however, large numbers of activated CD8 T cells entered the LP and the epithelium. This migration begins ∼48 h after immunization (25, 26; data not shown). Only ∼85–90% of this migration was β7 integrin dependent, as determined by comparison of migration of normal and β7 −/− OT-I cells. This was true in small intestinal LP and the IEL compartments of small and large intestine. This was a conservative estimate, as the adoptive transfer system may overestimate the significance of β7 integrins due to competition between normal and mutant cells. That migration to the IEL compartment required, for the most part, β7 integrins suggested that either α4β7 interaction with basement membrane allows CD8 cells to enter the epithelium from the LP or the inhibition of entry to the LP resulted in decreased availability of cells for entry into the IEL population. Further analysis of other adhesion molecules will be necessary to answer this question. The demonstration of some β7-independent migration indicated that other adhesion molecules are capable of directing migration to the LP and the epithelium. Although α4β7 is thought to be the major participant in contact, rolling, arrest, and diapedesis of lymphocytes homing to LP ( 3 ), we previously demonstrated that β2 integrins and intracellular adhesion molecule 1 are important for the establishment of the CD8αβ LP and IEL populations ( 39 ). These findings suggest that the β2 integrins, perhaps LFA-1, may be able to direct some level of homing to intestinal effector sites. Perhaps other adhesion molecules, such as l -selectin, are also involved in β7-independent homing to mucosa. In contrast to the well-described functions of the α4β7 integrin, the function of the αEβ7 integrin in vivo remains unknown. The only known ligand for αEβ7 is E-cadherin, which is highly expressed by intestinal and other epithelial cells ( 19 , 40 ). Because IEL half-life is much less than that of epithelial cells, this finding supported the hypothesis that IEL are retained in the epithelium via an αEβ7–E-cadherin interaction. As α4 integrin–deficient mice have normal IEL numbers ( 41 ), whereas β7 integrin–deficient mice have reduced IEL numbers ( 27 ), these results also support the possibility that αEβ7 is involved in some aspect of IEL migration or retention. However, the data shown here do not support this concept. The absence of the αE integrin chain on OT-I cells had no effect on their ability to migrate to the LP or the epithelium after primary activation, indicating that all β7-mediated migration was α4β7 dependent. Moreover, OT-I cells with or without αE expression were retained equally well in the epithelium for up to 3 wk. Indeed, the long-lived cells expressed low levels of β7 integrins, suggesting that α4β7 was also not involved in long-term retention of IEL in the epithelium. Therefore, other adhesive molecules must be involved in allowing long-lived IEL to be maintained in the epithelium, perhaps via interactions with the basement membrane rather than the epithelial cells. Given the in vitro data, it seems likely that the αEβ7 integrin plays an adhesive role not measured by our system. What remains unknown is the resulting functional outcome of such an interaction. | Study | biomedical | en | 0.999999 |
10330443 | The mIgM construct: a VDJ region containing the canonical anti-(4-hydroxy-3-nitro-phenyl) acetyl membrane (NP) V H 186.2 was cut from plasmid CμB1-8.24, containing the V region of the hybridoma B1-8 ( 17 ), using EcoRI and XbaI. It was then ligated to an XbaI-EcoRI fragment containing the IgH intronic enhancer region from plasmid Cμ19.7.1 ( 17 ). The BglII-XhoI fragment of pICEM-Cμ (containing Cμ1-4, and membrane and secreted exons) was replaced by the corresponding region from pSV5-Cμm, from which the secreted exon and polyadenylation site had been deleted ( 18 ). The EcoRI-XhoI fragment of the resulting plasmid, pICEM-Cμm, was then inserted into pBKS-II. The VDJ-enhancer unit was then excised as an EcoRI fragment and inserted at the EcoRI site of calf intestinal phosphatase (CIP)-treated pBKS-Cμm; correct orientation was confirmed by restriction digest. The resulting V H 186.2–mIgM construct (termed mIgM hereon) encodes membrane-bound, but not secreted, IgM a . The mIgM construct was tested by transfection into the CH1 cell line, which expresses IgM b using λ light chain. Although FACS ® analysis confirmed the presence of NP-binding IgM a on transfectants, no IgM a was detectable by ELISA in culture supernatant. Transgenic (Tgic) mice resulting from microinjection of the purified DNA construct were initially identified by Southern blot analysis. Mice bearing the mIgM transgene (Tg) were initially backcrossed with MRL/ lpr mice four times, fixing homozygosity for lpr . The resultant Tg-positive mice were then backcrossed to the J H D/ lpr strain to fix homozygosity of the J H D mutation, a neo insertion in the J H locus (fixation occurred after backcross one). This eliminated the expression of endogenous Ig heavy chains. All subsequent Tg-positive mice were generated by backcrossing to the J H D/ lpr strain. Through this breeding strategy, all the Tgic animals used in this study had >98.9% MRL genes. The resulting mIgM.MRL/MpJ- Fas lpr mice (termed mIgM mice hereon) have functional B cells that do not secrete appreciable Ig (see below). All animals used in this study were obtained from our colony at the Yale University School of Medicine (New Haven, CT) and were housed under specific pathogen-free conditions. All Tgic mice and controls analyzed in this study were aged 24 wk or more. A PCR detecting the rearranged V H 186.2 V(D)J segment of the mIgM Tg was used to identify the Tg-positive mice. The oligonucleotides used for this PCR were V H 186.2 5′ (5′-TGCTCTTCTTGGCAGCAAC-3′ [5′ primer]) and V H 186.2 3′ (5′-TGAGGAGACTGTGAGAGTG-3′ [3′ primer]). Amplification conditions were 94°C for 2 min and 35 cycles of 30 s each at 94°C, 54°C, and 72°C, followed by a 7-min incubation at 72°C. Homozygosity for lpr was detected by PCR as previously described ( 13 ). The following mAbs were used as FACS ® reagents in this study: CD19 (1D3-biotin; PharMingen ); B220 (RA3-6B2-FITC); CD4 (H129.19-CyChrome; PharMingen ); CD44 (Pgp-1-FITC); CD62L (Mel-14–biotin); and anti– mouse Fcγ receptor (2.4G2). Streptavidin-conjugated PE (Molecular Probes) was added as a secondary step for the biotinylated reagents. Pgp-1, Mel-14, and RA3-6B2 were purified from hybridoma supernatants on protein G columns ( Amersham Pharmacia Biotech ) after ammonium sulfate precipitation, and were conjugated as described ( 19 ). The antibodies were verified by comparison with commercially available antibodies with the same specificities. The following mAbs were used as standard controls for the ELISAs: T183 (IgM; Sigma Chemical Co. ), 4G7 (IgG1; reference 19 ), Hy1.2 (IgG2a; reference 20 ), Pl9-10 (IgG2b), Pl9-11 (IgG3), Pl9-10 (anti-double stranded [ds]DNA; reference 21 ), 400tμ23 (RF; reference 19 ), and LG4-1 (anti-chromatin; reference 22 ). 23.3 culture supernatant was used as the source of anti-NP IgG2a for the rheumatoid factor ELISAs ( 19 ). Polyclonal anti–mouse κ conjugated to FITC (Southern Biotechnology Associates, Inc.) was used in the immunofluorescence assays to detect glomerular Ig deposition. FACS ® analysis was conducted as previously described ( 16 ). For anti-Ig ELISA, Falcon 96-well, flat-bottomed plates ( Becton Dickinson ) were coated overnight at 4°C with the primary antibody (antiisotype). Plates were then blocked with 1% BSA in PBS for 1 h at room temperature. Serum dilutions along with the standard were allowed to incubate for 2 h. The secondary antibody (antiisotype conjugated to alkaline phosphatase; Southern Biotechnology Associates, Inc.) was then incubated in the plate for 1 h at room temperature. For autoantibody ELISA, the assays were conducted in the same manner as indicated above. However, the following were used to coat the plates with autoantigen: poly- l -lysine and purified calf thymus DNA (anti-dsDNA), NP/anti-NP IgG2a complexes (RF), and purified calf thymus chromatin (antichromatin). The secondary antibody was goat polyclonal anti–mouse κ conjugated to biotin, followed by streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Inc.). The colorimetric data from the pNPP substrate was quantified using Bio Kinetics Reader EL 340 (Bio-Tek Instruments). DeltaSoftIII software (BioMetallics, Inc.) was used to analyze this data. Kidneys were bisected and fixed in 10% buffered formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin. For immunofluorescent evaluation, kidney halves were lightly fixed in a 0.7% paraformaldehyde-lysine-periodate solution overnight at 4°C. Then, each sample was incubated in a 30% sucrose phosphate buffer at room temperature for >2 h and frozen in OCT compound (Tissue-Tek). 7-μm sections were cut using a Cryocut 1800 cryostat (Reichert-Jung) and stained overnight with an anti–mouse κ conjugated with FITC (Southern Biotechnology Associates, Inc.). Fluorescence was visualized using a Nikon Optiphot microscope ( Nikon , Japan) at 200× or 400× and was photographed with an exposure time of 5–6 s on Elite ASA 400 film ( Eastman Kodak Co. ). The severity of nephritis and vasculitis was graded based on a semiquantitative scale using the parameters described in Table I of reference 23 . In brief, a 0–4+ scale was used for each compartment (glomerular, interstitial, and vascular) with the pathology graded according to specified criteria as absent, mild, moderate, or severe. For comparative purposes, all of the tissue sections were scored by one observer (M.P.M.), who was blinded to their origin, in three sessions for the unmanipulated mice. A proportion of samples was reread a second time, with generally excellent concordance (<1 point average difference). In rare cases of disagreement, the sections were evaluated a third and final time and the average of the scores was used. Mortality curves were plotted using the Kaplan-Meier method and examined for significance using the Mantel-Cox logrank test. All other statistical tests used the nonparametric Mann-Whitney U statistic. The statistical analyses were conducted using StatView 4.5 (Abacus Software) for the Macintosh. P < 0.05 was considered significant. Tgic mice with the mIgM construct, which lacked the secreted exons, were created as described in Materials and Methods. A diagram of the Tg is shown in Fig. 1 . Mice bearing the mIgM Tg were backcrossed at least seven times to the B cell–deficient J H D strain, placing the Tgs onto the autoimmune MRL/ lpr background. This also established homozygosity of the J H D mutation, which prevented the development of B cells expressing endogenous Ig. As shown in Fig. 2 , the mIgM Tg restored B cell maturation. The percentages of B cells in the Tg mice were comparable with those in wild-type animals (average spleen percentage: B cell–intact − 15 ± 7%, mIgM – 16 ± 8). J H D mice at 4–6 mo of age have reduced lymphoid organ weights and cell numbers compared with B cell–intact animals ( 13 , 16 ). Table I lists the cell numbers and organ weights of the mIgM Tgic animals along with age-matched B cell–intact and B cell–deficient MRL/ lpr mice. Splenic weight and cell number were greater in mIgM mice than in B cell–deficient animals (weight: 2.5-fold increase, P < 0.02; cell number: 2.2-fold increase, P < 0.002). Since B cells generally comprise only 15–20% of splenocytes in older MRL/ lpr mice, this 2.2-fold difference in cell number cannot be accounted for simply by B cells per se. Most of the increased cell number is attributable, instead, to T cells, consistent with our previous reports on the effect of B cells on T cell activation and expansion. Although significantly greater than J H D/ lpr mice, splenomegaly in the mIgM Tgics was decreased when compared with control MRL/ lpr mice. Similarly, in the lymph nodes restoration of B cells in the Tgic mice had no effect on weight and cell number. The phenotype of partial restoration of T cell accumulation mediated by the Tgic mice most likely reflects the restricted repertoire enforced by a single V H in the Tg. A partial or reduced disease phenotype has been found in several other conventional Ig or TCR Tgic strains associated with the partial repertoire restriction imposed by allelic exclusion by the Tgs ( 24 , 25 ). The majority of T cells in MRL/ lpr mice have an activated/memory phenotype ( 16 , 26 ). However, in B cell–deficient MRL/ lpr animals, the percentage of naive cells is increased, whereas the percentage of memory T cells is decreased ( 16 ). Furthermore, B cells are required for the accumulation of most memory T cells since there are 5–10 times as many memory T cells in B cell–intact mice compared with B cell–deficient mice. In principle, B cells could be exerting this effect on memory T cell development either directly (e.g., via presentation of [auto]antigens) or indirectly (e.g., via autoantibody-mediated inflammation causing the release of autoantigens). To distinguish between these possibilities and to determine the mechanism by which B cells promote spontaneous T cell activation, we analyzed CD4 + T cells from the spleens of mIgM mice for expression of CD44 and CD62L . The T cell activation profiles of mIgM animals resembled B cell–intact mice rather than B cell–deficient mice. When compared with J H D animals, the percentages of memory cells (CD44 high , CD62L low ) were significantly greater in mIgM mice . Furthermore, percentages of naive cells (CD44 low , CD62L high ) were reduced by 90% ( P < 0.002). The number of memory CD4 + T cells in the mIgM mice was greater (5.5-fold increase, P < 0.003) than that of B cell–deficient animals and was similar to the B cell–intact, control MRL/ lpr mice . As in the spleen, the percentage of naive cells in the lymph nodes was decreased and the percentage of memory cells was increased in mIgM Tgic mice compared with B cell–deficient animals (data not shown). However, there was no statistical difference in memory CD4 + cell number since lymph nodes were not generally enlarged in the mIgM mice. For CD8 + T cells, a similar pattern of naive cell reduction and memory cell augmentation was observed (data not shown). Since the accumulation of memory and activated T cells proceeded efficiently in the absence of secreted antibody (see below) in the mIgM mice, we conclude that B cells directly promote T cell activation and accumulation, rather than indirectly via antibody-mediated tissue damage. MRL/ lpr mice characteristically develop spontaneous nephritis , whereas B cell–deficient J H D mice do not ( 13 ). mIgM mice, on the other hand, have significant cellular infiltrates in the renal interstitium and around the vessels, despite the inhibition of secreted Ig . These infiltrates were predominantly composed of T cells (data not shown). Renal disease in the glomeruli, interstitium, and vessels was scored blindly to assess severity . IN and vasculitis scores of the mIgM mice were greater than those of the J H D animals (interstitium, P < 0.03; vessels, P < 0.03) and comparable to those of the MRL/ lpr mice. There were no statistically significant differences in glomerular scores between the J H D and mIgM strains, both of which were lower than MRL/ lpr mice ( P < 0.02). Nevertheless, focal lesions that resembled glomerular atrophy were observed in some glomeruli of the mIgM mice , whereas these lesions were never observed in the J H D strain . This picture is different than the typical proliferative GN picture seen in mild to moderate MRL/ lpr disease and may reflect a previously unappreciated antibody-independent mode of glomerular disease perhaps related to adjacent interstitial or vascular disease. In our colony, 50% mortality for B cell–intact MRL/ lpr mice occurred at 32 wk ( n = 203) . The J H D strain, with markedly reduced nephritis, vasculitis, and T cell activation, had a significantly greater life span . The mortality of mIgM mice (50% at 56 weeks, n = 71) was accelerated when compared with the J H D mice , demonstrating that the restoration of B cells in the absence of circulating, soluble autoantibody has a direct and relevant effect on disease expression. However, in keeping with the fact that restoration of B cells with a restricted repertoire leads to milder nephritis and somewhat less T cell activation than in wild-type MRL/ lpr animals, mIgM mice also had prolonged survival compared with the control MRL/ lpr strain . To confirm the absence of secreted antibody in the mIgM strain, serum Ig and autoantibody levels were measured . Some animals had no detectable Ig as expected . However, others had trace quantities of some Ig isotypes; these concentrations were 100– 1,000 times lower than MRL/ lpr controls. Notably, there were mIgM animals that did not have any detectable serum Ig, yet developed renal disease. Finally, there was no significant total anti-dsDNA, RF, or antichromatin detected in the serum for most of the mIgM mice . Using immunofluorescence, we examined the kidneys of two mIgM mice that had detectable circulating Ig. In both, there were no observable Ig deposits in the glomeruli, interstitium, or vessels, consistent with the low to absent levels of serum Ig and autoantibodies. These mIgM kidneys were indistinguishable in this regard from kidneys of the B cell–deficient, negative controls, whereas MRL/ lpr mice showed intense staining, as expected . SLE is a complex disease that may have multiple pathogenic manifestations. In addition to the classic GN, vasculitis, IN, arthritis, and skin disease are often seen. The key finding of this work is that in the absence of circulating Ig and renal antibody deposition, the mIgM strain developed IN, vasculitis, and focal glomerular atrophy. Thus, these studies demonstrate for the first time an antibody-independent mechanism for renal and vascular disease in a murine model of SLE. Disease promoted by B cells in the absence of Ab is biologically relevant in that IN, vasculitis, and, importantly, mortality were all enhanced by B cells alone. To the extent that these results are generalizable to other murine models and to human SLE, the data suggest a wider view of lupus pathogenesis, which is generally thought to be solely antibody mediated ( 1 ). Renal cellular infiltrates in MRL/ lpr mice, which are not observed in the J H D strain, contain significant numbers of T cells (Chan, O.T.M., and M.J. Shlomchik, unpublished observation, and references 6 , 27 ). These cells are restored in mIgM mice . It is important to emphasize that interstitial disease and vasculitis are prominent features in diseased kidneys of many SLE patients ( 7 , 9 , 12 ). Indeed, interstitial injury may correlate best with overall outcome. Moreover, cellular infiltration is a prominent feature in other SLE manifestations, such as some skin lesions and sialoadenitis ( 10 , 28 ). It is worth noting that cellular infiltration in the MRL/ lpr model is not due to the Fas lpr mutation since Fas-intact MRL/MpJ-+ Fas-lpr /+ Fas-lpr mice also develop cellular infiltrates (references 3 and 5 , and Chan, O.T.M., unpublished data). Although overall statistical analysis of blind readings in a system that emphasized generalized disease did not show a difference in glomerular disease, this masks the fact that focal glomerular lesions were seen in mIgM mice . Focal glomerular lesions have never been noted in J H D mice ( 13 ). The nature of these lesions has not yet been defined; they could be due to adjacent vascular damage, periglomerular infiltrates, and/or local release of toxic cytokines ( 8 , 29 ). However, it seems that in the absence of marked glomerular disease, an antibody-mediated component is missing from the mIgM mice, as might have been predicted. The relationship of IN and GN has been controversial and the subject of speculation in the literature ( 30 ); but, until now, linkage could not be addressed in any experimental way in either humans or murine models. The present data show that IN and vasculitis can proceed without severe GN, but that some elements of GN might be exacerbated or even caused by surrounding cellular infiltrates as suggested by focal glomerular necrosis observed. In fact, several recent studies of experimental GN models that were thought to be solely antibody mediated have now demonstrated an important role for T cells ( 31 – 34 ). From all these studies, one could propose that there is synergy and cooperation among B and T cells to mediate a variety of pathogenic outcomes and that T cells do play a direct role in mediating disease that was previously thought to be only antibody dependent. The mIgM Tgic mice accumulated memory T cells in their spleens at levels similar to that of wild-type animals, in contrast to B cell–deficient mice. Thus, T cell activation is antibody-independent, but exactly how B cells promote T cell activation and cellular infiltration into tissues such as kidney and vessels is not completely clear. However, it is likely that B cells primarily act as autoantigen-presenting cells for the amplification of autoreactive T cells. In this scenario, autoreactive T cells may initially be activated by other APCs, such as dendritic cells; however, as B cell autoimmunity progresses and expanded clones of self-reactive B cells accumulate ( 35 , 36 ), these cells become increasingly important APCs for T cells ( 37 – 41 ). B cells are known to be extraordinarily efficient APCs for antigens that can bind to their surface Ig ( 42 – 44 ). Indeed, B cells that accumulate in MRL/ lpr mice chiefly have specificity for self-IgG (RFs) or DNA/histone ( 36 ). Such B cells bind particles (either immune complexes or nucleosomes) that are likely to contain multiple proteins, which could stimulate self-reactive T cells. Thus, these B cells are well suited to obtain T cell help. As certain autoreactive B cells expand and dominate the B cell repertoire, their relative importance in promoting disease via T cell activation (possibly including breaking peripheral T cell tolerance) ( 45 , 46 ) and autoantibody secretion would escalate. This might represent an element of a positive feedback circuit that leads to fulminant disease. Mamula, Janeway, and colleagues first suggested that B cells might be important autoantigen-presenting cells and might promote the breakdown of peripheral T cell tolerance ( 45 – 47 ). Using a cross-immunization scheme in normal mice, they were able to elicit autoreactive T cells in a fashion that was likely to be B cell dependent. They also suggested that this might be a mechanism for epitope spreading, a common phenomenon in both lupus and organ-specific autoimmunity. These studies were conducted in normal mice via immunization with heterologous proteins in Freund's adjuvant. Our work now lends support to the idea that these mechanisms are actually operating in spontaneously autoimmune animals. Although we favor a role for B cells as APCs, we cannot formally rule out that B cells are also acting as producers of cytokines that promote T cell activation or pathology. B cells are known to produce cytokines under certain circumstances ( 48 – 51 ). However, since B cells are not considered major producers of cytokines, it is difficult to envision B cells as supplying rate-limiting quantities of cytokines, particularly in a scenario in which there is massive T cell activation. In addition, immunohistochemical analyses revealed few B cells in the proximity of the renal infiltrates of MRL/ lpr mice (data not shown), making it very unlikely that B cells are the source of cytokines responsible for direct parenchymal injury. Although IN and vasculitis were marked in mIgM mice compared with their B cell–deficient MRL/ lpr counterparts, the median renal disease scores of the mIgM mice were not as high as those of the control MRL/ lpr mice. Similarly, mIgM mice had substantially accelerated mortality compared with B cell–deficient MRL/ lpr mice; but these Tgic mice had delayed mortality when compared with the wild-type. The lack of circulating autoantibody could certainly contribute to milder disease. Particularly for IN and vasculitis, it is likely that milder disease in mIgM mice is also due to a reduced Ig repertoire resulting from the fixed heavy chain Tg. It is possible that this restriction limited the generation of autoreactive B cells in the Tgic animals, thereby inhibiting the full potential of the B cell compartment to recognize self-antigens, activate them, and present these autoantigens to T cells. We predict that disease would have been more severe had a full Ig repertoire been expressed, but thus far the technology to produce such a mouse is not readily at hand. The importance in SLE of cell-mediated autoimmune pathogenesis revealed by our studies is reminiscent of present views of organ-specific autoimmune diseases such as diabetes. It is intriguing that B cell deletion also inhibited diabetes in the nonobese diabetic (NOD) mouse ( 52 – 54 ). This result was interpreted as due to lack of APC function, a point that was not actually proven but could potentially be demonstrated directly using our Tgs on the NOD background. In any case, the similarities suggest that organ-specific and systemic autoimmunity may not need to be distinguished as much on the basis of pathogenic mechanism, but rather in extent of autoimmune targeting. Our studies further support the rationale for directly targeting B cells in the therapy of SLE ( 13 , 16 ) and, by extension of this logic, to other autoimmune diseases. | Study | biomedical | en | 0.999996 |
10330444 | Antibodies used included rabbit polyclonal serum directed against the cytoplasmic domain of Syt I (a gift from Dr. T.C. Sudhof, Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, TX), mAbs directed against the NH 2 -terminal region of Syt II (a gift from Dr. M. Takahashi, Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan), and polyclonal antibodies against cathepsin D ( Calbiochem ). Bone marrow-derived mast cells (BMMCs) were obtained as previously described ( 28 ). In brief, femoral bone marrow cells from 6-wk-old BALB/c mice were cultured in 50% WEHI-3 cells conditioned medium. Culture medium was changed weekly, and nonadhering cells were used for further growth. After 3 wk, at least 99% of the cells were identified as mast cells by toluidine blue staining. Rat peritoneal mast cells (RPMCs) were obtained from Wistar rats by peritoneal lavage, and purified as previously described ( 29 ). In brief, a suspension of washed peritoneal cells was layered over a cushion of 30% Ficoll 400 ( Pharmacia Biotech Inc. ) in buffered saline and 0.1% BSA and centrifuged at 150 g for 15 min. The purity of mast cells recovered from the bottom of the tube was >90%, as assessed by toluidine blue staining. RBL-2H3 cells (hereafter termed RBL cells) were maintained in adherent cultures in DMEM supplemented with 10% FCS in a humidified atmosphere of 6% CO 2 at 37°C. RNA was isolated from trypsinized RBL cells collected by centrifugation at 400 g for 5 min, and from brains that were rapidly excised from 150–200-g Sprague-Dawley rats killed by CO 2 suffocation and then exsanguinated. Total RNA was isolated on a guanidine thiocyanate/CsCl gradient, extracted twice with phenol/chloroform, and then ethanol precipitated. The RNA was dissolved in 0.1% diethyl pyrocarbonate–treated water, quantified by measuring absorbance at 260 nm, evaluated for degradation by agarose-formaldehyde gel electrophoresis, and frozen until used. The mRNA was isolated from total RNA by oligo-dT cellulose chromatography [Poly(A)Pure; Ambion], and 2 μg was reverse transcribed by 125 U of Moloney's murine leukemia virus–reverse transcriptase (New England BioLabs) in a 50 μl reaction containing 2.5 μg each of (dT) 18 and random octamers, 1 mM of each dNTP, and 40 U of RNAsin ( Promega ) at 37°C for 30 min, 42°C for 30 min, and 50°C for 15 min. The first round of nested PCR was performed with 1 μl of AmpliTaq ( Perkin-Elmer Cetus) in 100 μl reaction buffer supplemented with 1.5 mM MgCl 2 , 10% (vol/vol) DMSO, 1 μM of each primer, 50 μM of each dNTP, and 1 μl of the reverse transcription reaction as template. The four primers correspond to RNA sequences encoding portions of Syt proteins schematized in Fig. 1 A, and their sequences were: A, TCWGACCCYTAYGTCAARRTCT; B, AGACCCARGTGCACMGGAAGAC; C, SYCYTTSACRTAGGGRTCTGA; D, GGGGTTSAGSGTGTTCTTCTT. For the first round of PCR, six cycles of 94°C for 1 min ramping to 49°C in 3 min, 49°C for 1 min, 72°C for 1 min were followed by 24 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min, with a final extension at 72°C for 6 min. 1 μl of the PCR product obtained with RBL cell cDNA and the A and D primers was used for template in a second round of PCR identical to the first except that the initial six cycles with low annealing temperature were not included. The product of the second reaction using the B and C primers was purified by agarose gel electrophoresis, then ligated into the pCR-II vector (Invitrogen). DH5α cells were transformed with the ligation mixture and colonies were selected for sequencing. Vectors containing the PCR-cloned Syt fragments were linearized with NotI for SP6-directed synthesis of riboprobes or with BamHI for T7-directed cRNA synthesis. cRNA hybridization controls were generated in a 50 μl reaction containing 3 μg of template DNA, 1.6 U/μl RNAsin, 10 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 1 mM of each NTP, 2.5 μM [ 3 H]UTP (45 Ci/mmol; Amersham ), and 100 U T7 RNA polymerase ( New England Biolabs ) in the manufacturer's buffer. Reactions were incubated for 4 h at 37°C, 10 U DNAse I was added, and the incubation was continued for another 20 min, and then cRNA was purified on a Nick-Spin column (Ambion). Riboprobes were transcribed in a 20 μl reaction using 1 μg of template DNA, 25 μM α-[ 32 P]CTP (800 Ci/mmol; Amersham ), 2 U/μl RNasin, 10 mM DTT, 0.1 mg/ml BSA, 0.5 mM each of other NTPs, and 10 U SP6 RNA polymerase ( New England Biolabs ) in the manufacturer's buffer for 1 h at 40°C. After treatment with 5 U of DNAse I, full-length transcripts were isolated by gel purification in 5% acrylamide/8 M urea gels. RNase protection assays were performed using the RPA II kit (Ambion). Cognate cRNA (0.1 pmol) and yeast RNA were used as positive and negative controls. Each experiment contained 1 pmol riboprobe and varying amounts of RBL cell RNA supplemented with yeast RNA to complete a total of 40 μg RNA. Hybridization was carried out overnight at 45°C. Protected probes were electrophoresed through 5% acrylamide/8 M urea gels and visualized by autoradiography. Mast cells (10 6 ) derived from different sources (RPMCs, BMMCs, and RBL-2H3) were washed in PBS and resuspended in 30 μl of lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM NaVO 4 , 1 mM PMSF, and 10 μg/ml leupeptin) and centrifuged at 12,000 g for 15 min at 4°C. The cleared supernatants were mixed with 5× Laemmli sample buffer to a final concentration of 1×, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting. For the preparation of brain homogenate, whole brain from a Wistar rat was homogenized in PBS at 4°C using a Polytron (Kinematica, GmbH, Switzerland; 20 s, setting 7). Aliquots (5–10 μg protein) were mixed with 5× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting. RBL cells (7 × 10 7 ) were washed with PBS and suspended in homogenization buffer (0.25 M sucrose, 1 mM MgCl 2 , 800 U/ml DNase I [ Sigma Chemical Co. ], 10 mM Hepes, pH 7.4, 1 mM PMSF, and a cocktail of protease inhibitors [ Boehringer Mannheim , Germany]). Cells were then disrupted by 3 cycles of freezing and thawing followed by 20 passages through a 21-gauge needle. Unbroken cells and nuclei were removed by sequential filtering through 5- and 2-μm filters (Poretics Co.). The final filtrate was then centrifuged for 10 min at 500 g and the supernatant loaded onto a continuous, 0.45–2.0 M sucrose gradient (10 ml), which was layered over a 0.3 ml cushion of 70% (wt/wt) sucrose and centrifuged for 18 h at 100,000 g . Histamine was assayed fluorimetrically after condensation in alkaline medium with o -phthalaldehyde ( 30 ). LDH activity was assayed using LDH reagent according to the manufacturer's instructions (Merck Diagnostica, Germany). RBL-2H3 cells were seeded in 24-well plates at 2 × 10 5 cells per well and incubated overnight in a humidified incubator at 37°C. The cells were then washed three times in Tyrode's buffer (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, and 0.1% BSA) and stimulated in the same buffer with the indicated concentrations of the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA; Calbiochem ). Secretion was allowed to proceed for 30 min at 37°C. Aliquots from the supernatants were taken for measurements of released β-hexosaminidase activity. Cells in control wells were lysed by addition of 0.1% Triton X-100 to determine the total enzyme content. For FcεRI induced secretion, cells were passively sensitized by overnight incubation with DNP specific monoclonal IgE (SPE7, a gift of Dr. Z. Eshhar, the Weizmann Institute of Science, Rehovot, Israel), washed three times in Tyrode's buffer, and then stimulated with the indicated concentrations of the antigen, DNP-BSA. Activity of the released β-hexosaminidase was determined by incubating aliquots (20 μl) of supernatants and cell lysates for 90 min at 37°C with 50 μl of the substrate solution consisting of 1.3 mg/ml p-nitrophenyl- N -acetyl-β- d -glucosaminide ( Sigma Chemical Co. ) in 0.1 M citrate pH 4.5. The reaction was stopped by the addition of 150 μl of 0.2 M glycine, pH 10.7. OD was read at 405 nm, in an ELISA reader. Results were expressed as percentage of total β-hexosaminidase activity present in the cells. To assay the release of cathepsin D, supernatants of cells, stimulated as above, were concentrated in VivaSpin concentrators with a 10 kD cut-off (VivaScience, UK). The concentrates were mixed with 5× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE and immunoblotting with anti–cathepsin D antibodies. For measurement of serotonin release, cells were incubated overnight with 2 μCi of [ 3 H]5-hydroxytryptamine (NEN), washed, and stimulated as above. Aliquots from the supernatants were taken for measurement of radioactivity. Samples (normalized according to protein content or number of cells) were separated by SDS-PAGE using 10 or 12% polyacrylamide gels. They were then electrophoretically transferred to nitrocellulose filters. Blots were blocked for 3 h in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) containing 5% skim milk followed by overnight incubation at 4°C with the indicated primary antibodies. Blots were washed three times and incubated for 1 h at room temperature with the secondary antibody (horseradish peroxidase–conjugated goat anti–rabbit or anti–mouse IgG; Jackson Research Labs.). Immunoreactive bands were visualized by the enhanced chemiluminescence method according to standard procedures. A full-length rat Syt II cDNA (provided by Dr. T.C. Sudhof) was subcloned into the EcoRI site of the pcDNA3 expression vector (Invitrogen) both in the sense and antisense orientations. RBL-2H3 cells (8 × 10 6 ) were transfected with 20 μg DNA of pcDNA3-Syt II or pcDNA3 alone, by electroporation (0.25 V, 960 μF). Cells were immediately replated in tissue culture dishes containing growth medium (supplemented DMEM). G418 (1 mg/ml) was added 24 h after transfection and stable transfectants were selected within 14 d. Primers were chosen from the conserved C2 domains that averaged 91% identity with known Syt isoforms. An initial round of PCR with four different primer pairs did not yield any visible product in reactions containing RBL cell cDNA, even though abundant product was obtained from brain cDNA using two different primer pairs . When the PCR product of RBL cell cDNA with primers A and D was then used as template in a second round of PCR, the nested reaction with primers B and C yielded abundant product of the predicted size of 365 bp . Sequencing the inserts of 21 colonies of subcloned PCR product yielded 10 colonies encoding a fragment of Syt II, 9 colonies encoding Syt III, and 2 colonies encoding Syt V. These findings were supported by the results of restriction digestions of the PCR product with multiple frequent cutting enzymes (data not shown) that were consistent with the presence of these three isoforms and did not indicate the presence of additional isoforms based on the known sequences of Syt isoforms. RNase protection assays were then performed to quantitatively assess the expression of Syt isoforms in RBL cells. Syt II was the most abundant , and serial dilution of mRNA used to protect the Syt II probe indicated that this isoform was approximately fivefold more concentrated in RBL cells than in Syt III. The Syt V isoform was not protected even when mRNA was present at a level at least 10-fold higher than that which measurably protected the Syt II probe , suggesting that Syt V mRNA is present in RBL cells at a concentration less than one-tenth the level of Syt II. Because Syt II, which shares the highest homology with the predominant neural isoform Syt I ( 31 ), was the most abundant isoform, we chose to focus this study on Syt II. We next examined the expression of the Syt II protein using specific antibodies (mAb 8G2B, directed against the NH 2 terminus of Syt II). A single immunoreactive protein was detected in RBL cells . Immunoreactivity in RBL cells (M r ∼80 kD) had less mobility on SDS-PAGE than immunoreactivity in the brain . Nevertheless, an 80-kD Syt II–immunoreactive protein was also detected in lysates from fully differentiated, connective tissue–type, RPMCs and primary murine BMMCs . These size differences in Syt II may thus arise from tissue-specific posttranslational modifications. To study the functional role of Syt II, we stably transfected RBL cells with neural Syt II cDNA and selected clones with increased levels (approximately twofold) of Syt II expression for further studies. Notably, transfection with neural Syt II cDNA resulted in overexpression of the same 80-kD Syt II–immunoreactive protein, strengthening the concept that the increased apparent M r of RBL-Syt II was due to tissue-specific posttranslational modifications. Overexpression of Syt II had no effect on the spontaneous release of the SG-associated enzyme, β-hexosaminidase ( 32 ). In the absence of any stimulus, both control cells (empty vector-transfected) and cells overexpressing Syt II released up to 5% of their total β-hexosaminidase . However, in contrast to transfection with Syt I ( 27 ), overexpression of Syt II failed to potentiate Ca 2+ -dependent exocytosis evoked by a Ca 2+ ionophore alone , or in the presence of phorbol ester . Instead, a mild inhibition could be observed when the cells were triggered with low (<10 μM) concentrations of the Ca 2+ ionophore. The fact that Syt II, unlike Syt I, is endogenously expressed in RBL cells enabled us to extend these results by investigating the effect of reducing the level of Syt II expression on exocytosis. To this end, cells were stably transfected with Syt II cDNA subcloned in the antisense orientation, resulting in substantially reduced levels of Syt II expression (15, 6, 47, and 6% of control levels) . Clones expressing the lowest levels (6–15%) were chosen for further analyses. In these cells (Syt II − ), the basal, spontaneous release of β-hexosaminidase was not affected, revealing that Syt II was not acting as a limiting fusion clamp. However, β-hexosaminidase release triggered by a Ca 2+ ionophore alone or with phorbol ester was markedly (by up to fivefold) potentiated. Physiologically, exocytosis in mast cells can be triggered by antigen- induced aggregation of the high-affinity receptors (FcεRI) for IgE ( 33 ). To investigate the involvement of Syt II in controlling FcεRI-mediated exocytosis, antigen-induced secretion was studied in the Syt II − and Syt II + cells. Secretion of β-hexosaminidase was unaffected in the Syt II + cells but was significantly elevated (by fourfold) in the Syt II − cells . Taken together, these results suggest that Syt II negatively regulates release of β-hexosaminidase, whether triggered by Ca 2+ ionophore or by an immunological trigger. To understand the opposite effects exerted by the transfected Syt I and Syt II proteins, we investigated their distribution in RBL cells using a continuous sucrose gradient. All of the Syt II immunoreactivity present in either the control or the Syt II– transfected cells comigrated with 60% of the β-hexosaminidase activity, present in fractions 6–13 at ∼0.75 M sucrose . These fractions did not include the histamine-containing SGs, which migrated at fractions 16–23 at 1.3 M sucrose and included the remaining β-hexosaminidase activity . Histamine was also found at the top of the gradient , but this probably reflected the contents of SGs that were released during cell disruption. Therefore, these results indicate that, unlike transfected Syt I , the endogenous and transfected Syt II proteins were not targeted to the histamine-containing SGs, but to a different intracellular compartment. The presence of β-hexosaminidase in fractions 6–13 of the sucrose gradient suggested the presence of a lysosomal organelle distinct from the histamine-containing SGs. Indeed, fractions 6–13 also contained procathepsin D, M r 53 kD, the precursor of the lysosomal protease cathepsin D . Lysosomes were recently shown to behave as Ca 2+ -regulated exocytic vesicles ( 34 ). Since β-hexosaminidase is distributed between histamine-containing SGs and procathepsin D–containing lysosomes in RBL cells, it was important to determine whether secretion of the content of the latter compartment is negatively regulated by Syt II. To address this question, we examined whether Syt II could modulate release of the lysosomally processed form of cathepsin D (mature cathepsin D, M r ∼43 kD) ( 35 ). Concentrating the cell supernatants by 20-fold allowed the detection of cathepsin D in supernatants from Ca 2+ ionophore- or antigen-triggered cells . The amount of secreted mature cathepsin D was significantly inhibited or increased in the Syt II + or Syt II − cells, respectively . Ca 2+ ionophore was more effective than the immunological stimulus in the Syt II + cells, but did not differ significantly in Syt II − cells. The precursor form of cathepsin D (53 kD) was detected in supernatants of both triggered and nontriggered cells (data not shown), reflecting the constitutive release of unprocessed cathepsin D ( 34 ). These results demonstrate that mast cell activation triggers exocytosis of a lysosomal fraction distinct from histamine-containing SGs, and that mobilization of this compartment depends substantially on the expression level of Syt II. We have also evaluated the effects of Syt II on the triggered release of serotonin, to exclusively monitor exocytosis of SGs ( 36 ). Overexpression of Syt II had no significant effect on serotonin release triggered by either secretagogue . However, reducing its level of expression in the Syt II − cells had a small but significant stimulatory effect . Previous studies have already alluded to the possibility that Syt isoforms may serve the role of general Ca 2+ sensors, controlling regulated exocytosis also in nonneuronal secretory cells ( 23 , 25 , 26 ). We and others have previously shown that mast cells express Syt and SNAREs that probably function to control mediators released from these cells ( 27 , 37 ). Here, we demonstrate that RBL cells endogenously express at least three distinct isoforms of Syt, including Syt II, III, and V. Syt II was identified both by RNAase protection assays and at the protein level, on the basis of its immunoreactivity with specific antibodies . However, in contrast to its location on SVs or SGs in neurons or endocrine cells, in the RBL cells, Syt II cofractionates with the lysosomal fraction rather than with the histamine-containing SGs . Furthermore, transfection of the RBL cells with neural Syt II cDNA resulted in overexpression of Syt II and its targeting to the same fraction . Mast cells belong to immune cells of the hemopoietic lineage, where an intimate connection exists between lysosomes and SGs ( 38 ). The SGs of mast cells include, in addition to their secretory cargo of vasoactive amines (e.g., histamine and serotonin), lysosomal enzymes such as β-hexosaminidase, β-glucuronidase, arylsulfatase, and carboxypeptidases ( 32 ), as well as lysosomal integral membrane proteins (LIMPs) ( 39 ). Therefore, mast cell SGs can be defined as secretory lysosomes. Nevertheless, in consistence with previous data ( 40 , 41 ), our data indicate that in addition to the lysosomal, amine-containing SGs, mast cells also contain lysosomes, which lack biogenic amines and with which Syt II is associated . Such amine-free lysosomes were previously reported to resist cell triggering by immunologic or Ca 2+ ionophore stimulation ( 40 , 41 ). Whether the two populations of granules are sequentially formed and by what mechanism selective retention of the nonsecretory lysosomes is achieved, remained unknown. We now demonstrate that mast cells can, to some extent, release also their lysosomal pool of hydrolases, upon both an immunologic and a Ca 2+ ionophore trigger. In this process both lysosomal enzymes, which are distributed between both SGs and lysosomes, such as β-hexosaminidase, as well as hydrolases localized exclusively to the amine-free lysosomal fraction, such as cathepsin D , are released. However, this release is inhibited by overexpression of Syt II and markedly potentiated by reducing the level of Syt II expression . Recently, three types of granules were ultrastructurally distinguished in IFN-γ–treated mast cells (Table I ). Type I and type II granules were both labeled by a fluid phase endocytic marker and both contained MHC class II as well as lysosomal markers ( 42 ). These results have therefore suggested their position in the endocytic pathway, similarly to lysosomal compartments ( 42 ). Serotonin was localized to type II and type III granules, of which the latter type did not internalize the fluid phase endocytic marker, nor did it contain MHC class II ( 42 ). Based on these results, it was suggested that a fusion event between type I (amine-free lysosomes) and III (e.g., SGs) granules may account for the formation of type II granules ( 42 ). Our results are compatible with this model and define Syt II as the molecular entity, which may control this fusion event and effect selective retention of the nonsecretory lysosomes during cell activation . Furthermore, this model predicts that downregulation of Syt II should also indirectly affect SG exocytosis by facilitating the fusion event between the amine-free lysosomes and SGs. Indeed, we found that suppression of Syt II level of expression also moderately potentiates serotonin release . The molecular mechanism by which Syt II inhibits lysosome exocytosis is currently unknown. Syts fall into three distinct classes that for syntaxin binding require either high Ca 2+ concentrations (200 μM) (class A) or low Ca 2+ concentrations (<1 μM) (class B), or do not bind syntaxin in a Ca 2+ -dependent manner (class C) ( 20 ). Syt II, the major RBL isoform, and Syt V, the least abundant isoform, are class A proteins. However, although Ca 2+ concentrations measured in neurons during an action potential are high enough to support Ca 2+ -dependent interaction of class A Syts with syntaxins, the rise of intracellular Ca 2+ concentration in mast cells induced by FcεRI cross-linking rarely exceeds 1 μM and would not be predicted to support such interaction ( 43 ). Calcium-dependent Syts negatively regulate neuronal exocytosis at basal Ca 2+ concentrations ( 44 ), whereas positive effects on exocytosis are observed only at elevated Ca 2+ concentrations and are thought to depend on interaction with syntaxin ( 20 ). In the mast cells Syt II seems to increase the Ca 2+ requirements for lysosomal exocytosis, since Ca 2+ ionophore is far more effective than immunologic stimulation in triggering cathepsin D release from control cells, but both are equally potent in Syt II − cells . It is of great interest that Syt II appears to be used in mast cells as a negative regulator of Ca 2+ -dependent exocytosis and of a subclass of secretory vesicles, and is the first example to our knowledge. Syt II inhibitory function appears to be linked to its lysosomal association since Syt I, although highly homologous to Syt II, potentiated Ca 2+ -dependent exocytosis of SG when transfected into the RBL cells, alongside its SG targeting ( 27 ). The reasons for this differential targeting of Syt I and Syt II remain unknown. Although not proven here, the remaining Syt isoform expressed in RBL cells, Syt III, which is a class B protein, would be an adequate candidate to serve as the positive regulator of SG exocytosis, whose action is mimicked by transfected Syt I. In conclusion, our findings provide unequivocal evidence for an active role of Syt II in negatively controlling Ca 2+ -regulated lysosomal exocytosis. This observation extends the function of Syt II to regulation of exocytosis of secretory organelles exclusive to SVs or SGs. Specifically, in mast cells regulation by Syt II may have important implications on their function as APCs in host defense mechanisms, as this process requires uptake and lysosomal processing of antigens, followed by presentation of MHC class II–peptide complexes on the mast cell surface ( 10 , 45 ). Our model predicts that the cellular level of Syt II could be up- or downregulated to determine the balance of mast cell effector function between the secretion of inflammatory mediators from SG exocytosis and the presentation of antigen by externalization of MHC II–containing lysosomes. Syt II may thus play a central role in controlling the physiological functions of mast cells. | Study | biomedical | en | 0.999996 |
10330445 | The following chemicals and reagents were used and were purchased from the sources indicated: RPMI 1640, AIM-V medium, Lipofectamine, and G418 ( GIBCO BRL ); the eukaryotic expression vector pcDNA3.1 (Invitrogen); anti– HLA-DR1 mAb (One Lambda); and anti-IgM antibody conjugated with FITC (Vector Labs., Inc.). CD4 + TILs were cultured from a subcutaneous metastasis resected from patient 1363. T cell clones or lines were grown in AIM-V medium containing 10% human AB serum and rIL-2 (1,000 IU/ml; Chiron). Melanoma cell lines and EBV-transformed B cell lines were maintained in RPMI 1640 with 10% FCS. COS-7, 293, 293IMDR1, and 293IMDR4 cell lines were grown in the DMEM containing 10% FCS. The T cell clones or cloids were generated by limiting dilution methods (at 1 cell/well) from the CD4 + TIL1363 cell line. After 12 d, the T cell clones were expanded in AIM-V medium containing 6,000 IU/ml IL-2. To obtain an optimal expansion, we used the OKT3 expansion method as previously described ( 15 , 16 ). In brief, on day 0, 5 × 10 4–5 T cells were cocultured with HLA-DR1 + PBLs (PBL/T cell ratio, 500:1) and 586 EBV B cells (EBV/T cell ratio, 100:1) in 25 ml RPMI 1640 containing 11% human sera, 30 ng/ml OKT3 antibody, and antibiotics. On day 1, IL-2 was added at a final concentration of 180 IU/ml. The medium was changed with fresh medium containing 11% human sera and 180 IU/ml IL-2 on day 5. The medium was then changed every 3 d. On days 12–14, T cells were harvested, counted, and cryopreserved. Melanoma cell lines 397mel, 1088mel, 1359mel, 1363mel, and 1558mel and EBV-transformed B cell lines 586EBV, 1363EBV, 1359EBV, and 1558EBV were established in our laboratory and cultured in RPMI 1640 containing 10% FCS. Total RNA was extracted from 1363mel using Trizol reagent from GIBCO BRL . Poly(A) RNA was purified from total RNA by polyATract system ( Promega Corp. ) and converted to cDNA using a cDNA construction kit ( GIBCO BRL ) with an oligo-dT primer. To create an invariant chain (Ii) fusion library, an Ii fragment (amino acids 1–80) was inserted into a mammalian expression vector, pEAK8 (Edge BioSystem), to generate a pTi80 vector. The cDNA inserts were then ligated to pTi80, and cDNA libraries were electroporated into DH10B cells ( GIBCO BRL ). Plasmid DNAs for cDNA library pools were prepared from bacteria, each consisting of ∼100 cDNA clones. DNA transfection and GM-CSF assays were performed as previously described ( 15 , 16 ). In brief, 200 ng of cDNA pools was mixed with 2 μl of Lipofectamine in 100 μl of serum-free DMEM for 15–45 min. The DNA/Lipofectamine mixture was then added to the 293IMDR1 (5 × 10 4 ) cells and incubated overnight. The following day, cells were washed twice with AIM-V medium. CD4 + TIL1363 cells were added at a concentration of 5 × 10 4 cells/well in AIM-V medium containing 120 IU/ml of IL-2. After 18–24 h of incubation, 100 μl of supernatant was collected and GM-CSF concentration was measured in a standard ELISA assay (R&D Systems). For testing peptide recognition, 1363EBV cells were incubated with peptides at 37°C for 90 min, then washed three times with AIM-V medium containing 120 IU/ml of IL-2. T cells were added and incubated for an additional 18– 24 h. In some experiments, peptides were directly incubated with CD4 + TIL1363 for 90 min, then washed three times. 5 × 10 4 T cells were then mixed with the peptide-binding T cells and incubated for an additional 18–24 h. 100 μl of supernatant was collected for GM-CSF assay. Total RNA was isolated using Trizol reagent from GIBCO BRL . Total RNA from human normal tissue was purchased from Clontech . 20 μg of total RNA was subjected to electrophoresis in a 1.2% formaldehyde agarose gel and transferred to a nylon membrane. DNA fragments of the LDLR-FUT (low density lipid receptor–GDP- l -fucose:β- d -galactosidase 2-α- l -fucosyltransferase) fusion gene for probe A, the LDLR-specific probe B, and the FUT-specific probe C were labeled with [α- 32 P]CTP by the random priming method. Prehybridization and hybridization were performed according to the QuickHyb protocol (Stratagene). Membranes were washed twice with 2× SSC/0.1% SDS at room temperature for 15 min and twice with 0.1× SSC/0.1% SDS at 60°C for 30 min. The autoradiography was performed at −70°C. Genomic DNAs were isolated from tumor and EBV-B cell lines using a DNA Isolation Kit ( Boehringer Mannheim ), and digested with BamHI, HindIII, or EcoRI. The digested DNA fragments were separated from an 0.8% agarose gel, and transferred to nitrocellulose membrane. Membranes were prehybridized for 1 h, then hybridized with [α- 32 P]CTP– labeled probes using QuickHyb solution (Stratagene). After washing twice with 2× SSC/0.1% SDS at room temperature for 15 min and twice with 0.1× SSC/0.1% SDS at 65°C for 15 min, the membrane was exposed to an x-ray film at −70°C. Total RNA was extracted from tumor cell lines as described above. 5′ RACE (rapid amplification of cDNA ends) was performed according to the manufacturer's procedure ( GIBCO BRL ). PCR products were cloned into the TA cloning vector (Invitrogen). Recombinant DNA was prepared and used for DNA sequencing analysis using an automatic sequencer (Applied Biosystems, Inc.). The peptides were synthesized by a solid-phase method using a peptide synthesizer (model AMS 422; Gilson Co., Inc.). Some peptides were purified by HPLC and had >98% purity. The mass of some peptides was confirmed by mass spectrometry analysis. Identification and characterization of peptides reactive with CD4 + TIL1363 were conducted as previously described ( 15 , 16 ). CD4 + TIL1363 cells were generated from a melanoma metastasis of patient 1363 and were found to recognize the autologous tumor cell line 1363mel, but did not recognize autologous EBV-B cells , other EBV-B cells, MHC class II–matched or –mismatched tumor cell lines, nor 293-expressing DR molecules . Furthermore, CD4 + TIL1363 cells were capable of recognizing cell lysates of 1363mel pulsed onto DR-matched EBV B cells, but did not recognize cell lysates derived from other tumors, EBV-B cells, or fibroblasts (data not shown). These results suggested that TIL1363 recognized a unique tumor antigen from the autologous tumor. T cell recognition was specifically blocked by an mAb against HLA-DR, but not by mAbs against HLA-DQ or MHC class I . FACS ® analysis of 1363mel indicated that only HLA-DR1 was expressed in 1363mel , which was consistent with the result of HLA genotyping analysis. These results suggested that CD4 + TIL1363 recognized a tumor antigen presented by HLA-DR1. Although many MHC class I–restricted tumor antigens have been identified by the expression cloning approach, this conventional expression approach cannot be applied to isolating genes encoding MHC class II–restricted Ags, because of differences in the endogenous and exogenous Ag presentation pathways. It has been reported that Ii-fused antigens could be endogenously processed and presented to CD4 + T cells ( 17 – 20 ). Our strategy is to target Ii fusion proteins translated from the Ii fusion library to the endosomal/lysosomal compartment for efficient antigen processing and presentation. Although EBV-B and dendritic cells are professional APCs, they were poorly transfectable and cDNA libraries could not be efficiently introduced into these APCs. Thus, we generated 293IMDR1 cells by introducing cDNAs encoding DRα, DRβ, DMA, DMB, and Ii into 293 cells, a transformed human kidney embryonic cell line, and used them as “professional” APCs. This novel approach has been successfully used to isolate a mutated CDC27 as an MHC class II–restricted tumor antigen recognized by CD4 + T cells ( 20a ). To isolate the gene encoding a tumor antigen recognized by CD4 + TIL1363, we constructed a cDNA library with the fusion of a targeting sequence of Ii in the 5′ end of cDNAs derived from 1363mel. cDNA subpools with ∼100 cDNA clones per pool were prepared. The 1363 cDNA library was introduced into 293IMDR1 expressing DMA, DMB, Ii, and DR1 molecules. After screening a total of 3.5 × 10 5 cDNA clones, we identified >10 positive cDNA pools that conferred T cell recognition by CD4 + TIL1363 when transfected into 293IMDR1. The individual positive clones were then isolated from the positive cDNA pools and tested for recognition by CD4 + TIL1363. Representative data is shown in Fig. 2 . DNA sequencing analysis showed that cDNA clone 7 encoded a fusion protein consisting of 363 amino acids . A database search revealed that the first 880 nucleotides were identical to a published sequence of the LDL receptor ( 21 , 22 ). However, the DNA sequence in the 3′ end of cDNA clone 7 was found to be identical to the previously published sequence of FUT with the exception of a one-nucleotide (G) deletion at position 1049 ( 23 ). Furthermore, the LDLR sequence in the 5′ end of the cDNA was fused to the intron/exon 3 of the FUT sequence in an antisense orientation such that the translational reading frame of the LDLR fusion protein continued for an additional 131 amino acids until a stop codon was reached. Therefore, the LDLR-FUT fusion protein contained the first five ligand binding repeats of LDLR followed by a novel polypeptide of 131 amino acids at the COOH terminus. A database search did not reveal any sequence homologue to the 131 amino acid polypeptide. To confirm if T cell recognition was restricted by HLA-DR1, the cDNA clone 7 was transfected into both 293IMDR1 and 293IMDR4 and then tested for recognition by CD4 + TIL1363. To our surprise, CD4 + TIL1363 cells were capable of recognizing both HLA-DR1 and DR4 positive cell lines transfected with cDNA clone 7, but not with other control cDNA clones (data not shown). FACS ® analysis revealed that TIL1363 expressed a high level of HLA-DR1 molecules on the cell surface, suggesting that these T cells may function as APCs . To further explore how TIL1363 recognized an antigen expressed by 293IMDR4 cells lacking DR1 molecules, we tested the possibility that T cells may capture an antigen from cell supernatants of cells transfected with cDNA clone 7, and process and present antigenic peptides to each other. Results presented in Fig. 4 showed that CD4 + TIL1363 cells were capable of recognizing an antigen derived from the cell culture supernatants of COS-7, 293, and 293IMDR4 cells transfected with cDNA clone 7 and supernatants from 1363mel. Recognition of the supernatants by CD4 + TIL1363 cells were blocked by an anti-DR antibody, but not by a control antibody. Treatment of the cDNA clone 7–transfected COS-7, 293, and 293IMDR4 cells with the fixative agent PFA alone or PFA combined with the anti-DR antibody completely abrogated the ability of conferring T cell recognition , suggesting that blocking antigen secretion from cells transfected with cDNA clone 7 abolished antigen recognition by T cells. T cell recognition of 1363mel by CD4 + TIL1363 was partially inhibited by PFA. This may be due to the existence of different subsets of CD4 + T cells in the TIL1363 population that recognized additional antigens presented by 1363mel. These results indicated that COS-7, 293, and 293IMDR4 cells transfected with cDNA clone 7, as well as the untransfected 1363mel, secreted the LDLR-FUT fusion protein into the culture medium, which was in turn presented by MHC class II–positive T cells to themselves. To determine whether the fusion protein was a consequence of a chromosomal rearrangement, we did Southern blot analysis using the LDLR-FUT fusion cDNA as probe A . No difference was found in the DNA band pattern of 1363mel genomic DNA digested with either HindIII or EcoRI, compared with genomic DNA derived from other cell lines digested with the same enzymes. However, an additional band was observed in 1363mel genomic DNA digested with BamHI when compared with the DNA patterns of 293, SK23, 1359mel, 624mel, and 586mel . These results suggested that a DNA rearrangement occurred in 1363mel. To further evaluate if the fusion cDNA was expressed in 1363mel, Northern blot analyses were performed. Two unique bands were detected only in 1363mel by the fusion cDNA probe A . No corresponding bands were observed from any other tumor cell lines or normal tissues tested . Using specific DNA probes derived from either LDLR cDNA or FUT , we found that probe B, which was specific for LDLR, detected two identical bands observed in the 1363mel RNA sample using probe A, but not in the RNA samples of 1363EBV, 1359mel, or 293 . However, the DNA probe C, which was specific for FUT, only detected a single band, suggesting that the 5′ portion of two RNAs from 1363mel was the same and contained the LDLR fragment, but the 3′ portion was different, which might result from an alternative splicing. Both LDLR and FUT had been mapped on human chromosome 19, but LDLR was located on 19p and FUT on 19q ( 24 , 25 ). In Fig. 6 we propose a possible gene fusion resulting from a chromosomal rearrangement. The LDLR gene was fused to the FUT gene in an opposite direction to LDLR. The fusion junction was located between the exon 4 of LDLR and the intron/exon 3 of FUT. Two RNA species were generated, probably by an alternative splicing. To determine the antigenic epitopes recognized by CD4 + TIL1363, we first tested whether CD4 + TIL1363 recognized 293IMDR1 transfected with the wild-type LDLR cDNA. No T cell recognition was observed with the LDLR-transfected 293IMDR1, suggesting that T cell epitopes were located either in the new polypeptide translated from the sequence of FUT in an antisense direction or in the junction region of the fusion protein. 34 15-mer peptides overlapping by eight amino acids were synthesized based on the predicted amino acid sequence of the new polypeptide derived from the FUT sequence, and were tested for T cell recognition. Two of these peptides were found to be recognized by CD4 + TIL1363 . These two peptides shared eight amino acids (WRRAPAPG). Further truncations of the peptides from either the NH 2 or COOH terminus defined Trp (W) at amino acid position 315 and Ala (A) at amino acid position 320 as critical amino acids required for T cell recognition . All the peptides containing the core sequence WRRAPA were recognized by CD4 + TIL1363. One 9-mer peptide LRFP 312–320 (PVTWRRAPA), the shortest peptide used in this experiment, was found to be active in the stimulation of T cells . Further truncations of this 9-mer peptide LRFP 312–320 from the NH 2 terminus resulted in a loss of the ability to stimulate CD4 + TIL1363 for GM-CSF release (data not shown). We also tested T cell recognition of several truncated forms of the 15-mer peptide (WRRAPAPGAKA) from the COOH terminus, and found that the shortest active peptide was the 9-mer peptide LRFP 315–323 (WRRAPAPGA) . Therefore, these studies defined two 9-mer peptides, LRF 312–320 and LRFP 315–323 , recognized by CD4 + TIL1363. These two peptides shared a core sequence WRRAPA, and Trp (W) at amino acid position 315 may serve as the P1 anchor residue for MHC binding, conforming to the HLA-DR1 binding motif ( 26 ). The minimal peptide length is 9-mer, and peptides shorter than 9-mer failed to activate CD4 + TIL1363 for GM-CSF release even though they contained the core sequence . Peptide titration experiments showed that both LRFP 312–320 and LRFP 315–323 peptides exhibited a very similar peptide binding avidity to DR1 and could be detected by T cells at 100 ng/ml peptide concentration . In this study we identified a novel tumor antigen recognized by CD4 + TIL1363 using a novel genetic strategy. Although a biochemical approach has been used to identify MHC class II–restricted tumor antigens, it is limited to the CD4 + T cells that are capable of recognizing tumor lysates pulsed onto APCs such as EBV B cells. Several CD4 + T cell lines established in our laboratory recognized whole tumor cells, but did not recognize cell lysates of autologous tumor due to either low amounts of a particular antigen or low efficient uptake by APCs (data not shown). We recently cloned a mutated CDC27 as a tumor antigen recognized by CD4 + TIL1359 using this novel cloning strategy ( 20a ). Here, we demonstrate that a secreted protein encoded by a LDLR-FUT fusion cDNA is a tumor antigen recognized by CD4 + TIL1363. Despite the fact that a special cell line expressing MHC class II was not required in this case, this genetic approach can be useful for the isolation of genes encoding MHC class II–restricted tumor antigens recognized by CD4 + T cells. The LDLR-FUT fusion gene was generated by a chromosomal rearrangement as evidenced by our Southern and Northern blot analyses . Human LDL receptor is a cell surface glycoprotein that regulates plasma cholesterol levels. The ligand-binding domain of LDLR comprises seven imperfect repeats in the extracellular region of the protein. Each domain consists of ∼40 amino acids. LDL receptors specifically bind LDL, the receptor–lipoprotein complex is internalized by the cells via coated pits and vesicles, and the entire LDL particle is delivered to lysosomes, where it is disassembled by enzymatic hydrolysis, releasing cholesterol for further cellular metabolism ( 24 ). More than 250 LDLR mutations including deletions, insertions, and single point mutations have been reported to affect LDLR function associated with familial hypercholesterolemia ( 24 ). The FUT gene product regulates the expression of the H antigen mainly on erythrocyte membranes. Koda et al. reported that the FUT gene was expressed in gastric cancer and ovarian cancer cells ( 23 ). Although the fusion protein may still have the ability to bind LDL, we do not know the biological significance of the fusion protein resulting from a chromosomal rearrangement in 1363mel at this time. There were several unique features associated with the fusion antigen. (a) To our knowledge, this is the first report that a secretory protein functions as a tumor antigen recognized by CD4 + T cells. Most antigens identified as MHC class I–restricted tumor antigens are membrane proteins and the previously identified MHC class II–restricted tumor antigen, tyrosinase, is a membrane protein as well ( 14 ). (b) It was previously reported that CTLs could be generated in vitro against the junction region of an oncogenic fusion protein resulting from a chromosomal translocation ( 27 , 28 ). We show here that natural CD4 + T cells from the patient 1363 were capable of recognizing a melanoma antigen consisting of a fusion protein, which allowed us to detect a chromosomal rearrangement in chromosome 19 in 1363mel. The chromosomal rearrangement observed may be a consequence of intrachromosomal recombination between the Alu repeats in the LDLR and FUT genes ( 24 ). Lehrman et al. reported that deletions in the LDLR gene by recombination of Alu repeats produced a truncated, secreted LDLR protein ( 29 ). (c) DR1-positive TIL1363 cells were capable of presenting the LDLR-FUT fusion protein to each other though it is not clear whether the fusion protein directly bound to the DR1 molecules on TIL1363 for recognition. Alternatively, TIL1363 may capture the antigen from the culture medium and process and present it to each other by the exogenous antigen presentation pathway. Our results indicate that CD4 + TIL1363 recognized a unique fusion antigen expressed in 1363mel . Several CD4 + TILs have been established and characterized in our laboratory. Most CD4 + TILs recognized unique antigens, but not nonmutated shared antigens, as is the case for the majority of the MHC class I–restricted tumor antigens recognized by CD8 + CTLs that have been characterized ( 1 – 3 ). Tyrosinase is the only shared human melanoma antigen identified thus far and is recognized by an HLA-DR4–restricted CD4 + TIL1088 ( 14 ). Pieper et al. reported a mutated triosephosphate isomerase as a tumor antigen recognized by CD4 + TIL1558 ( 30 ). The point mutation of triosephosphate isomerase created a T cell epitope that required a lower peptide concentration necessary for T cell reactivity compared with the wild-type peptide. Recently, we identified a mutated CDC27 as a tumor antigen recognized by a DR4-restricted CD4 + TIL1359 ( 20a ). CDC27 is an important component of the anaphase promoting complex and plays a role in cell cycle regulation ( 31 , 32 ). Interestingly, the mutation itself in CDC27 did not constitute a T cell epitope, rather it affected the posttranslational modification (phosphorylation) and protein trafficking, which led to targeting of the mutated CDC27 protein to the MHC class II presentation pathway. Peptides identified from the LDLR-FUT fusion by CD4 + TIL1363 cells were located in the new polypeptide translated from the FUT sequence in an antisense orientation. These T cell epitopes were present in the 1363mel tumor cells, but not in the normal cells. The requirement for peptides to be recognized by CD4 + TIL1363 was the antigenic peptides containing a core sequence (WRRAPA) with a minimal length of 9 residues. Although many tumor cells are MHC class II negative, the specific role of CD4 + T cells in antitumor immunity has been demonstrated in a MHC class II–negative tumor model ( 33 ). Moreover, in addition to providing help for CD8 + CTLs ( 34 ), CD4 + T cells may play a far broader role in orchestrating the host response to tumor ( 35 ) and autoimmune diseases ( 36 , 37 ). Our studies indicate the importance of incorporating CD4 + reactive antigens in immunotherapy strategy. Definition of these MHC class II–presented antigens may provide new opportunities for developing more effective immunotherapies against cancer. | Other | biomedical | en | 0.999997 |
10330446 | Mouse pTα cDNA probe was amplified by reverse transcriptase (RT)-PCR as described ( 13 ) and used to screen a 129/SvJ mouse bacterial artificial chromosome genomic library (Genome Systems). A positive clone encompassing >100 kb of pTα locus was isolated and used for restriction mapping with oligonucleotide probes and for subcloning into plasmid vectors. The sequencing and mapping data were consistent with those reported previously ( 16 ). For reporter assays, a 5′ pTα fragment spanning 1.2 kb up to the PstI site within the known 5′ untranslated region (UTR) ( 16 ) was subcloned into the HindIII site of the promoterless LacZ ( Escherichia coli β-galactosidase) reporter vector pβgal-Basic ( Clontech ). An 8-kb XhoI-BglII 5′ fragment was subcloned into XhoI-BglII sites of this construct to create a 9-kb XhoI-PstI 5′ pTα fragment upstream of LacZ. Promoter and enhancer mapping were carried out by in-vector deletions of the above constructs or by subcloning of fragments upstream of the pTα promoter. The BstEII-MluNI enhancer fragment was subcloned into pBluescript and sequenced using T3 and T7 primers. As heterologous promoters, we used the CMV immediate early promoter and the SV40 early promoter (pβgal-Promoter vector; Clontech ) subcloned upstream of LacZ in the pβgal-Basic vector. As control enhancers, we used 0.6-kb mouse CD3δ enhancer (reference 17 ; a gift of Dr. Cox Terhorst, Harvard Medical School) and 0.55-kb human CD2 enhancer fragment ( 18 ). To examine enhancer activity in various cell lines, 4.3-kb NheI-BglII and 0.35-kb BstEII-NarI pTα enhancer fragments were subcloned upstream of the SV40 promoter in the pβgal-Promoter vector. For deletion analysis of the pTα enhancer, fragments were amplified by PCR using Pwo DNA polymerase (Roche Molecular Biochemicals) and subcloned into the KpnI-BglII sites of the pβgal-Promoter vector. Site-directed mutagenesis was performed using the QuikChange kit (Stratagene). All of these constructs were verified by sequencing using a primer within the SV40 promoter. A panel of T cell lymphomas, either derived in this laboratory from various knockout mouse strains or obtained from the American Type Culture Collection, was screened for the expression of pTα mRNA by Northern hybridization and RT-PCR (data not shown). The pTα-positive lines included LR1 ( atm −/− rag-2 −/− ), 642 ( p53 −/− ), and 799 ( atm −/− p53 −/− ); the pTα-negative lines included LR2 ( atm −/− rag-2 −/− ), EL4, and BW5147. Other cell lines included 1105, a B cell lymphoma from a c-myc– transgenic animal; MEL, a mouse erythroleukemia line; and NIH3T3 mouse fibroblasts. All lines were cultured in DMEM with 10% FCS, l -glutamine, and 2-ME. Cells were harvested and lysed in reticulocyte standard buffer (10 mM Tris/HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl 2 ) with 0.5% NP-40. Nuclei were washed and resuspended in reticulocyte standard buffer and treated with twofold dilutions of DNase I (Roche Molecular Biochemicals) for 5 min at room temperature. The reaction was stopped with a buffer containing 1% SDS, 50 mM EDTA, and proteinase K, and DNA was extracted, digested with appropriate enzymes, and analyzed by Southern hybridization. For the comparison between different cell lines, the amount of DNase was calibrated for each line and three concentrations were chosen: (i) the minimal DNase concentration producing a visible downshift of DNA fragments after restriction digest, (ii) a twofold lower concentration, and (iii) no DNase. The following genomic probes were used: a 0.46-kb PstI-NcoI fragment encompassing pTα exon 2 (probe 1); a 0.5-kb XhoI-HpaI fragment 10 kb 5′ of exon 1; and a 0.4-kb EcoRI-NcoI fragment 7 kb 5′ of exon 1 (probe 2). The construct containing a 9-kb pTα 5′ fragment upstream of LacZ and SV40 intron/Poly(A) sequences in the pβgal-Basic vector was digested with XhoI/SalI and gel-purified to remove the vector backbone. DNA was microinjected into fertilized oocytes from FVB mice, and transgenic founders were bred with wild-type FVB mice. A 0.25-kb AspI-PstI probe from the 3′ end of the 9-kb pTα fragment hybridized with 5- and 1.6-kb EcoRV fragments from the endogenous pTα gene and the transgene, respectively, and was used for transgene copy number determination by PhosphorImager (Molecular Dynamics) quantitation. Total RNA was extracted from various organs of 4-wk-old F1-transgenic mice, and Poly(A) RNA was prepared from 250 μg total RNA using biotin-oligo(dT)/streptavidin magnetic beads (Roche Molecular Biochemicals). Transgene expression was analyzed by Northern hybridization with a 5′ 1.3-kb fragment of LacZ gene. An NcoI fragment spanning 1.6 kb 5′ of the pTα first exon was subcloned in the reverse orientation into the NcoI site of pSL301 vector and linearized with EcoRV. A 0.4-kb α-[ 32 P]dUTP–labeled riboprobe was synthesized using T3 RNA polymerase and hybridized with 20 μg total RNA from the indicated cell lines. After digestion with RNase T 1 and A, the protected fragments were resolved on a 6% denaturing sequencing gel in parallel with a DNA sequencing ladder as a marker. Cells were transferred into 6-well plates and transfected with 1–2 μg DNA/well in duplicate, using lipid reagents according to the manufacturers' instructions. Cell lines LR1, BW5147, MEL, and NIH3T3 were transfected using Fugene 6 reagent (Roche Molecular Biochemicals); cells from cell lines LR2 and 642 were transfected using Superfect reagent (Qiagen). LacZ reporter vectors containing no promoter (pβgal-Basic) or SV40 promoter (pβgal-Promoter) were used as controls in all experiments. After 24 h, cells were harvested and β-galactosidase activity was measured using a chemiluminescent assay ( Clontech or Tropix) in the scintillation counter. The results represent mean cpm ± range of duplicate transfections. Note that these are arbitrary units depending on the assay conditions; in particular, the assay from Tropix was found to be 10–100 times more sensitive than the assay from Clontech . For crude nuclear extract preparation, cell nuclei were isolated by hypotonic lysis, and proteins were extracted with a buffer containing 0.4 M NaCl. Each binding reaction (15 μl) contained 10 fmol (10 5 cpm) γ-[ 32 P] dATP–labeled double-stranded oligonucleotide probe, 3–5 μg nuclear protein extract, 0.5 μg poly(dI-dC), and, where indicated, a 100-fold excess of unlabeled oligonucleotide or 2 μg Ab. Binding was performed for 20 min at room temperature in a final concentration of 50 mM NaCl; 20 mM Tris/HCl, pH 7.5; 1 mM EDTA; 1 mM dithiothreitol; and 10% glycerol. Protein–DNA complexes were separated on 4% nondenaturing polyacrylamide gels in 0.5× Tris/borate/EDTA buffer and visualized by autoradiography. Oligonucleotide probes 1–6 spanning the core enhancer are depicted . Other probes included consensus oligonucleotides for YY1 ( Santa Cruz Biotechnology ), Sp1 ( Promega Corp. ), and Ikaros (IK-BS2; reference 19 ), and an lck proximal promoter −365/−328 probe (lckB; reference 20 ). Anti-YY1 mAb and anti-Sp1 and -Sp3 polyclonal Ab were purchased from Santa Cruz Biotechnology . Anti-ZBP89 rabbit antiserum was provided by Drs. Juanita Merchant and David Law (University of Michigan, Ann Arbor, MI) and was used as protein A–purified IgG fraction. Purified anti-CD3 mAb 145-2C11 was used as a negative control. Potential transcription factor binding sites were analyzed using the TRANSFAC database and software ( http://transfac.gbf.de ; reference 21 ). Because regulatory genomic regions often display increased sensitivity to DNase I treatment, we searched for such DNase-hypersensitive sites (DHS) in the pTα locus of T cell lymphomas manifesting or lacking pTα expression. We first used a probe corresponding to pTα exon 2 in conjunction with several restriction digests shown in Fig. 1 A. No strong DHS correlating with pTα expression were detected within or downstream of the pTα gene . A nonspecific DHS was detected 4 kb downstream of the last exon in all cell lines tested, confirming the validity of the analysis . In contrast, a specific site (DHS 1) was found immediately upstream of the first exon in pTα-positive but not pTα-negative T cell lines . To examine the regions further upstream of the pTα gene, we initially used a probe detecting a 10-kb BglII fragment 5′ of the gene (data not shown). This preliminary analysis suggested the presence of a specific DHS within the region; however, a nonspecific DHS immediately 3′ to the probe precluded more precise mapping. To better localize the potential second DHS, we used a more downstream fragment as a probe . As shown in Fig. 1 C, these experiments revealed the presence of two closely located DHS, collectively referred to as DHS 2, ∼4–4.5 kb upstream of the first pTα exon specifically in pTα-positive cell lines. Thus, the genomic region 5′ of pTα harbors at least two specific DHS and is likely to play a major role in the regulation of pTα expression. To verify that putative regulatory regions upstream of the pTα gene were sufficient for pre-T cell–specific gene expression, we created transgenic mice carrying the marker gene LacZ under the control of a pTα 5′ fragment. The transgene contained 9 kb of pTα 5′ region, including both DHS and a part of the known 5′ UTR, upstream of LacZ and SV40 intron and Poly(A) signal. The heterozygous progeny of transgenic founders were analyzed for transgene expression by Northern hybridization with a LacZ probe. Of six transgenic lines analyzed, two lines manifested detectable LacZ expression in the thymus. Both lines expressed LacZ in the thymus but not in the spleen; the line carrying fewer copies of the transgene (four copies) was analyzed in more detail. As shown in Fig. 2 , the marker gene was expressed exclusively in the thymus but not in the spleen, lymph nodes, or other organs, with a pattern and abundance comparable to that of the endogenous pTα gene. Thus, a 9-kb 5′ genomic fragment of the pTα gene supported thymus-specific transgene expression in two transgenic mouse lines. We therefore conclude that this fragment contains all information necessary for pTα expression in thymocytes. Because of its position 5′ of the first exon, the first DHS (DHS 1) was likely to reflect the activity of a proximal promoter. To confirm this notion, we analyzed the 5′ transcriptional start of the pTα gene by RNase protection assay, using a probe spanning 0.4 kb immediately upstream of the translation start site . Fig. 3 B shows the presence in T cell lymphomas of heterogeneous pTα transcripts initiated within 100–200 bp of ATG, with the most abundant transcript corresponding to a short 5′ UTR of ∼124 bp. This position is only 5 bp 5′ of the longest pTα cDNA clone ( 16 ) and might represent a major transcription start site. To examine the promoter function of the sequences adjacent to the identified transcription start site, a 1.3-kb fragment spanning this region was subcloned upstream of a LacZ (β-galactosidase) reporter gene and transfected into the T cell line LR1. This early passage cell line, derived from an atm −/− rag-2 −/− thymoma, has an immature T cell phenotype (Thy-1 hi CD4 lo CD8 lo CD2 lo CD25 − CD44 − TCR-β − pTα + ) and can be transfected using lipid reagents. As shown in Fig. 3 C, the pTα upstream fragment manifested a relatively weak orientation-dependent promoter activity. Using 5′ deletions of the fragment, the promoter function was localized to a 0.15-kb PpuMI-PstI fragment containing 115 bp 5′ of the putative transcription start site. Although the low activity of pTα promoter precluded the analysis of its function in other T cell lines, the promoter was inactive in an erythroleukemia cell line, MEL and in NIH3T3 fibroblasts (not shown). Thus, DHS 1 apparently corresponds to a short proximal promoter that might be specific at least for lymphoid cells. To search for the possible distal enhancers in the pTα locus, we examined the effect of larger pTα genomic fragments in a transient transfection assay with LR1 T cells. As shown in Fig. 4 , a 9-kb 5′ pTα fragment, used in the transgene as described above, was more active than a 0.5-kb 5′ fragment containing the core promoter. Using in-vector deletions of the 9-kb region, we first localized the enhancer activity to a 2.8-kb XbaI fragment. By creating a series of nested 5′ and 3′ deletions of this fragment (data not shown), this activity was further mapped to a 0.35-kb region between BstEII and NarI sites, 4 kb upstream of the pTα promoter. Fig. 4 demonstrates that this fragment and a smaller 0.25-kb BstEII-MluNI fragment were fully active as transcriptional enhancers. In contrast, 5′ or 3′ truncations at a PstI site within this region significantly reduced the enhancer function. Importantly, these mapping data are consistent with the location of DHS 2, suggesting that the described pTα enhancer is at least one regulatory region corresponding to DHS 2. Fig. 4 also demonstrates that pTα enhancer increased transcription when placed in either orientation and at a variable distance from the promoter. To examine its activity on heterologous promoters, the larger 4.3-kb and the smaller 0.35-kb fragments containing the pTα enhancer were subcloned upstream of the SV40 early promoter. As shown in Fig. 5 A, both pTα enhancer fragments significantly increased the activity of the SV40 promoter in LR1 T cells. The activity of pTα enhancer in LR1 cells was stronger than that of previously described T cell–specific enhancers of CD3δ and CD2 (not shown) genes and was also observed with another strong heterologous promoter, the CMV immediate early promoter (data not shown). Thus, the identified upstream enhancer of the pTα gene appears as a powerful, bona fide transcriptional enhancer, functioning irrespective of distance, orientation, or the corresponding promoter ( 22 ). To examine the cell and stage specificity of the pTα enhancer, the enhancer/SV40 promoter constructs were introduced into a pTα-negative T cell line, BW5147. As shown in Fig. 5 A, the large enhancer fragment was completely inactive, whereas the small, 0.35-kb fragment produced only a minor increase similar to the CD3δ enhancer. Similar results were obtained with smaller enhancer fragments (not shown). This was not due to a limiting transfection efficiency, as the CMV promoter produced a strong reporter activity in these cells. A similarly low activity of the pTα enhancer was observed in nonlymphoid MEL and NIH3T3 cells (data not shown). In another series of experiments, the same reporter constructs were introduced into pTα-positive (642) or -negative (LR2) T cell lines. These early passage T cell lymphomas could be transfected with a comparable low efficiency by the same protocol. Fig. 5 B demonstrates that the pTα enhancer increased the promoter activity in 642 cells but was scarcely functional in LR2 cells compared with the control CD3δ enhancer. Altogether, these data suggest, but do not prove, that the pTα enhancer is preferentially active in pre-T cells as compared with mature T cells or nonlymphoid cells. The sequence of the 0.25-kb BstEII-MluNI enhancer fragment was determined and is shown in Fig. 6 A. Nested 5′ and 3′ deletions of this region were produced by PCR using the indicated primers, subcloned into the SV40 promoter/ LacZ reporter vector, and assessed for their activity in LR1 cells . This analysis revealed a core enhancer of 149 bp, defined by primers F2 and R2. Further deletions in this region significantly decreased the enhancer activity; the regions between F2/F3, R2/R3, and R4/R5 appeared particularly important. These data are consistent with the deleterious effect of truncations at the PstI site within the core enhancer region . Next, we examined nuclear proteins binding to the pTα enhancer by electrophoretic mobility shift assay (EMSA) using nuclear extracts from several pTα-positive or -negative T cell lines, a B cell line, and an erythroleukemia cell line. As probes, we used six double-stranded oligonucleotides spanning the core enhancer or larger promoter and enhancer DNA fragments. This analysis revealed multiple distinct nuclear factors interacting with the core enhancer; however, we were unable to detect any DNA– protein complexes appearing specifically in pTα-expressing T cell lines. The core enhancer sequence contained a potential binding site (CCAT; reference 23 ) for the transcription factor YY1. Indeed, probe 3, containing the putative YY1 site, formed a complex, found in all cells examined, that could be specifically competed by YY1 consensus oligonucleotide . Furthermore, Fig. 7 shows that the factor binding to both probes could be supershifted by anti-YY1 Ab, thus confirming its identity as YY1. To explore the functional role of this interaction, the YY1 binding site was mutated so that the mutant sequence was unable to compete with YY1 binding (not shown). As shown in Fig. 6 C, the YY1 site mutation resulted in a minor, but consistent, increase in enhancer activity. Thus, the core pTα enhancer appears to interact with YY1 transcription factor, which might contribute to the repression of its activity. It was reported previously that the thymocyte-specific lck proximal promoter contains a G-rich site that appears critical for the promoter function and binds a T cell nuclear factor (factor B) ( 20 ). Because the pTα enhancer sequence features two similar C-rich stretches, we examined the factors binding to these sites and their possible relation to factor B of the lck promoter. Enhancer probes 2, 5, and 6 produced a similar pattern of nuclear complexes and effectively competed with each other for binding (not shown); we therefore concluded that the 5′ and 3′ C-rich sites bind the same nuclear factors. Fig. 8 demonstrates that enhancer probe 2, encompassing the 5′ C-rich site, formed two distinct complexes in T cell nuclear extracts. The upper complex was specifically competed out by an Sp1 consensus probe, whereas the lower complex was competed out by the G-rich B site of lck promoter (lckB); moreover, the labeled lckB probe formed a single complex of similar size. Antibody supershift experiments confirmed that the upper complex consisted of Sp1 and Sp3 transcription factors . Thus, the C-rich enhancer regions bind Sp1 family proteins and another protein identical to the B complex of lck promoter. A recently cloned zinc finger transcription factor, ZBP-89 (BFCOL1, BERF-1), was shown to bind long, G-rich stretches in several promoters ( 24 – 26 ) and, therefore, represented a good candidate for the observed binding activity. We tested this possibility and found that anti–ZBP-89 Ab specifically blocked the formation of the lower complex with probe 2 and of the major complex with lckB probe . Thus, ZBP-89 appears to interact with two sites within the pTα enhancer core and with a critical site of the lck proximal promoter. These observations suggest a possible role for ZBP-89 in thymocyte-specific gene expression. This study was aimed at establishing a model to study the regulation of pre-T cell–specific gene transcription. The regulation of genes expressed at the early stages of B cell development has been extensively studied, and stage-specific transcription factors such as EBF (early B cell factor) have been identified ( 27 ). In contrast, the mechanisms of stage-specific gene expression in T cells are less well understood. Certain transcription factors appear to regulate specific stages of T cell development, as illustrated by the role of LKLF (lung Kruppel-like factor) in the maintenance of mature T cell quiescence ( 28 ). A clear example of reciprocal transcriptional regulation in early versus mature T cells is the alternative promoter usage at the lck tyrosine kinase gene ( 5 ). In particular, the lck proximal promoter was proven to function specifically in immature thymocytes ( 20 ) and has been extensively used to target transgene expression to early T cells. In another case, a thymocyte-specific enhancer was found in the third intron of Thy-1 gene ( 29 ). Furthermore, stage-specific silencers and enhancers were described in the CD4 ( 6 , 7 ) and CD8 ( 8 – 10 ) loci, respectively. Despite this progress, the molecular basis of an apparently common pattern of pre-T cell– specific transcription is obscure. The expression of the pTα gene has been examined in great detail and was shown to occur specifically in pre-T cells ( 13 , 14 ). Recently, the expression of an alternatively spliced pTα isoform (lacking the extracellular Ig domain) was described ( 30 , 31 ) and proposed to occur in mature T cells as well as in pre-T cells ( 30 ). This isoform was also observed in the original analysis of pTα expression but was not detected in mature T cells ( 14 ). Similarly, we could not detect its expression in the spleen or in pTα-negative T cell lines (Reizis, B., unpublished results). Therefore, the expression of a shorter pTα isoform in mature T cells is likely to occur, if at all, only in a specialized minor population of T cells or at extremely low levels. Thus, by and large, the pTα gene appears to be expressed in early T cells and as such represents a valuable model for stage-specific T cell gene expression. Although the mouse and human pTα genes have been extensively mapped and sequenced ( 16 , 31 ), the regulation of pTα gene expression has not been studied. We now report that mouse pTα gene transcription is regulated primarily by an upstream genomic region. It is possible, however, that additional genomic elements within, downstream of, or farther upstream of the gene might contribute to its regulation. In particular, possible locus control regions conferring position-independent transgene expression remain to be identified in the pTα locus; the nonspecific DHS 5′ and 3′ of the gene are candidates for such elements. Another likely regulatory region is the recently described sequence in the first pTα intron, which is conserved between mouse and human genes ( 31 ). We found that a fragment containing this sequence lacked any detectable enhancer activity in a transient transfection assay (data not shown); thus, it might function as a silencer or an element regulating chromatin accessibility. Further studies are required to delineate the complete hierarchy of pTα transcriptional regulation. In any case, our data indicate that the cell and stage specificity of pTα expression are fully determined by upstream elements. Within the pTα upstream region, we have identified a proximal promoter and an enhancer located 4 kb 5′ of the promoter. As in many T cell–specific genes, the promoter appeared relatively weak and is most likely insufficient for pTα expression. The pTα enhancer, on the other hand, manifested high activity in transient transfection assays and appeared to function preferentially in pTα-positive pre-T cell lines. It is possible, however, that additional elements in the vicinity of the enhancer contribute to its specificity. In this regard, it is noteworthy that DHS 2 actually consists of two sites separated by ∼0.3–0.4 kb; the precise nature of these sites is currently under investigation. In addition, the enhancer may require its cognate pTα promoter to achieve full specificity. Our analysis of the nuclear factors interacting with the core pTα enhancer suggests a preliminary model of its architecture . We could detect at least two distinct factors binding to the 5′ end of the sequence, and these interactions appear important for the enhancer function, as evidenced by the F2/F3 deletion . The two 5′ E boxes appear to have formed identical complexes, whereas the 3′ E box represents a consensus binding site for bHLH-ZIP transcription factors and might bind a distinct set of proteins. The enhancer features a perfect Ikaros binding site, and probe 4, spanning this site, formed a nuclear complex that was specifically inhibited by Ikaros consensus probe IK-BS2 ( 19 ) and vice versa; however, we were unable to confirm the identity of this factor using anti-Ikaros antiserum. Nevertheless, Ikaros protein isoforms or Ikaros-related factors might interact with this site in vivo and contribute to the repression of the enhancer by recruiting it to centromeric heterochromatin in pTα-negative cells ( 32 , 33 ). In addition, transcription factor YY1 binds to the middle portion of the enhancer and appears to repress its activity. YY1 is a multifunctional factor implicated in, among other things, the repression of tissue-specific genes such as Ig and globin genes, possibly due to the recruitment of corepressors such as histone deacetylases ( 34 ). Thus, the pTα enhancer may be subject to the complex regulation at the level of chromatin modification. The pTα enhancer contains two C-rich sites, and deletion of the 3′ site compromised the enhancer function . We found that these sites bind Sp1 and Sp3, the related ubiquitous transcriptional activator and repressor proteins, respectively ( 35 ). The true role of Sp proteins in the regulation of the pTα enhancer might be difficult to establish; indeed, the expression of many genes thought to be regulated by Sp1 was not affected in its absence ( 35 ). It should be noted, however, that Sp1 is abundantly expressed in the thymus ( 36 ) and therefore might play a specific role in thymocyte gene expression. In addition, these C-rich sites, as well as an important G-rich site in the proximal lck promoter, bind a common nuclear factor identified here as ZBP-89. The truncated clone of ZBP-89, htβ, was originally cloned as a zinc finger protein binding to the CACCC box in the human TCR-α promoter ( 37 ). Recently, the full length protein ZBP-89/ BFCOL1/BERF-1 was cloned as a protein binding to long, G-rich stretches in various promoters ( 24 – 26 , 38 , 39 ). This protein appears to be capable of both transcriptional activation ( 25 , 26 , 37 ) and repression ( 24 , 26 , 38 ), possibly depending on the DNA context and cell type; the precise function of ZBP-89 on the pTα enhancer remains to be established. Despite its apparently ubiquitous expression, we have found that ZBP-89 mRNA is expressed in the thymus at significantly higher levels than in any other organ tested, including the spleen (Reizis, B., unpublished results). Indeed, six out of eight cDNA clones corresponding to ZBP-89 in the mouse expressed sequence tag database are derived from the thymus library. In addition, the ZBP-89 complex was easily detectable by EMSA in T cell lines but not in nonlymphoid cells such as MEL. Together with the observed binding of ZBP-89 to the regulatory elements active specifically in early T cells, these observations suggest an important role of ZBP-89 in T cell development. A proposed model of the pTα core enhancer differs significantly from the core enhancers of TCR genes ( 3 ); in particular, it lacks obvious binding sites for T cell–specific transcription factors such as GATA3 or TCF1/LEF1. Furthermore, we were unable to detect enhancer-binding nuclear factors directly correlating with pTα expression in T cell lines. This may be due to the technical limitations of our approach, or it might reflect the requirement for additional regulatory sites as discussed above. Another possibility, however, is the existence of protein cofactors interacting with the enhancer-binding proteins and conferring cell and stage specificity on the enhancer. Indeed, the bHLH proteins, YY1 and Sp1, are known to be involved in complex interactions with other proteins, which are essential for their function on a particular regulatory region. Similarly, ZBP-89 is likely to undergo cell type–specific regulation by other proteins. Future studies using this and other models should delineate the precise mechanism of stage-specific gene expression in T cells. | Study | biomedical | en | 0.999998 |
10330447 | A retroviral expression vector LXSN coding for the wild-type TCR β chain (Vβ8.2-Jβ2.1) cDNA was used as template for mutagenesis. Deletion of the region corresponding to the 14–amino acid FG loop of the Cβ domain was performed by linking PCR. A 1:1 ratio of the products from PCR 1 (5′ oligo of Vβ8.2 GAATTCCTTGAGCTCAAGATGGGCTCCAGGCTCTTC [oligo A] and 3′ oligo spanning the deletion GTTCTGTGTGACCCCAT GGA AC TGCACT TGGCAGCG) and PCR 2 (5′ oligo spanning the deletion CAGTTCCATGGGGTCACACAGAACATCAGTGCAGAG and 3′ oligo containing the stop codon AGGATCCTCATGAGTTTTTTCTTTTGAC [oligo B]) was used as template for PCR 3 (oligo A and B). The PCR product was digested with EcoRI and BamHI and cloned into an EcoRI and BamHI–opened retroviral vector LXSN. Deletion (underlined amino acids 231–244) G LSEEDKWPEGSPKP V was then verified by DNA sequencing. Transgenic vectors were as described previously ( 8 ). Infectious retroviral stocks were generated by transfecting packaging cell lines GP+E-86 ( 9 ) with retroviral expression vectors LXSN (neomycin resistant) coding for wild-type or mutant TCR β chain, or vectors LXSP (puromycin resistant) coding for wild-type TCR α chain (Vα4-Jα47). The supernatants from appropriately selected packaging cell lines were used to infect TCR − hybridomas. The wild-type β or mutant β chain were first introduced into the hybridomas, and after neomycin selection (G418, 1 mg/ml) these lines were superinfected separately with TCR α chain as described previously ( 10 ). The cell lines were then cultured in IMDM supplemented with 2% FCS, G418, and puromycin (10 μg/ml). TCR expression was tested by FACS ® as soon as 4 d after selection. Stable transfectants were maintained in G418 and puromycin–containing medium. BALB/c and C56BL/6 mice were purchased from IFFA-Credo. The TCR-β knock-out mice have already been described ( 11 ), and were bred in our specific pathogen–free animal facility with the wild-type TCR-β or mutant TCR-β transgenic mice. Immunofluorescence stainings were done as described previously ( 12 ). Flow cytometric analysis was performed with a FACSCalibur™ equipped with CellQuest software ( Becton Dickinson ). The reagents used were mAbs biotinylated 145-2C11 (anti-CD3ε), PE-labeled RM4-5 (anti-CD4) and FITC-labeled H57-597 (anti-Cβ) ( 13 ), B20.1 (anti-Vα2), RR3-16 (anti-Vα3.2), B21-14 (anti-Vα8), and RR8-1 (anti-Vα11.1, 2) (all seven mAbs purchased from PharMingen ), Cy5-labeled 53-6.7 (anti-CD8), fluorescein-succinimidyl-ester (FLUOS)- labeled F23.1 (anti-Vβ8.1, 2, 3) ( 14 ), and second-step reagent streptavidin-allophycocyanin (APC) (Molecular Probes, Inc.). For T cell proliferation, 2 × 10 5 spleen cells were cultured in triplicate with various concentrations of staphylococcal enterotoxin B (SEB) and SEC 2 superantigens in 200 μl of IMDM supplemented with 10% FCS in 96-well flat-bottomed plates. Proliferative responses were assessed after 48 h of culture. Cultures were pulsed 8 h before harvesting with 1 μCi [ 3 H]TdR (40 Ci/nmol; Radiochemical Center, Amersham Pharmacia Biotech ), and incorporation of [ 3 H]TdR was measured by liquid scintillation spectrometry. Helper T cell responses were tested by immunizing mice (three per group) with 100 μg of NIP-OVA in CFA in the tail base. For control, mice received PBS in CFA . After 14 d, sera from immunized mice were pooled and tested for the presence of anti-NIP IgG by ELISA as described ( 15 ). Plates coated with 5 μg/ml of NIP-BSA and then blocked with PBS/1% BSA received dilutions of the sera. Binding of the anti-NIP IgGs was revealed by alkaline phosphatase–conjugated goat anti–mouse IgG (Southern Biotechnology Associates). Allogeneic killer cells were generated as described previously ( 8 ). In brief, 10 7 responders (H-2 b splenocytes from wild-type TCR-β or mutant TCR-β transgenic mice) were cultured with 10 7 x-irradiated stimulators (H-2 d splenocytes from BALB/c mice). After 5 d, various numbers of responder cells (numbers used to calculate the E/T ratios) were cultured with 10 4 Na 2 51 CrO 4 -labeled target LPS blasts. After 4 h, supernatant was harvested. Some wells contained only labeled targets with or without 0.01 M HCl/10% SDS containing medium to determine maximum and spontaneous release, respectively. Data are presented as percentage of killing = [(experimental release − spontaneous release)/(total release − spontaneous release)] × 100. To test whether the deletion of the complete 14–amino acid FG loop in the Cβ domain would be deleterious for the TCR assembly and surface expression, we transfected TCR − T cell thymoma 58 with retroviral vectors coding for either a control or a mutant β chain together with a wild-type α chain ( 10 ). To our initial surprise, the TCR surface expression was only slightly lower in the mutant case . However, we must point out that the observed 30–50% reduction in the surface expression represents a handicap in the TCR assembly which, although small, is real since we have used a very efficient retroviral transfection system that allows us to create bulk transformants containing thousands of individual clones and which, therefore, provides us with a reliable statistical average. Functional analyses of these transfectomas consistently showed that the cells transfected with the mutant TCR β chain responded slightly less (about threefold) to antigenic stimulation as exemplified here by the dose–response curves to influenza hemagglutinin peptide HA 110-119 or SEC 3 superantigen . To more rigorously assess the functional potential of the TCR containing the mutant β chain in normal physiological settings in vivo, we generated transgenic mice expressing either a wild-type or a loop-deleted version of the TCR β chain. The β transgenes, as in the above transfection studies, were derived from 14.3d T cell hybridoma expressing the TCR specific for influenza hemagglutinin peptide HA 110-119 in the context of I-E d MHC class II molecules ( 16 ). In fact, it was the very same β chain (Vβ8.2-Jβ2.1) whose three-dimensional structure was first solved, thus providing us with the inspiration for the current study ( 3 ). Two characteristics of the transgenic lines used here were considered essential for straightforward interpretation of the data. First, the level of α/β TCR expression was identical in both lines . Presumably the small handicap of the mutant β chain in the TCR assembly could be compensated by higher intracellular expression. Second, both transgenes were bred to TCR-β −/− background to avoid any contribution of endogenous β chains for the observed α/β T cell behavior ( 11 ). α/β T cell development proceeds undisturbed and similarly in both TCR β chain transgenic lines as shown by flow cytometric analysis of thymic and lymph node cells . Even the skewing into single positive CD4 thymocytes, as noted earlier for our wild-type TCR-β transgenic mice ( 8 ), occurs to the same extent in both lines. As predicted, mAb H57-597 (anti-Cβ ) does not bind to mutant TCR β chain . Interestingly, mAb F23.1 (anti-Vβ8.1, 2, 3 ) binds equally well to both β chains, whereas mAb MR5-2 (anti-Vβ8.1, 2 ) fails to react with the mutant, suggesting that the FG loop may form part of the MR5-2 epitope (not shown). Since the cellularity of thymi is normal in both cases, we assume that pre-TCR–mediated T cell expansion occurs normally in these mice. Peripheral T cell responses were measured in several types of assays, and none of them, to our disappointment, showed any significant differences between mice of the different transgenic lines. The in vitro responses to anti-TCR antibodies (not shown) and to SEC 2 and SEB superantigens were repeatedly similar in all mice tested . In addition, the in vivo CD4 + T cell responses measured by T cell help for hapten-specific IgG production were basically indistinguishable between control and mutant mice . Finally, α/β T cells from mutant TCR β chain transgenic mice made as vigorous cytotoxic T cell responses against allogeneic targets as their control counterparts . We also monitored the representation of four different Vα families by flow cytometry in peripheral T cells in order to reveal any subtle in vivo biases, but none were found (Table I ). In addition, limited DNA sequence analyses of Vα 2 and 8 families from single α/β T cells revealed no obvious “mutant”-specific features (data not shown). Thus far, we have found only a quantitative role in the TCR assembly process for the large solvent-exposed FG loop on the Cβ domain. In transfectants, the TCR will assemble in the absence of the loop in the β chain but slightly less efficiently compared with the wild-type structure. Of course, the reduced surface expression leads to somewhat impaired function. However, we were able to show in vivo that TCRs are functionally expressed at the same level with or without the FG loop, and we did not find any qualitative or quantitative differences in their activity. This finding seems to rule out the models where the FG loop has an absolute role in TCR signaling. However, the apparent absence of any effect in vivo could also be due to the fact that some subtle compensatory mechanisms have been turned on in vivo (but not in cell lines), e.g., TCR affinities could be modulated, or new carbohydrate structures on the Cβ domain could partially replace the FG loop functionally. Interestingly, all nonmammalian species studied to date, including birds, amphibians, reptiles, and fish, do not have the FG loop on their Cβ domain ( 18 ); hence, our in vivo findings may not be that surprising. | Study | biomedical | en | 0.999997 |
10352010 | Tyrosine-based signals constitute a family of degenerate motifs minimally defined by the presence of a critical tyrosine residue (see reference 22 and references therein). Most tyrosine-based signals conform to the consensus motifs YXXØ (Y is tyrosine, X is any amino acid, and Ø is an amino acid with a bulky hydrophobic side chain; reference 5 ) or NPXY (N is asparagine and P is proline; reference 6 ). YXXØ signals are currently the best understood from a structural standpoint and thus will be the primary subject of our discussion. YXXØ signals can be found within the cytosolic domains of all types of transmembrane proteins, including type I (e.g., lamp-1), type II (e.g., the transferrin receptor), and multi-spanning (e.g., CD63). They can be most easily identified within short cytosolic tails (i.e., <35 amino acid residues), although they have also been shown to exist within the large cytosolic domains of some signaling receptors (e.g., the epidermal growth factor receptor) and retroviral envelope glycoproteins (e.g., HIV-1 gp41). The presence of a sequence conforming to the YXXØ motif within a large cytosolic domain, however, is not necessarily predictive of sorting information since signals must be presented in an appropriate context to be active. In mammalian cells, virtually all YXXØ signals mediate rapid internalization from the cell surface. Some YXXØ signals can additionally mediate lysosomal targeting, localization to specialized endosomal-lysosomal organelles such as antigen-processing compartments, delivery to the basolateral plasma membrane of polarized epithelial cells or localization to the TGN (reviewed in reference 19 , 22 , 24 ). The multiple functions of YXXØ signals raise the question of how the same type of signal can mediate sorting to different cellular compartments. A hypothesis that has been put forth to explain the various roles of YXXØ signals is that they must interact selectively with a family of recognition molecules associated with different sites of protein sorting. Recent findings that YXXØ signals are capable of interacting with several AP complexes provide a framework for testing the validity of this hypothesis. Glickman et al. ( 14 ) pioneered the use of in vitro affinity-binding methods to study the interactions of the cytosolic tails of membrane receptors with AP complexes. In the course of these studies, they demonstrated a tyrosine-dependent interaction of the cytosolic tail of the cation-independent mannose 6-phosphate receptor with AP-2, a plasma membrane, clathrin-associated complex composed of two large subunits (α and β2), one medium subunit (μ2), and one small subunit (σ2) . Generalization of this biochemical approach to other transmembrane proteins, however, was hampered by the low affinity of the interactions in vitro. Further progress required the development of more sensitive protein interaction assays based on techniques such as the yeast two-hybrid system and surface plasmon resonance spectroscopy. The use of the yeast two-hybrid system, for instance, was instrumental in the identification of μ2 as a recognition molecule for YXXØ signals ( 29 ). Mutational and combinatorial analyses demonstrated that the Y residue is essential for binding to μ2 and cannot be effectively substituted even by the structurally related phenylalanine or phosphotyrosine residues ( 4 , 28 , 36 ). Leucine is the preferred residue at the Ø position, although isoleucine, phenylalanine, methionine, and, to a lesser extent, valine, are tolerated ( 4 , 27 , 28 ). Many residues are permitted at the X positions, although arginine and proline are favored at the second X position ( 4 , 27 , 28 ). All of these preferences are consistent with the requirements for optimal function of YXXØ signals in rapid internalization, and thus provide strong correlative evidence for the physiological role of YXXØ-μ2 interactions. Structure-function analyses of μ2 have established that this polypeptide has a bipartite structure with the NH 2 -terminal third of the molecule (amino acid residues ∼1–145) being involved in assembly with β2, and the remaining two-thirds (amino acid residues ∼164–435) in interactions with YXXØ signals ( 1 ) . In a landmark study, David Owen and Philip Evans ( 32 ) have recently solved the crystal structure of the YXXØ-binding domain of μ2 complexed to peptides containing either the YQRL signal from the protein TGN38 or the YRAL signal from the epidermal growth factor receptor. The YXXØ-binding domain of μ2 has a banana-shaped structure consisting of 16 β-sheet strands arranged into two subdomains . YXXØ signals bind in an extended conformation (rather than as a tight turn, as was previously believed) to a region of the molecule having pockets for both the Y and Ø residues. This mode of interaction, resembling a two-pronged plug fitting into a two-holed socket, is reminiscent of that of phosphotyrosine-containing motifs with SH2 domains ( 40 ), although the topographic features of the binding sites and the details of the interactions differ considerably. The aromatic ring of the critical Y residue is involved in hydrophobic interactions with μ2 residues F 174 and W 421 , as well as stacking on the guanidinium group of R 423 . In addition, the phenolic hydroxyl group of the Y residue is engaged in a network of hydrogen bonds with D 176 , K 203 , and R 423 of μ2 . These characteristics of the Y-binding pocket explain why phenylalanine and phosphotyrosine residues substitute poorly or not at all for tyrosine residues in the signals: phenylalanine residues would be unable to establish hydrogen bonds with residues at the bottom of the pocket, while phosphotyrosine residues would be too bulky to fit into the pocket and would elicit electrostatic repulsion by D 176 . Residues lining the Ø pocket include L 173 , L 175 , V 401 , L 404 , V 422 , and the aliphatic portion of K 420 . The hydrophobicity and flexibility of the side chains of these residues allow accommodation of different bulky hydrophobic side chains at the Ø position, with leucine providing the best fit. Although interactions through the Y and Ø residues provide the main means of attachment of signals to μ2, specific X residues at positions between the Y and Ø residues may contribute additional contact points. For example, the R residue at the second X position of the YQRL signal is engaged in hydrophobic interactions with W 421 and I 419 and hydrogen bonding with K 420 thus explaining the preference for R at this position ( 4 , 27 , 28 ). Neither NPXY-type signals ( 6 ) nor dileucine-based signals (another type of signal having a critical pair of bulky hydrophobic residues; reference 17 , 21 ) can be accommodated in the YXXØ-binding site of μ2 ( 32 ), in agreement with the failure to isolate peptides conforming to these motifs in combinatorial screens ( 4 , 27 ), as well as with the inability of these signals to compete with YXXØ signals for the sorting machinery in vivo ( 23 , 42 ). In fact, recent studies have shown that NPXY and dileucine-based signals bind to other recognition molecules, namely the terminal domain of clathrin ( 18 ) and the β subunits of AP-1 and AP-2 ( 15 , 34 ), respectively. The finding that the μ2 subunit of AP-2 interacts with YXXØ signals raised the possibility that analogous subunits of other AP complexes could similarly function in recognition of YXXØ. To date, three additional complexes structurally related to AP-2 have been described in mammals: AP-1, AP-3, and AP-4 . Each of these AP complexes contains a μ subunit that displays significant homology to μ2 over the entire sequence. μ1A (formerly called μ1; reference 25 ) is a component of the AP-1 complex in most cell types, whereas a closely related isoform, μ1B, may be a subunit of this complex in polarized epithelial and glandular cells ( 30 ). μ3A and μ3B are alternative components of AP-3 ( 10 , 37 , 38 ); μ3A is widely expressed, whereas μ3B expression is mainly restricted to cells of neuronal origin ( 33 ). Finally, μ4 (originally known as μ-ARP2; reference 41 ) is a subunit of the recently described AP-4 complex ( 9 ). Sequence alignments indicate that most of the μ2 residues directly involved in interactions with the Y and Ø residues of YXXØ signals are conserved in other AP μ family members . Indeed, μ1A, μ1B, μ3A, and μ3B have all been shown to interact with YXXØ signals, albeit with lower affinity relative to μ2 ( 10 , 27 – 30 , 34 , 39 ). The conservation of Y- and Ø-binding residues also extends to μ4, as well as to AP μ orthologs from nonmammalian organisms . This suggests that these molecules may also be capable of recognizing YXXØ signals. The identification of a family of proteins that interact with YXXØ signals supports the hypothesis that the functional specificity of these signals may be dictated by their selective interaction with different recognition molecules. As mentioned above, μ2 tolerates many different amino acid side chains surrounding the critical Y and Ø residues, although it prefers arginine at the second X position of the YXXØ signal ( 4 , 27 , 28 ). Similar analyses have revealed that μ1A and μ3A prefer non-polar and acidic residues, respectively, at that position ( 27 ). Although the functional significance of the μ1A preferences is unclear, μ3A preferences are suggestive of a role in lysosomal targeting since the signals of several proteins localized to lysosomes and lysosome-related organelles (e.g., CD63, lamp-2a, and GMP-17) contain acidic residues at positions adjacent to the tyrosine residue. Having just identified a family of YXXØ-recognition molecules, an important next question that needs to be addressed is: what sorting events are mediated by interaction of YXXØ signals with each of these molecules? AP-1 has been localized mainly to the TGN at steady state, where it is thought to mediate transport of lamp-1 and mannose 6-phosphate receptors to compartments of the endosomal-lysosomal system ( 13 , 16 ). Recent studies, however, have raised the possibility that AP-1 may be involved in protein sorting to the basolateral plasma membrane of polarized epithelial cells ( 12 , 31 ). As the only AP complex localized to the plasma membrane, AP-2 is an obvious candidate for mediating rapid internalization through recognition of YXXØ signals. Recently, Nesterov et al. have provided compelling evidence for a role of μ2 in this process using a dominant negative genetic approach ( 26 ). These investigators constructed a μ2 variant with mutations in D 176 and W 421 , which are critical elements of the YXXØ-binding site . This mutant μ2 was unable to bind YXXØ signals but competed with endogenous μ2 for incorporation into the AP-2 complex. Interestingly, overexpression of mutant μ2 inhibited internalization of the transferrin receptor ( 26 ), which is known to be mediated by the YXXØ-type signal YTRF ( 7 ). The intracellular localization of the AP-3 complex is not known with certainty, although published evidence suggests an association with endosomes and/or the TGN ( 8 , 10 , 37 , 38 ). Evidence for a role of AP-3 in sorting mediated by YXXØ signals has recently been obtained from the analysis of AP-3–deficient cells. These cells were either generated by using an antisense RNA methodology ( 20 ) or derived from two patients with Hermansky-Pudlak syndrome carrying mutations in the AP-3 β3A subunit ( 11 ). In both cases, the AP-3 deficiency resulted in increased routing of YXXØ-containing, lysosomal membrane proteins through the plasma membrane, thus suggesting a function for AP-3 in YXXØ-mediated targeting to lysosomes. In contrast, the trafficking of non-lysosomal membrane proteins having YXXØ signals (e.g., the transferrin receptor) was not noticeably altered ( 11 ). This differential effect, which is consistent with the preference of the AP-3 μ3A subunit for YXXØ signals found in lysosomal membrane proteins ( 11 , 27 , 39 ), lends support to the notion that selective interaction with AP complexes underlies the functional specificity of YXXØ signals. The fact that a substantial fraction of lysosomal membrane proteins are still targeted to lysosomes in AP-3–deficient cells ( 11 , 20 ) suggests that other AP complexes may provide alternative means of delivery to lysosomes. Perhaps this is a function of AP-1, or of the recently described AP-4 complex, which appears to be localized to the TGN or a neighboring compartment ( 9 ). In conclusion, the hypothesis advanced to explain the involvement of YXXØ signals in multiple sorting events can now be made more explicit: YXXØ signals are recognized with characteristic preferences by the medium (μ) subunits of several AP complexes. The factors that determine the fidelity of sorting processes in vivo, however, remain poorly understood. First, although each μ subunit displays preferences for certain X and Ø residues, there is nonetheless a significant overlap in sequence specificity ( 27 ). Contextual factors such as the position of the signal within the cytosolic domain ( 35 ), the oligomeric state of the transmembrane protein ( 3 ), and the presence of other signals in the cytosolic domain, may contribute to differential interactions with the AP complexes. Second, there still may be additional YXXØ-binding proteins to be discovered. As discussed above, μ4 is a likely candidate for one such molecule. Finally, transmembrane proteins moving along trafficking pathways may meet the AP complexes sequentially rather than simultaneously. This means that the trajectory followed by a protein, as well as potential biochemical modifications along the way, may determine which interactions actually take place. Further research will be needed to assess the contribution of these factors to the selectivity of sorting by YXXØ signals. With a solid molecular foundation now in place, however, we can anticipate rapid progress toward the decipherment of this protein sorting code. | Review | biomedical | en | 0.999997 |
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